Suppression of apoptotic and necrotic cell death by poliovirus

To determine an antiapoptotic activity of poliovirus type 1 (PV-1), we examined the effect of PV-1 infection on apoptosis that was induced in HEp-2 cells by the treatment with 1 M sorbitol. The virus did not induce apoptosis in the infected cells and could suppress both the fragmentation of chromosomal DNA and morphological cell and cell nuclei changes in the sorbitol-treated cells, indicating that PV-1 induces an antiapoptotic state. Comparison of the kinetics showed that this ability of the virus appeared in the infected cells at the time of progeny virus formation (maturation step of virus multiplication). Simultaneously with this antiapoptotic activity, PV-1 infection also suppressed non-apoptotic cell death induced by sodium chloride. Electron microscopic observation revealed that the cells killed by the sodium chloride treatment had undergone liquefactive necrosis, indicating that PV-1 can inhibit both apoptosis and necrosis. In addition, PV-1 can grow in the apoptotic cells, although the virus yield was reduced to a quarter of the yield in normal cells.


Growth characteristics of SHIV without the vpu gene

NM-3 is a prototype of chimeric virus between simian and human immunodeficiency viruses (SHIV). It grows in monkey lymphocytic cells in vitro and in vivo as well as in human cells. A mutant designated NM3-E65, which lacks expression of the entire vpu gene, was constructed from NM-3, and monitored for its replication property. Examination of growth properties in simian and human cells of SHIV, HIV, and their vpu mutants revealed that the vpu gene in the genome of the chimeric virus is functional, but non-essential for virus replication.

International Journal of Molecular Medicine


The Human Immunodeficiency Virus Type 1 Accessory Protein Vpu Induces Apoptosis by Suppressing the Nuclear Factor B dependent Expression of Antiapoptotic Factors

Human immunodeficiency virus (HIV) type 1 Vpu is an integral membrane protein with a unique affinity for betaTrCP (TrCP), a key member of the SkpI-Cullin-F-box E3 ubiquitin ligase complex that is involved in the regulated degradation of cellular proteins, including IkappaB. Remarkably, Vpu is resistant to TrCP-mediated degradation and competitively inhibits TrCP-dependent degradation of IkappaB, resulting in the suppression of nuclear factor (NF)-kappaB activity in Vpu-expressing cells. We now report that Vpu, through its interaction with TrCP, potently contributes to the induction of apoptosis in HIV-infected T cells. Vpu-induced apoptosis is specific and independent of other viral proteins. Mutation of a TrCP-binding motif in Vpu abolishes its apoptogenic property, demonstrating a close correlation between this property of Vpu and its ability to inhibit NF-kappaB activity. The involvement of NF-kappaB in Vpu-induced apoptosis is further supported by the finding that the levels of antiapoptotic factors Bcl-xL, A1/Bfl-1, and TNF receptor-associated factor (TRAF)1, all of which are expressed in an NF-kappaB-dependent manner, are reduced and, at the same time, levels of active caspase-3 are elevated. Thus, Vpu induces apoptosis through activation of the caspase pathway by way of inhibiting the NF-kappaB-dependent expression of antiapoptotic genes.


Lack of apoptosis in Sendai virus-infected HEp-2 cells without participation of viral antiapoptosis gene

Sendai virus (SeV) has been reported to induce apoptosis in many types of cells. In HEp-2 cells, however, it did not induce apoptosis in most of the infected cells under the conditions in which vesicular stomatitis virus induced massive apoptosis. The use of a novel technique, which allows the detection of viral antiapoptotic activity in the infected cells, showed that SeV does not have any antiapoptotic activity to interfere with the induction of apoptosis. Consistently, vesicular stomatitis virus-induced apoptosis was not interfered with by preinfection with SeV. These results indicate that the observed lack of apoptosis in these SeV-infected cells does not result from the suppression of apoptosis by viral antiapoptotic activity in the infected cells and suggest that, without activating a signaling pathway for the induction of apoptotic response in the infected cells, SeV can escape apoptosis of the cells, allowing long-term survival of the infected cells.


Analysis of the cell-dependent replication potentials of human immunodeficiency virus type 1 vif mutants

Eleven in-frame vif gene mutants of HIV type 1 produced in non-permissive cells were examined for their replication potentials in various CD4-positive and -negative cell lines. Virus replication for each mutant was monitored by using several single- and multiple-cycle infectivity assays. Except for a mutant with wild-type phenotype, most mutants were severely defective for replication in all the cell lines as expected from the producer cell-dependent functioning of Vif so far reported. In contrast, two mutants, which have mutations in the hydrophilic or effector regions of Vif were found to have target cell-dependent replication potentials. These results demonstrate the presence of a novel category of the vif mutants important for elucidation of the Vif function.


Cyclophilin A-Independent Replication of a Human Immunodeficiency Virus Type 1 Isolate Carrying a Small Portion of the Simian Immunodeficiency Virus SIV(MAC) gag Capsid Region

Hybrid viruses between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus strain mac (SIV(MAC)) are invaluable to various fields of HIV-1 research. To date, however, no replication-competent HIV-1 strain containing the gag capsid (CA) region of SIV(MAC) has been reported. To obtain the viable gag gene chimeric virus in an HIV-1 background, seven HIV-1 strains carrying a part of SIV(MAC) CA or a small deletion in the CA region were constructed and examined for their biological and biochemical characteristics. While all the recombinants and mutants were found to express Gag and to produce progeny virions on transfection, only one chimeric virus, which has 18 bp of SIV gag CA sequence in place of the region encoding the HIV-1 CA cyclophilin A (CyPA)-binding loop, was infectious for human cell lines. Although this chimeric virus was unable to grow in monkey lymphocytic cells like wild-type (wt) HIV-1 did, it grew much better than wt virus in the presence of cyclosporin A in a human cell line which supports HIV-1 replication in a CyPA-dependent manner. These results indicate that the transfer of a small portion of the SIV(MAC) CA region to HIV-1 could confer the CyPA-independent replication potential of SIV(MAC) on the virus.


A single amino acid change at Leu-188 in the reverse transcriptase of HIV-2 and SIV renders them sensitive to non-nucleoside reverse transcriptase inhibitors

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are selective for human immunodeficiency virus type 1 (HIV-1) and generally not effective on HIV-2 or simian immunodeficiency virus (SIV). Only SIVagm was found to be sensitive to NNRTIs. When the amino acid differences in RT between SIVmac and SIVagm were compared with the known amino acid substitutions of NNRTI-resistance variants of HIV-1, we came to consider that the amino acid residue Leu-188 of HIV-2 and SIVmac might be related to their resistance to NNRTIs. To test this hypothesis, we substituted Leu-188 to Cys or Tyr in HIV-2 and SIVmac, and examined sensitivity of the mutant molecular clones to NNRTIs. The L188Y mutant of HIV-2 became completely sensitive to delavirdine and efavirenz, while that of SIVmac was also significantly sensitive to these NNRTIs. We further isolated NNRTI-resistant variants from these mutant viruses and determined amino acid substitutions in RT. The roles of the observed substitutions in NNRTI-resistance were further confirmed by site-directed mutagenesis. Our study reveals the crucial role of L188 in the natural resistance of HIV-2 and SIVmac to NNRTIs. Furthermore, the observed substitutions in RT of HIV-2 and SIVmac support the common mechanism of action of NNRTIs against HIV-1, HIV-2 and SIV.


Isolation and characterization of simian immunodeficiency virus variants that are resistant to nonnucleoside reverse transcriptase inhibitors

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) act quite specifically on human immunodeficiency virus type 1 (HIV-1). In general, they are not effective on human immunodeficiency virus type 2 (HIV-2) or simian immunodeficiency virus (SIV). Only SIV strains from African green monkeys are sensitive to several NNRTIs. Here we isolated NNRTI- and 3TC-resistant SIVagm variants. Viruses resistant to delavirdine contained V112I and M231I substitutions, while those resistant to 3TC contained a M 185I substitution. These amino acids are highly conserved in HIV-1, HIV-2, SIVmac and SIVagm, and the M184I (M185I in SIVagm) substitution was observed in 3TC-resistant HIV-1 and SIVmac. The roles of the observed mutations in NNRTI-resistance of SIVagm and HIV-1 were further confirmed by site-directed mutagenesis. The present results have provided a new insight into the common mechanism of sensitivity of HIV- 1 and SIVagm to NNRTIs.


Recombinant p51 as Antigen in an Immune Complex Transfer Enzyme Immunoassay of Immunoglobulin G Antibody to Human Immunodeficiency Virus Type 1

An ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of antibody immunoglobulin G (IgG) to human immunodeficiency virus type 1 (HIV-1) has been developed using recombinant HIV-1 reverse transcriptase (rRT) as antigen. However, some disadvantages were noted in the use of rRT as antigen: rRT was produced only with low efficiency in widely used strains of Escherichia coli using a rather long DNA fragment (3,012 bp) of the whole HIV-1 pol gene, and it was impossible to produce fusion proteins of RT for simple purification, since rRT is a heterodimer of p66 and p51. In this study, recombinant HIV-1 p51 and p66 with Ser-Ser at the N termini (Ser-Ser-rp51 and Ser-Ser-rp66) were produced in E. coli as fusion proteins with maltose binding protein containing a factor Xa site between the two proteins and were purified after digestion with factor Xa. Ser-Ser-rp51 was produced in larger amounts and purified in higher yields with less polymerization than Ser-Ser-rp66. Polymerized Ser-Ser-rp66 tended to be precipitated on mercaptoacetylation for conjugation to beta-D-galactosidase (used as a label) and showed higher nonspecific and lower specific signals in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 than Ser-Ser-rp51. The signals for serum samples of HIV-1-seropositive subjects by immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 using Ser-Ser-rp51 as antigen (Y) were well correlated to those obtained using rRT as antigen (X) (log Y = 0.99 log X + 0.23; r = 0.99). Thus, the use of rp51 as antigen was advantageous over that of rp66 and rRT in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1.

Clinical and Diagnostic Laboratory Immunology


Cell-dependent gag mutants of HIV-1 are crucially defective at the stage of uncoating/reverse transcription in non-permissive cells

We have previously shown that some of the human immunodeficiency virus type 1 (HIV-1) gag matrix (MA), capsid (CA), and nucleocapsid (NC) mutants display host-cell-dependent replication potential, and that they are defective at the early phase of the virus replication cycle in non-permissive cells. To determine the defective replication stage of the cell-dependent mutants precisely, the processes of virus entry into cells and virus DNA synthesis were monitored by the highly sensitive enzyme-linked immunosorbent assay and polymerase chain reaction amplification analysis. The results obtained indicated that all the cell-dependent MA, CA and NC mutants are defective at the stage of uncoating/reverse transcription, and that a cellular factor(s) is involved in this process.


Mutational analysis of HIV-1 gag proteins

The mature Gag proteins of human immunodeficiency virus type 1 (HIV-1) are major components of infectious virions, and thought to carry out numerous functions throughout the HIV-1 replication cycle. We have recently generated numerous gag gene mutants of HIV-1 to genetically study the functions of the Gag proteins. Through the biological and biochemical analyses, our HIV-1 gag mutants have been grouped into early (defective for uncoating/reverse transcription), late (defective for virion release/maturation), and early/late (defective for both steps) mutants. Many mutants are found to efficiently inhibit the replication of wild-type virus. Worthy of note, there are some early mutants which show host cell-dependent replication potential.

International Journal of Molecular Medicine


Preparations of recombinant HIV-1 p66 antigen to improve the specificity of immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 reverse transcriptase

Recombinant HIV-1 p66 (rp66, a subunit of reverse transcriptase (RT), a heterodimer of p66 and p51) was produced in Escherichia coli in three different ways. First, rp66 was produced as a part of the fusion protein of lacZ protein and HIV-1 pol protein consisting of three components: protease (p10), RT (p51/p66), and integrase (p31), and was released from the fusion protein by the protease (pol-rp66). Second, rp66 with Ser-Ser at the N-terminus was produced as a fusion protein with maltose-binding protein containing a factor Xa site between the two proteins (MBP-Ser-Ser-rp66) and was released from the fusion protein by factor Xa (Ser-Ser-rp66). Third, rp66 with Met-Gly at the N-terminus was produced in transformed cells (Met-Gly-rp66). The recombinant proteins were purified from sonic extracts of transformed cells by ammonium sulfate fractionation and various column chromatographies. MBP-Ser-Ser-rp66 and Met-Gly-rp66 were readily purified in sufficient amounts for labeling with 2, 4-dinitrophenyl groups and beta-D-galactosidase from E. coli, but pol-rp66 and Ser-Ser-rp66 were not for enzyme-labeling. Ser-Ser-rp66 was not only polymerized but also degraded to considerable extents. The purified preparations were labeled with 2,4-dinitrophenyl groups and beta-D-galactosidase and were tested in immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 RT using serum samples from 600 HIV-1 seronegative and 30 HIV-1 seropositive subjects. Among various combined uses of the two labeled preparations, the uses of 2,4-dinitrophenylated MBP-Ser-Ser-rp66 and pol-rp66 with beta-D-galactosidase-labeled Met-Gly-rp66 showed the highest (99.8%) and the second highest (99.5%) specificities, which were higher than that with the labeled preparations used in the previous study (98. 0%).


Physiological significance of apoptosis in animal virus infection

In contrast to insect viruses, animal viruses can produce considerable amounts of progeny virus in cells undergoing apoptosis. Nevertheless, viruses in general have acquired the ability to escape apoptosis of infected cells. These facts indicate that the role of apoptosis in virus infection is different in insect virus and animal virus, although both viruses need to avoid apoptosis of the infected cells for a viral life cycle in nature. In animal virus infection, the primary role of apoptosis is considered not to be a premature lysis of the infected cells (and the following abortion of virus multiplication) but to allow the dying cells to be phagocytosed by macrophages. This phagocytosis is able to prevent dysregulated inflammatory reactions at the site of virus infection and to initiate a specific immune response against the infected virus.


Host cell-dependent replication of HIV-1 mutants with deletions in gp41 cytoplasmic tail region is independent of the function of Vif


MHC-I expression in HTLV-1-positive and -negative cells

The expression level of major histocompatibility class I (MHC-I) and the extent of down-regulation of MHC-I after an anti-MHC-I antibody treatment in numerous human T-cell leukemia virus type 1 (HTLV-1)-positive and -negative lymphocytic cell lines were examined. While there was no clear correlation between the expression level of MHC-I and the presence of HTLV-1 genome, a relatively low level of MHC-I down-regulation was generally induced in HTLV-1-positive cells by the antibody. The results may suggest the potential involvement of MHC-I in HTLV-1 leukemogenesis.


Regulation of cell cycle and apoptosis by human immunodeficiency virus type 1 Vpr.

Biological effects of HIV-1 Vpr on CD4(+) cells were studied by an infection system. High-titered HIV-1 stocks pseudotyped with vesicular stomatitis virus G protein were prepared and used to inoculate into CD4(+ )T cells at high multiplicity of infection. Both cell- and virion-associated Vpr were demonstrated to arrest the cell cycle at the G2/M phase, and to induce cell apoptosis. Of note, morphologically apoptotic cells were shown to be arrested at the G2/M stage. No appreciable effect of Vpr on the anti-Fas antibody-mediated apoptosis was observed in this system.


Cell-dependent requirement of human immunodeficiency virus type 1 gp41 cytoplasmic tail for Env incorporation into virions.

Growth kinetics in lymphocytic H9 and M8166 cells of two mutants of human immunodeficiency virus type 1 (HIV-1) with deleted gp41 cytoplasmic tails were examined. While the mutant viruses designated CTdel-44 and CTdel-144 were able to grow in M8166 cells, they were unable to grow in H9 cells. Transfection and single-round infectivity assays demonstrated that they are defective in the early phase of viral replication in H9 cells. Analysis of the mutant virions revealed drastically reduced incorporation of Env gp120 (compared with the incorporation of wild-type virions) in H9 cells but normal incorporation in M8166 cells. These results indicate that the HIV-1 cytoplasmic tail of gp41 determines virus infectivity in a cell-dependent manner by affecting incorporation of Env into virions and suggest the involvement of a host cell factor(s) in the Env incorporation.


Characterization of apoptosis induced by sorbitol: a unique system for the detection of antiapoptotic activities of viruses

The treatment of HEp-2 cells with sorbitol induced massive apoptosis rapidly. This method for inducing apoptosis is very useful to detect antiapoptotic activity of viruses as well as viral genes. Commitment to death occurred immediately upon incubation with sorbitol, even in the presence of pancaspase inhibitor, Z-VAD-FMK. Apoptosis is also induced by other polyhydric alcohols with more than four hydroxyl groups, but not induced by glycerol or ethylene glycol. Sorbitol treatment on ice did not induce apoptosis either. These results suggest that this induction of apoptosis does not result simply from high osmotic pressure but probably by the interaction of solutes through their physical nature (such as hydrophobicity) with the plasma membrane of the cells.


Nef-Induced Major Histocompatibility Complex Class I Down-Regulation Is Functionally Dissociated from Its Virion Incorporation, Enhancement of Viral Infectivity, and CD4 Down-Regulation

The N-terminal alpha-helix domain of the human immunodeficiency virus type 1 (HIV-1) Nef protein plays important roles in enhancement of viral infectivity, virion incorporation of Nef, and the down-regulation of major histocompatibility complex class I (MHC-I) expression on cell surfaces. In this study, we demonstrated that Met 20 in the alpha-helix domain was indispensable for the ability of Nef to modulate MHC-I expression but not for other events. We also showed that Met 20 was unnecessary for the down-regulation of CD4. These findings indicate that the region governing MHC-I down-regulation is proximate in the alpha-helix domain but is dissociated functionally from that determining enhancement of viral infectivity, virion incorporation of Nef, and CD4 down-regulation.


The H9/M8166 tropism of various HIV-1 mutants is determined by distinct cellular factors

We have previously shown that a number of human immunodeficiency virus type 1 (HIV-1) mutants generated in vitro display a replication-defect in a cell-dependent manner. Of the mutants of this category, those of gag, vif, and env mutants do not grow at all in lymphocytic H9 (non-permissive) cells, but quite well in M8166 (permissive) cells. To determine whether the cell-dependent growth of the mutants is functionally related to each other, a number of double mutants were constructed, and monitored for their replication and biochemical property in various cells. The results obtained have indicated that there are multiple cellular factors responsible for the growth phenotype. Together with our previous findings on the host cell-dependent mutants of HIV-1, it is concluded that number of distinct cellular factors are involved in the various steps in HIV-1 replication cycle.

International Journal of Molecular Medicine


Nef-induced major histocompatibility complex class 1 down-regulation is functionally dissociated from its virion incorporation, enhancement of virul infectibity, and CD4 down-regulation.


Small Amino Acid Changes in the V3 Loop of Human Immunodeficiency Virus Type 2 Determines the Coreceptor Usage for CXCR4 and CCR5

HIV-2 GH-1 is a molecular clone derived from an AIDS patient from Ghana. In contrast to the prototypic molecular clone ROD, GH-1 exhibits a narrow range of target cell specificity. By an infectious assay using HeLa-CD4 cells stably transfected with an HIV-1 LTR-beta-galactosidase reporter gene and transiently expressing various cloned chemokine receptors, we have examined the coreceptor usage of GH-1. In contrast to ROD, which uses principally CXCR4, GH-1 was found to use mainly if not exclusively CCR5 but not CXCR4. The distinct coreceptor usage of these two molecular clones allowed us to further map the region of gp120 that is important for the coreceptor specificity. By constructing a series of chimeric viruses between GH-1 and ROD, we have demonstrated that the C-terminal half of the V3 loop region of gp120 determines the differential coreceptor usage between GH-1 and ROD, and only a few amino acid differences in this region appear to be able to shift the specificity between CCR5 and CXCR4. Notably, the shift in the coreceptor usage from CCR5 to CXCR4 is associated with an increase in the net positive charge in the V3 region.


Pseudotyping human immunodeficiency virus type 1 by vesicular stomatitis virus G protein does not reduce the cell-dependent requirement of Vif for optimal infectivity: functional difference between Vif and Nef

The functions of Vif and Nef in human immunodeficiency virus type 1 (HIV-1) infection have some similarities: Vif- and Nef-dependent enhancement of HIV-1 replication is cell type-specific, and defective mutations in these genes result in restricted proviral DNA synthesis in infected cells. It has recently been shown that pseudotyping HIV-1 by the envelope glycoprotein of vesicular stomatitis virus (VSV-G) targets HIV-1 entry to an endocytic pathway and suppresses the requirement of Nef for virus infectivity. In this study, we examined whether VSV-G pseudotyping suppresses the requirement of Vif for HIV-1 infectivity. It was found that pseudotyping HIV-1 by VSV-G did not compensate for the Vif function. Together with the findings that Vif does not influence virus binding/entry and virion incorporation of Env, it is concluded that Vif enhances HIV-1 infectivity at the post-entry step(s) independently of the Env function by a different mechanism to that of Nef.


Comparison of an antiviral activity of recombinant consensus interferon with recombinant interferon-α-2b

To avoid possible uncertainty in comparing biological activities of interferon samples from different sources where interferon concentrations were determined independently, we prepared chromatographically pure preparations of consensus interferon and interferon-alpha-2b (one of the two commercially available recombinant alpha interferons). We revealed that consensus interferon has a stronger antiviral activity than interferon-alpha-2b, although the effects of these two recombinant interferons on the cellular macromolecule synthesis are at similar levels.


Induction of Apoptosis in Herpesvirus saimiri-Immortalized T Lymphocytes by Blocking Interaction of CD28 with CD80/CD86

Biochemical and Biophysical Research Communications


Cell-dependent replication potentials of HIV-1 gag mutants

An infectious molecular clone of human immunodeficiency virus type 1 (HIV-1), designated pNLaiKH, which is tropic for both lymphocytic and monocytic cells, was constructed. To study the early function of HIV-1 Gag proteins in two types of cells, the mutations known to give host cell-dependent early defects were introduced into pNLaiKH, and the replication potentials and defective replication sites in the cells of the resultant mutants were monitored. All mutants grew in some lymphocytic cells, but not at all in monocytic cells. A nucleocapsid mutant was found to be defective at an early replication phase in all the cell lines to various extent, as expected. In contrast, a matrix mutant and a capsid mutant displayed a replication defect in a producer-cell-dependent manner. These results demonstrated that complex interactions of cell factors and Gag proteins are involved in an early process of HIV-1 replication.


Antiapoptotic activity of herpes simplex virus type 2: the role of US3 protein kinase gene

In order to determine the ability of herpes simplex virus type 2 (HSV-2) to suppress apoptosis, we examined the effect of HSV-2 infection on apoptosis induced in HEp-2 cells by treatment with 1 M sorbitol. Although a wild-type strain of HSV-2 induced apoptosis in a significant fraction of the infected cells, HSV-2 could suppress sorbitol-induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV-1), indicating that HSV-2, like HSV-1, has an antiapoptosis gene. Characterization of the cells infected with a US3-deletion mutant of HSV-2 revealed the necessity of a US3 gene in the antiapoptotic activity of this virus.


Inhibition of HIV/SIV Replication by Dominant Negative Gag Mutants

There are several major strategies against HIV/AIDS. Of these, the gene therapy is a novel, challenging, and promising one. The target genes, which have been extensively studied for the potential gene therapy of HIV/AIDS, include those of cellular and viral origins. Especially, trans-dominant negative Tat, Rev, Env, Pol, and Gag mutants of HIV have currently attracted considerable attention. In this brief review, we summarize the nature of the HIV/SIV mutants of this category and discuss their future use for gene therapy with special reference to the dominant negative Gag mutants of HIV-1.


Effects of SIVmac Infection on Peripheral Blood CD4+CD8+T Lymphocytes in Cynomolgus Macaques

We have previously reported that CD4+CD8+ double-positive (DP) T cells with a resting memory phenotype exist in a substantial proportion of peripheral blood lymphocytes of adult cynomolgus macaques. In this study, we examined the effects of simian immunodeficiency virus of macaque (SIVmac) infection on DP T cells. In vitro, SIVmac239 nef-open (239) and its nef-deletion mutant replicated well in both CD4+CD8- and DP T cells. However, when the macaques were infected with 239, DP, but not CD4+CD8-, T cells were transiently increased in parallel with cell activation and viral replication, followed by depletion within 1 month postinfection. Interestingly, the nef gene was required for depletion but not for the increase and activation of DP T cells. These data suggest that the pathogenic SIV infection may downmodulate production and/or blood circulation of DP T cells by a Nef function-related mechanism(s) different from that for the depletion of CD4+CD8- T cells.


Host cell-dependent replication of HIV-1 gag MA, CA, and NC mutants are independent of the functions of Vif and Vpu

We have previously shown that some gag gene mutants of human immunodeficiency virus type 1 (HIV-1) display a replication-defect in a cell-dependent manner. We and others have also demonstrated that the requirement of vif and vpu genes for HIV-1 replication is cell-dependent. To determine whether the cell-dependent growth of the HIV-1 gag mutants is related to the functions of Vif and Vpu, double mutants of gag-vif and gag-vpu were constructed, and monitored for their replication in various cell lines. The results obtained showed that the mutations in gag do not affect the cell-dependent functions of Vif and Vpu.

International Journal of Molecular Medicine


The Cellular Kinase Binding Motifs (PxxP and RR) in Human Immunodeficiency Virus Type 1 Nef Protein Are Dispensable for Producer-Cell-Dependent Enhancement of Viral Entry

We have recently demonstrated that human immunodeficiency virus type 1 (HIV-1) Nef is required for enhancing viral infectivity by increasing the efficiency of viral entry in a producer-cell-dependent manner, suggesting the possible involvement of a cellular factor(s) in the enhancement of viral entry. Moreover, it has been reported that a proline-rich (PxxP) motif and an Arg-Arg (RR) motif in HIV-1 Nef bind to the SH3 domain of the Src-family tyrosine kinase Hck and to a serine/threonine kinase, respectively. To address whether these cellular kinase binding motifs, PxxP and RR, could be involved in virus producer-cell-dependent enhancement of viral entry, we constructed two nef mutant proviral clones in which these motifs were mutated. The results show that the HIV-1 Nef PxxP motif, which significantly influenced viral infectivity, and the RR motif, which modestly affected viral infectivity, were both dispensable for enhanced viral entry, thus suggesting that another interaction of Nef with a cellular factor(s) is involved in the efficiency of viral entry.


Cell-dependent function of HIV-1 Vif for virus replication

It has been well established that the Vif protein of human immunodeficiency virus type 1 (HIV-1) acts late in the viral life cycle and increases the infectivity of the progeny virions in a producer cell-dependent manner. The virions produced in the absence of Vif in non-permissive cells (Delta Vif) are defective for a step(s) before and/or during reverse transcription. In this review, the functional and structural analyses of these virions including our new data are summarized.


Compatibility of Vpu-like Activity in the Four Groups of Primate Immunodeficiency Viruses


Mutational Analysis of Human Immunodeficiency Virus Type 1 Vif Gene

Mutations were introduced into scattered regions of the HIV-1 vif gene. The twelve in-frame mutants generated were evaluated for the replication potentials in cells by transfection and infection experiments. All the mutants produced a normal level of progeny virions upon transfection, indicating the absence of the late function of HIV-1 Vif protein. The infectivity of virions obtained was monitored in H9 cells, which are non-permissive for HIV-1 without the Vif function. Most of the mutations in various parts of the vif gene, including those in the three conserved regions among HIV/SIV, abrogated the infectivity of the virus. In contrast, the cysteine residue at position 133, which was reported to be critical for viral infectivity, was found not to be essential. In addition, the C-terminal eight amino acid residues (185-192) in the Vif protein could be deleted with no effects on viral growth potential.


Cell-dependent functional roles of HIV-1 Nef for virus replication


Activation of HIV-1 enhancer sequence by vaccinia virus

To investigate whether vaccinia virus (VV) can augment gene expression of human immunodeficiency virus type 1 (HIV-1), co-transfection experiments were carried out in which recombinant plasmids containing various portions of the HIV-1 long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) gene were transfected into cultured cells. A high level of enhancement in CAT activity directed by the HIV-1 LTRs containing the enhancer sequence was observed in cells infected with VV, as in the cells infected with type 1 herpes simplex virus (HSV-1). The sequence responsible for this augmentation of CAT activity was different from that recognized by HIV-1 Tat. These data clearly demonstrated that VV transactivates HIV-1 LTR through a mechanism distinct from that of activation by HIV-1 Tat.


Rev-dependent expression of three species of HIV-1 mRNAs

The expression of structural and accessory genes of human immunodeficiency virus type 1 (HIV-1) except for nef requires a viral regulatory protein Rev. Rev-dependency of the expression of structural (gag, pol and env), regulatory (tat and rev), and accessory genes (vif, vpr, vpu and nef) has been investigated by various systems, and it has been demonstrated that unspliced (encodes gag and pol) and singly-spliced (env-vpu, vif and vpr) viral mRNAs are differentially dependent on the function of Rev. In this review, the function of HIV-1 Rev in relation to these findings is discussed.


Growth characteristics of T-cell tropic HIV-1 vpu gene mutants in human peripheral blood mononuclear cells

A mutant designated NL-E65, which lacks the expression of entire vpu gene, was constructed from T-cell tropic wild-type (wt) human immunodeficiency virus type 1 (HIV-1) clone and monitored for its replication property in human cells, along with a mutant NL-Ss which expresses a C-terminal truncated Vpu. The mutant NL-Ss could grow in two cell lines and in all peripheral blood mononuclear cell (PBMC) preparations to some extent, with kinetics similar to those of wt virus. Likewise, the mutant NL-E65 exhibited a replication property typical to the vpu mutant in the two cell lines and in all PBMC cultures, growing at a low level. Along with the results previously reported, these data indicate that HIV-1 Vpu is dispensable for virus replication in any of the types of cells so far tested.

The Journal of Medical Investigation : JMI


Exchangeability of accessory Vif and Vpu proteins between various HIV/SIVs

International Journal of Molecular Medicine


Rapid formation of the immune complexes on solid phase in the immune complex transfer enzyme immunoassay for HIV-1 p24 antigen and antibody IgGs to HIV-1

In order to perform the immune complex transfer enzyme immunoassays for HIV-1 p24 antigen and antibody IgGs to HIV-1 p17, reverse transcriptase and gp41 antigens as rapidly as possible, methods for rapid formation of the immune complexes on solid phase are described. HIV-1 p24 antigen was reacted with monoclonal anti-p24 Fab'-beta-D-galactosidase conjugate at a high concentration and subsequently with polystyrene beads coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-biotinyl bovine serum albumin-affinity-purified anti-p24 Fab' conjugate. Antibody IgGs to HIV-1 were reacted with polystyrene beads coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-HIV-1 antigen conjugates and subsequently with HIV-1 antigen-beta-D-galactosidase conjugates. The periods of time used for the formation of the immune complexes comprising the three components on the polystyrene beads (15-30 min) were much shorter than those used in the previous immune complex transfer enzyme immunoassays (90-300 min), and the sensitivities of the present and previous immune complex transfer enzyme immunoassays were similar. The detection limit of the HIV-1 p24 antigen by the present and previous methods were similar (3 to 10 zmol/assay).


Ultrasensitive and rapid enzyme immunoassay (thin aqueous layer immune complex transfer enzyme immunoassay) for antibody IgG to HIV-1 p17 antigen

The immune complex transfer enzyme immunoassay for antibody IgG to HIV-1 p17 antigen was performed in two different ways (the present immunoassays I and II) within shorter periods of time than previously reported. In the present (simultaneous) immunoassay I, antibody IgG to HIV-1 p17 antigen in 10 microL of serum samples was incubated simultaneously with 2,4-dinitrophenyl-maltose binding protein-recombinant p17(rp17) fusion protein and rp17-beta-D-galactosidase conjugate in a total volume of 22 microL for 10 min to form the immune complex comprising the three components. The reaction mixture was incubated with a polystyrene bead of 6.35 mm in diameter coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 5 min in a styrol test tube (13.3 x 54 mm and 2.1 g) to trap the immune complex. After washing, the polystyrene bead was incubated with 30 microL of epsilonN-2,4-dinitrophenyl-L-lysine solution in a polystyrene tube (12 x 75 mm) coated with affinity-purified (antihuman IgG gamma-chain) IgG for 10 min to transfer the immune complex. In the present (sequential) immunoassay 11, a polystyrene bead of 6.35 mm in diameter coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-maltose binding protein-rp17 fusion protein was incubated in a styrol test tube (13.3 x 54 mm and 2.1 g) sequentially with antibody IgG to HIV-1 p17 antigen in 10 microL of serum samples in a total volume of 16 microL for 5 min and subsequently with rp17-beta-D-galactosidase conjugate in a volume of 10 microL for 5 and 10 min. The immune complex formed on the polystyrene bead was transferred to a polystyrene tube coated with affinity-purified (antihuman IgG gamma-chain) IgG for 5 and 10 min in the same way as in the present immunoassay I. During the incubations, the styrol test tubes containing the polystyrene beads and reaction mixtures were shaken, and the polystyrene test tubes were rotated with shaking, so that the polystyrene beads were rotated randomly, and small drops (16 to 30 microL) of the reaction mixtures evenly contacted all parts of the solid phase surfaces during the incubations, though only small parts of the solid phase surfaces were contacted at one time. The intent was to continuously mix thin aqueous layers of the reaction mixtures covering the solid phase surfaces with the rest of the reaction mixtures. (Therefore, these immunoassays were called thin aqueous layer immunoassays.) beta-D-Galactosidase activity bound to the polystyrene tubes was assayed by fluorometry for 30 and 60 min. The present immunoassays I and II, in which only 15 to 25 min were used for the immunoreactions, were as sensitive if not more so than the previous immune complex transfer enzyme immunoassay requiring 150 min for the immunoreactions. In these earlier immunoreactions, the immune complex comprising the three components formed by 30 min incubation was trapped onto two polystyrene beads (3.2 mm in diameter) coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 60 min, and was then transferred to two polystyrene beads (3.2 mm in diameter) coated with affinity-purified (antihuman IgG y-chain) IgG for 60 min in a total volume of 150 microL. Furthermore, the present (sequential) immunoassay 11 (and probably I) could become approximately 10 times more sensitive by assaying bound beta-D-galactosidase activity for a longer period of time (10 h), since beta-D-galactosidase activity, bound nonspecifically in the presence of serum samples from HIV-1 seronegative subjects, was considerably low.


Induction of apoptosis in HEp-2 cells by infection with herpes simplex virus type 2

Although herpes simplex virus type 1 (HSV-1) does not induce apoptosis in infected HEp-2 cells, herpes simplex virus type 2 (HSV-2) did induce apoptosis in a small but significant fraction of the same cells. Apoptosis was not observed in Vero or HeLa cells infected with HSV-2. In addition, HSV-2 infection in the presence of cycloheximide induced extensive apoptosis of HEp-2 or HeLa cells.


The tropism of human and simian immunodeficiency viruses (HIV and SIV) determined by sequences other than env has been studied. The restriction of HIV-1 replication in monkey cells was demonstrated to be regulated by viral non-env sequence. Likewise, the gag-pol region of SIVagm (virus of the African green monkey) genome was found to be responsible for growth restriction in human cells of the virus. No viral DNA synthesis was detected in cells nonpermissive for the viruses. In addition, a number of HIV-1 gag gene mutants, which have an early defect in viral replication cycle and direct no viral DNA synthesis in some cells, exhibited a phenotype of host range mutant. Taken together, it can be concluded that the viral tropism associated with the uncoating/ reverse transcription process does exist in HIV/SIV replication. Furthermore, many of the accessory gene mutants of HIV/SIV exhibit host cell-dependent replication property. In this review, we summarize these examples of non-env tropism of HIV/SIV.


Functional roles of HIV accessory proteins for viral replication

Numerous lentiviruses, including human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) of causative agents of human AIDS as representative, have been recently isolated from various species of primates. The fundamental and most prominent feature of the viruses is the presence of a number of accessory genes in their genomes. Extensive biological and biochemical studies have demonstrated that the accessory gene products are not essential for viral replication at least in certain types of cells. Quite surprisingly, some of these accessory proteins are absolutely non-essential in any types of cells so far examined. In this brief review, our systematic genetic studies on the importance of the accessory proteins of HIV-1 and HIV-2 for viral replication are described and discussed.


Enhancement of human immunodeficiency virus type 1 infectivity by Nef is producer cell-dependent

The growth kinetics of wild-type and nef mutant viruses of human immunodeficiency virus type 1 were comparatively analysed in several human CD4+ cell lines. Delayed replication of nef mutant virus was observed in all cell lines examined. To determine the stage in the virus replication cycle that is affected by Nef, a single-round replication assay was performed. Initially, the expression of marker genes in transfected cells was examined in order to study the role of Nef in the late phase of infection. The results obtained indicated that Nef is dispensable during the transcription to virion production stage. Next, the effect of Nef on the early phase was investigated with a single-round infection. It was demonstrated that Nef is required in the early phase of the virus replication cycle, from virion adsorption to integration. Finally, the infectivity of virus stocks prepared from four cell lines was determined. The relative infectivity of the nef mutant from the four cell lines differed. Taken together, we conclude that Nef acts via modulation of viral particles to enhance virus infectivity in a cell-dependent manner.


Producer Cell-Dependent Requirement of the Nef Protein for Efficient Entry of HIV-1 into Cells

A proviral nef gene mutant of human immunodeficiency virus type 1 (HIV-1) was evaluated for its defective early replication step. Virus stocks were prepared from six CD4-positive and -negative cell lines transfected with wild-type (wt) or the nef mutant clone and inoculated into two target CD4-positive cell lines to monitor the efficiency of viral entry process. The nef mutant virions produced in one cell line exhibited a severe defect in the entry process, although those produced in the other five cell lines were only slightly less efficient than the wt virions at entering into cells. These results have demonstrated that the HIV-1 Nef is critical for efficient viral entry in a producer cell-dependent manner.

Biochemical and Biophysical Research Communications


Suppression of HIV replication by dominant negative mutants of HIV-1

Various gag mutants of human immunodeficiency virus type 1 (HIV-1) generated in vitro were evaluated for their potentials to suppress the replication of wild-type (wt) virus. A single-round of wt virus replication in the presence of various mutant proteins was quantitatively monitored by transfection and infection experiments. Out of 38 mutants examined, 15 were demonstrated to interfere with the replication of wt HIV-1 at early and/or late viral replication phase. Some of these mutants were also effective against the replication of wt HIV-2. In this review, we focus on the mutants, which are able to act against a wide variety of HIV, and are very useful for future gene therapy against AIDS.

International Journal of Molecular Medicine


The HIV-1 Vpr displays strong anti-apoptotic activity

Mutations in the human immunodeficiency virus type 1 (HIV-1) vpr gene only slightly reduce the replication rate of the virus. To study the role of HIV-1 Vpr in biological effects on cells, HEp-2 cells, which express HIV-1 Vpr constitutively but at a low level, were established. While control HEp-2 cells underwent apoptosis when incubated with sorbitol, the morphological and biochemical apoptotic changes were inefficiently induced in the HIV-1 Vpr-expressing cells by the same treatment. These results clearly indicate that HIV-1 Vpr has anti-apoptotic activity, and raise the possibility that Vpr acts as a weak activator of virus replication through anti-apoptosis.


Early Function of HIV-1 Gag Proteins Is Cell-Dependent

Various gag gene mutants of human immunodeficiency virus type 1 (HIV-1) were monitored for their replication potentials and defective replication sites in various CD4-positive T-cell lines. Some matrix, capsid, and nucleocapsid mutants displayed a replication defect in a cell-dependent manner. The single-round replication assays demonstrated that these mutants were defective at an early infection phase also in a cell-dependent way. These results indicated that interaction of a cell factor(s) and Gag proteins is involved in an early process of HIV-1 replication.


Suppression of HIV-2 Replication by HIV-1gagMutants

Gag gene mutants of human immunodeficiency virus type 1 (HIV-1) were analyzed for their potentials of inhibiting the replication of wild-type (wt) HIV-2, the second AIDS virus, in a single-round of viral replication. Of twenty-two HIV-1 gag mutants examined, seven were found to efficiently interfere with the replication of wt HIV-2. Some mutants, which can suppress the replication of wt HIV-1, did not show this inhibitory effect. These mutants were defective at the late phase of viral replication. A mutant designated NL-C1a was demonstrated to be very effective against the replication of HIV-1 and HIV-2 in monocytic cells as well as in lymphocytic cells.


The Potential of Various HIV-1 Mutants to Inhibit the Replication of Wild-Type Virus

We have previously demonstrated that many of gag mutants of human immunodeficiency virus type 1 (HIV-1) inhibited the replication of wild-type (wt) HIV-1. In this study, various HIV-1 mutants were systematically analyzed with respect to their ability to suppress the replication of wt HIV-1. Sixteen mutants of all eight HIV-1 genes other than gag were evaluated for their inhibitory effects. Only an env mutant designated NL-Hi efficiently interfered with the replication of wt HIV-1 in a single round of infection. The NL-Hi did not affect the late replication processes of wt virus, including transcription, translation, and assembly/release. Virions produced in the presence of the mutant Env were defective for the viral entry process in the early phase of HIV-1 replication cycle.


Induction of MHC-II DR expression on circulating CD8+ lymphocytes in macaques infected with SIVmac239 nef-open but not with its nef-deletion mutant

We examined the expression kinetics of activation antigens CD38 and MHC-IIDR (DR) on circulating CD8+ lymphocytes in rhesus macaques infected with pathogenic simian immunodeficiency virus strain SIVmac239 nef-open (239) or its nonpathogenic nef-deletion mutant (delta nef). In the longitudinal study, we found for the first time the induction of DR expression on CD8+ lymphocytes in 239-infected macaques. The induction of DR was in parallel with an increasing viral load and a decreasing CD4+ lymphocyte level. In the macaques with the high viral load and low CD4 level, a considerable proportion of the DR+CD8+ subpopulation was CD69+, indicating an activated state. On the other hand, no significant increase in the DR+CD8+ subpopulation level was observed in delta nef-infected macaques. These data indicate that the evaluation of activation markers such as DR and/or CD69 on circulating CD8+ cells may be valuable as a surrogate marker in the SIV-macaque model.


Acceleration of virus-induced apoptosis by tumor necrosis factor

The multiplication of vesicular stomatitis virus in HeLa cells was inhibited by treating the cells with tumor necrosis factor (TNF). Comparison of the kinetics of virus multiplication and that of virus-induced apoptosis in the TNF-treated cells revealed that the antiviral effect of TNF is accompanied by a rapid induction of apoptosis in the cells upon infection, suggesting that TNF can inhibit virus multiplication by accelerating an apoptotic response in the infected cells.


Selective induction of β7 integrin expression on lymphocytes undergoing apoptosis in lymphoid tissues

It has been previously shown that the beta 7 chain of integrin forms heterodimers with the alpha 4 or alpha E chain, which plays essential roles in lymphocyte homing to mucosal lymphoid tissues. The aim of this study was to re-evaluate the possible role of the beta 7 integrin other than lymphocyte homing. We prepared spleen and lymph node lymphocytes from biopsied specimens from macaque monkeys and examined for the reactivity with a monoclonal antibody specific for the beta 7 chain. As a result, a minor population of the lymphocytes with a smaller size, which were in the early stage of apoptosis, was found to express a higher level of the beta 7 integrin than a majority of the lymphocytes with a normal size. Interestingly, the apoptotic lymphocytes expressed neither alpha 4 nor alpha E chains, suggesting that the beta 7 chain on these cells may be associated with an undefined alpha chain. These findings indicate that in the lymphoid tissues the shrunken lymphocytes undergoing apoptosis selectively express a unique beta 7 integrin.

Biochemical and Biophysical Research Communications


Simian T cell leukemia virus type I-induced malignant adult T cell leukemia-like disease in a naturally infected African green monkey: implication of CD8+ T cell leukemia

Spontaneous T cell leukemia was found in an African green monkey (Cercopithecus aethiops, AGM) naturally infected with simian T cell leukemia virus type I (STLV-I). The hematological features and the evidence for monoclonal integration of provirus DNA in the leukemic cells revealed that the leukemia was an ATL-like disease. The expression of surface markers on the leukemic cells indicated that they were defined as an activated CD8+ T cell subset. Together with the finding that seven in vitro spontaneously STLV-I-transformed cell lines were CD4-CD8+, it is likely that CD8+ T cells are transformed by STLV-I in AGMs, in contrast with human ATL. Finally, we assessed characteristics of the CD8 chains on these transformed cells. The result indicated that the leukemic cells expressed only the alpha chains but not the beta chains. However, in the case of in vitro-transformed cell lines the expression pattern of the CD8 chains varied in individual monkeys. Thus, STLV-I may preferentially transform CD8+ (both alphaalpha+ and alphabeta+) T cells in AGMs.


Mapping the genetic determinants of human immunodeficiency virus type 2 for cell tropism and replication efficiency

Two distinct infectious molecular clones of human immunodeficiency type 2 (HIV-2) were analyzed for their biological properties in six cell lines. Fourteen chimeric and ten mutant viruses were constructed from these two viral genomes to localize the genetic determinants responsible for the phenotypes. Growth property of the viruses in the cell lines, together with the biochemical data, showed that a major determinant for the viral tropism resides in the env gene. In addition, in some cell lines, the accessory genes vif and nef affected the efficiency of virus replication. Thus, like HIV-1, mutations in the auxiliary and env genes of HIV-2 contributed much to the differences in virological characteristics.


Suppression of HIV-1 replication in peripheral blood mononuclear cells by fasudil

Fasudil is a potent inhibitor for various protein kinases such as myosin light chain kinase and protein kinase C. It has been used as a drug for improvement of intracranial vasospasm and following ischaemic diseases. In this report, we demonstrate that fasudil suppressed the replication of human immunodeficiency virus type 1 (HIV-1) in mitogen-activated peripheral blood mononuclear cells. Our finding shows that fasudil may be useful as a new and distinct chemotherapeutic agent against HIV-1 infection.

The Journal of Medical Investigation : JMI


Inhibition of HIV Replication by Capsid Mutant C6b

A Gag capsid mutant of human immunodeficiency virus type 1 (HIV-1) designated C6b was biologically and biochemically characterized with respect to its ability to suppress the replication of wild-type (wt) HIV. The C6b efficiently interfered with the replication of wt HIV-1 in the cleavage of Gag precursor, and also in the early replication process before or during viral DNA synthesis after viral penetration. The C6b Gag appeared to be unable to form chimeric multimers with HIV-2 Gag and failed to inhibit the replication of wt HIV-2.


Complete Inhibition of SIVmac Replication by its Capsid Mutants


Gag Pol Region Determines the Tropism of SIVagm for Human Cells

Simian immunodeficiency virus isolated from African green monkeys (SIVagm) does not grow in many of human cell lines such as CEM x 174, H9, and MT-4, but could replicate in some human cell lines. Sequence of SIVagm responsible for its narrow host range was determined by making and monitoring growth potential of chimeric clones between SIVagm and human immunodeficiency virus type 1 (HIV-1). The results obtained indicated that the gag-pol region determines the observed narrow host range. By monitoring virus DNA synthesis and progeny virion production, the defect(s) of SIVagm in the replication in the restricted cells was demonstrated to be located at early phase.


Comparative Analysis of Human and Macaque Monkey CD4: Differences in Formaldehyde Lability and Conformation

Here we characterized macaque monkey CD4 by flow cytometry. The results showed that relatively lower fluorescence intensity was observed depending on the monoclonal antibodies (mAbs) used for staining; Leu-3a exhibited four-fold lower intensity than Nu-Th/i, and that formaldehyde fixation dramatically reduced fluorescence intensity of macaque CD4+ cells stained with Leu-3a but not of human cells. Nu-Th/i is therefore preferable for the analysis of macaque CD4. Pretreatment of either mAb inhibited the other mAb binding to human CD4. On the contrary, Nu-Th/i inhibited Leu-3a binding but Leu-3a poorly blocked Nu-Th/i binding to the macaque CD4. These results indicate that Leu-3a and Nu-Th/i epitopes are conserved in macaque CD4 but Leu-3a epitope is conformationally cryptic and/or fragile, resulting in the lower affinity. Amino acid sequence alignment of CD4 domain 1 shows that the substitutions outside the linear Leu-3a epitope may determine these characteristics of macaque CD4.


Functional Domain Mapping of HIV-1 Gag Proteins

A series of human immunodeficiency virus type 1 (HIV-1) proviral gag gene mutants carrying bacterial CAT gene were constructed and monitored for the expression of reverse transcriptase and CAT in a highly sensitive single-round replication assay system to determine the defective replication phase in lymphocytic cells. All the mutants displayed no abnormality in the process of transcription and translation at late replication stage. In contrast, some matrix, capsid, and p6 mutants were defective at final phase, that is, assembly and virion release. Most of the mutants including nucleocapsid mutants, which showed normal phenotype at late stage, were defective at early replication phase. From the functional domain map thus obtained, it is evident that HIV-1 Gag proteins are required for both early and late replication phases.

Biochemical and Biophysical Research Communications


Induction of apoptosis by herpes simplex virus type 1

Although herpes simplex virus type 1 (HSV-1) does not induce apoptosis in infected HEp-2 cells, in the presence of cycloheximide infection induced apoptosis with characteristic morphological changes as well as endonucleosomal DNA cleavage. The induction of apoptosis without de novo protein synthesis suggests that a structural protein of the HSV-1 virion is responsible for the observed apoptosis.


HIV-1 capsid mutants inhibit the replication of wild-type virus at both early and late infection phases

In-frame mutations were introduced into various portions of the human immunodeficiency virus type 1 (HIV-1) gag gene, and potentials of the mutants to suppress the replication of wild-type HIV-1 were monitored. In contrast to results obtained with matrix and nucleocapsid mutants, almost all capsid mutants blocked HIV-1 replication completely in single-round replication assays. A capsid mutant designated C6b was demonstrated to be one of the most efficient inhibitors for HIV-1 reported to date, and to be effective at both early and late viral replication phases. T-cells, which are engineered to express the C6b Gag in response to HIV-1 infection, were perfectly resistant to HIV-1.


Cleavage of Gag precursor is required for early replication phase of HIV-1

A mutant of human immunodeficiency virus type 1 (HIV-1), which is deficient for Gag precursor cleavage and noninfectious, was characterized with respect to its defective step in the viral replication phase. Upon transfection, the mutant produced a normal level of progeny virions as monitored by electron microscopy and RNA hybridization. Single-round replication assay demonstrated, in contrast, that the mutant was defective at the early phase of the replication cycle. Furthermore, no viral DNA was detected in the cells infected with the mutant. Taken together, it is concluded that maturation of Gag precursor protein of HIV-1 is required for an early event(s) before or during a coupled process of uncoating/reverse transcription.


Protection of monkeys vaccinated with vpr- and/or nef-defective simian immunodeficiency virus strain mac/human immunodeficiency virus type 1 chimeric viruses: a potential candidate live-attenuated human AIDS vaccine

Two simian immunodeficiency virus strain mac (SIVmac)/human immunodeficiency virus type 1(HIV-1) chimeric viruses (SHIVs), designated NM-3 and NM-3n, with env derived from HIV-1 and defective vpr (plus defective nef for NM-3), were inoculated into seven macaques. These macaques were transiently or persistently infected and most of them produced long-lasting neutralizing antibodies and Env-specific killer T cells to HIV-1 with no AIDS-like symptoms. When they were challenged with another SHIV with intact vpr and nef (designated NM-3rN), all were protected as judged by virus recovery, DNA detection by PCR and antibody responses. Anti-HIV-1 Env-specific killer T cells were considered to have played a major role in this protection, but a non-specific defence mechanism as well as specific immunity also appeared to be involved. Thus, these two non-pathogenic SHIVs induced long-lasting protective immunities in macaques, suggesting the possibility of gene-defective SHIVs as attenuated live vaccines for human use.


Functional Analysis of vif Genes Derived from Various Primate Immunodeficiency Viruses

Replication property in cells of human and simian immunodeficiency viruses (HIVs and SIVs) lacking intact vif gene was evaluated. Of 10 vif mutants constructed in vitro of the major four HIV/SIV groups, only those derived from HIV-1 and HIV-2/SIVmac displayed replication defect. The cell lines non-permissive for the vif mutants of HIV-1 and SIVmac were found to be different. To determine whether Vif is exchangeable between HIV-1 and SIVmac, chimeric virus clones with respect to the vi gene were constructed and virus replication in the cells non-permissive for the vif mutant viruses was monitored. Productive infection in these cells of chimeric viruses clearly indicated that Vif is functionally exchangeable, and that Vifs of different virus origin act through a similar mechanism.


Susceptibility of nude mice carrying the Fv-4 gene to Friend murine leukemia virus infection

Fv-4 is a mouse gene that dominantly confers resistance to infection with Friend murine leukemia virus (F-MuLV) (S. Suzuki, Jpn. J. Exp. Med. 45:473-478, 1975). Despite complete resistance to ecotropic MuLV infection in mice carrying the Fv-4 gene, it is known that cells carrying the resistance gene in tissue culture do not always show resistance as extensive as that in vivo (H. Yoshikura and T. Odaka, JNCI 61:461-463, 1978). To investigate the immunological effect on resistance in vivo, we introduced the Fv-4 gene into BALB/c nude mice (Fv-4-/- nude[nu/nu]) by mating them with Fv-4 congenic BALB/c mice (Fv-4r/r nude+/+) and examined the susceptibility of the F2 progeny to F-MuLV. All BALB/c nude mice without the Fv-4 gene (Fv-4-/- nude[nu/nu]) were permissive to F-MuLV and developed erythroleukemia within 2 weeks after virus inoculation. The BALB/c nude mice with the Fv-4 gene (Fv-4r/r nude[nu/nu]) did not develop leukemia, and no or little virus was detected in the spleen 7 weeks after virus inoculation. The resistance to F-MuLV was dominant in (Fv-4 congenic BALB/c x BALB/c nude) F1 mice with the Fv-4r/- nude(nu/+) genotype as strictly as in (Fv-4 congenic BALB/c x BALB/c) F1 mice with the Fv-4r/- nude+/+ genotype. However, almost all BALB/c nude mice with the Fv-4r/- nude(nu/nu) genotype developed the disease within 7 weeks, and the virus was detected in all of their spleens even in the mice without leukemia. These results show that the resistance caused by the Fv-4 gene is recessive in nude mice and dominant in BALB/c mice. Some immunological effects, perhaps cell-mediated immunity, may play important roles in the resistance to F-MuLV infection in vivo in addition to the dosage effect of the Fv-4 product.


Epitope mapping of murine monoclonal antibodies against human immunodeficiency virus type 1 Nef

It has shown that the human immunodeficiency virus type 1 (HIV-1) Nef protein has the high antigenicity in HIV-1 seropostive individuals. We newly obtained seven monoclonal antibodies (mAbs). To identify the antigenic determinants of HIV-1 Nef protein against murine, epitope mapping of the mAbs was performed by enzyme-linked immunosorbent assay (ELISA) by using several recombinant truncated Nef fusion proteins, that were expressed in Esherichia coli, and synthetic peptides. The results showed that mAbs A6, A7, F2, F3, F4, F8 and E5 recognized epitopes on Nef protein located at amino acid residues 18-26, 28-45, 115-137, 128-137, 115-126, 128-137, and 170-181, respectively.


Growth ability of auxiliary gene mutants of human immunodeficiency virus types 1 and 2 in unstimulated peripheral blood mononuclear cells

Mutational studies on the Vif, Vpr, Vpu, Vpx, and Nef genes of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) were performed to evaluate their biological functions in natural target cells. For this purpose, replication properties of mutant viruses derived from HIV-1 NL strain and HIV-2 GH strain in unstimulated peripheral blood mononuclear cells were determined. Vif- viruses of both HIV-1 and HIV-2 did not grow at all in these cells. Similarly, no replication of HIV-2 Vpx- mutant was detected. In contrast, both of Vpr- and Nef- viruses of HIV-1 and HIV-2, and Vpu- virus of HIV-1 grew quite well in the cells. These results show, together with the data previously reported, that only Vif and Vpx are essential for HIV replication in primary blood cell cultures.


In vitro cytocidal effects of human immunodeficiency virus type 1 Nef on unprimed human CD4+ T cells without MHC restriction

We have previously shown that the C-terminal region of human immunodeficiency virus type 1 (HIV-1) Nef antigen present on the outer surface of virus-infected cells has an affinity for uninfected T cells and that the Nef protein is responsible for T cell death. To exclude completely the possibility of MHC restriction of this cytotoxic activity, the in vitro cytotoxic potential of HIV-1 Nef against various CD4+ T cell lines as well as naive T lymphocytes was investigated using a baculovirus expression system. Insect cells expressing myristoylated Nef on their cell surface were shown to kill a proportion of CD4+ T cells within 8 h. However, N-terminal truncated and unmyristoylated Nef proteins were not present on the outer surface of insect cell membranes and failed to show any killing activity. Monoclonal antibodies against the C-terminal region of Nef inhibited cytolysis. Thus, we conclude that specific Nef-mediated cytolysis is induced by contact with unprimed CD4+ T lymphocytes without MHC restriction.


Clustered localization of oligomeric Nef protein of human immunodeficiency virus type 1 on the cell surface

We studied human immunodeficiency virus type 1 (HIV-1) Nef protein biochemically and histologically. HIV-1 Nef, derived from baculosystem and from cells infected with HIV-1, formed homomeric monomers, dimers, trimers, and further polymers. These oligomers were non-covalently associated. In cells infected with HIV-1, Nef molecules were clustered at the cell surface as well as cytoplasm. Our previous results have indicated that the Nef on the surface of cells infected with HIV-1 is cytotoxic against uninfected CD4+ T cells. Thus, it is very likely that the HIV-1-mediated cytotoxic reaction is due, at least in part, to the clustered localization of oligomeric Nef on the cell surface.


Human immunodeficiency virus type 1 Nef protein on the cell surface is cytocidal for human CD4+ T cells

We have previously shown that the carboxyl-terminal region of human immunodeficiency virus type 1 (HIV-1) Nef antigen present on the outer surface of virus-infected cells has affinity for uninfected T cells. Here, the in vitro cytotoxic potential of HIV-1 Nef on the T cell surface against CD4+ T cells was investigated in detail. Human T cells expressing Nef on the cell surface by transfection with non-infectious mutant HIV-1 proviruses were demonstrated to kill CD4+ T cells efficiently. Furthermore, it was shown that the carboxyl-terminal portion of Nef was cytotoxic for CD4+ T cells and that monoclonal antibody against the carboxyl-terminal region of Nef inhibited Nef induced-cytolysis. Thus, we concluded that Nef protein on CD4+ T cells may play an important role in the specific loss of CD4+ T lymphocytes during HIV-1 infection.


Soluble Nef antigen of HIV-1 is cytotoxic for human CD4+ T cells

We have previously shown that Nef-gene 10 fusion protein induces marked growth arrest of human primary CD4+ T cells. Here, in vitro cytostatic and cytotoxic activities of human immunodeficiency virus type 1 (HIV-1) Nef against CD4+ T cells were extensively investigated. Growth of human CD4+ cells was inhibited significantly just by the addition of purified full-length Nef to cultures. When Nef was cross-linked by anti-Nef antibodies, it became very cytocidal for CD4+ T cells. A high percentage of sera from HIV-1-infected individuals contained soluble Nef. Thus, soluble Nef in vivo may play an important role in immunodysfunction of CD4+ T lymphocytes in HIV-1 infection.


Possible origin of murine AIDS (MAIDS) virus: conversion of an endogenous retroviral p12gag sequence to a MAIDS-inducing sequence by frameshift mutations

The murine AIDS (MAIDS) virus has a unique sequence in its p12gag region, which is responsible for MAIDS development. A transcript hybridizing with this sequence is expressed in normal C57BL/6 mice. The transcript, designated Edv, has been previously cloned and sequenced (Y. Kubo, Y. Nakagawa, K. Kakimi, H. Matsui, K. Higo, L. Wang, H. Kobayashi, T. Hirama, and A. Ishimoto, J. Gen. Virol. 75:881-888, 1994). Compared with the nucleotide sequence of the helper LP-BM5 ecotropic virus, the pathogenic replication-defective MAIDS virus has a 16-bp deletion and a 1-bp insertion in the 5' and 3' regions of the p12gag sequence, respectively, and the Edv transcript contains only a 3-bp deletion. Therefore, the amino acid sequence of the defective MAIDS virus p12gag region is not homologous to that of the helper virus and the Edv transcript because of the frameshift. To determine whether the amino acid sequence resulting from the frameshift is critical for MAIDS development, we constructed chimeric viruses that contained the p12gag regions of the helper virus and the Edv transcript, respectively, with and without the same frame as the defective MAIDS virus by the artificial frameshift mutations. The mutant viruses with the frameshift mutations induced MAIDS in inoculated mice, but the viruses without the mutations did not. These results suggested that the MAIDS virus was generated by frameshift mutations in the p12gag region of Edv or a related sequence.


Genomic and biological alteration of a human immunodeficiency virus type 1 (HIV-1)-simian immunodeficiency virus strain mac chimera with HIV-1 Env recovered from a long-term carrier monkey

A macaque monkey infected with NM-3, a human immunodeficiency virus type 1 (HIV-1)-simian immunodeficiency virus strain mac (SIVmac) chimeric virus with env, rev, tat and vpu derived from HIV-1 and LTR, gag, pol, vif and vpx derived from SIVmac, became a long-term carrier (more than 2.8 years). This monkey produced neutralizing antibodies to the original NM-3 as well as to the parental HIV-1. The virus recovered at 116 weeks replicated more rapidly and productively in macaque peripheral blood mononuclear cells than the original virus. The recovered virus was not neutralized either by antibodies raised early in the monkey or by a neutralizing monoclonal antibody that recognizes the V3 loop of HIV-1 Env, whereas both the early antibodies and the monoclonal antibody neutralized the original NM-3. Analysis of the virus genomic population revealed a few common mutations in the V3 region that caused amino acid changes. These data are consistent with the hypothesis that the virus escaped from the early antibodies and that the observed mutations contributed to this, as with HIV-1-infected humans. The observed mutations could equally well be the result of adaptation to simian cells. These results suggest that the HIV-1-SIVmac chimeric virus will be useful for investigating genetic variation of HIV-1 env and alteration of biological properties in vivo in relation to the host immune response.


Evidence for direct association of Vpr and matrix protein p17 within the HIV-1 virion

Vpr is one of the auxiliary proteins of HIV-1 and is selectively incorporated into the virion by a process involving the C-terminal p6 portion of the Gag precursor Pr55. Vpr and the matrix protein p17 are the components of the viral preintegration complex and appear to play important roles in the nuclear transport of proviral DNA in nondividing cells. In the present study, we have demonstrated by coimmunoprecipitation experiments that Vpr associates with matrix protein p17 but not with capsid protein p24 within the HIV-1 virion. Experiments employing the yeast two-hybrid GAL4 assay for protein-protein interactions also demonstrated a direct association between Vpr and the C-terminal region of matrix protein p17. Association of Vpr and the matrix protein p17 within the mature virion is consistent with their collaborative role in the nuclear transportation of the viral preintegration complex in nondividing cells such as macrophages.


Genetic complementation between replication-defective mutants of HIV-1 and SIVagm

To investigate the functional complementation of essential genes for virus growth between HIV-1 and SIVagm derived from African green monkeys, we co-transfected replication-defective molecular clones containing mutations in gag, pol, env, tat or rev, and monitored transient complementation by reverse transcriptase assay (RT), cytopathic effect (CPE) and immunofluorescence assay (IFA). The following results were obtained: 1) No complementation was observed in combinations of the gag and pol mutants. 2) The rev mutant of HIV-1 was minimally complemented by other SIVagm mutants, although the rev mutant of SIVagm was significantly complemented by other HIV-1 mutants. 3) Among all combinations tested, the env mutant of HIV-1 was the most effectively complemented by SIVagm mutants. 4) CPE was mostly absent in combinations of the env mutant of SIVagm and the gag, pol, or tat mutants of HIV-1, although there were significant positive results in RT and IFA assays. These findings provided basic information about the functional compatibility of pathogenic HIV-1 and nonpathogenic SIVagm which will be useful for generating chimeras of these two viruses.


Functional analysis of simian immunodeficiency virus SIVagm long terminal repeat

We have previously shown that long terminal repeats (LTRs) derived from various isolates of SIVAGM share a unique functional property. In the absence of viral Tat, all SIVAGM LTRs act as much more efficient promoters than any of the other LTRs derived from representative primate immunodeficiency viruses. In the presence of Tat, however, SIVAGM LTRs are activated relatively inefficiently. To map the elements that confer these features on the SIVAGM LTR, a number of deletion mutants were constructed, and their promoter activities were determined using a bacterial CAT gene as a marker. The results obtained indicated that various elements located in the U3 region may contribute to the high basal promoter activity and that no negative elements are present in the region. The Tat-responsive sequence TAR was localized to the R region as observed for the other LTRs. A mutant carrying a single nucleotide deletion in this region completely lost responsiveness to Tat protein.


Both SU and TM Env proteins are responsible for monkey cell tropism of simian immunodeficiency virus SIVmac

While simian immunodeficiency virus (SIV) derived from an infectious molecular clone pMA239 is tropic and pathogenic for monkeys, the virus derived from another infectious clone pMA142 does not replicate in monkey cells. To determine genetic sequences responsible for this tropism, a series of recombinant clones were constructed from pMA142 and pMA239. The determinant in pMA239 was mapped within regions encompassing the env gene. Viruses, which carry the 239 env gene encoding surface and/or transmembrane proteins, were tropic for monkey cells.


Targeting of chrolamphenicol acetyltransferase to human immunodeficiency virus particles via Vpr and Vpx

Vpr and Vpx are the auxiliary proteins of human immunodeficiency viruses (HIVs) selectively incorporated into mature viral particles. We showed that the bacterial chloramphenicol acetyltransferase (CAT) fused to the N-terminus of HIV-1 Vpr, HIV-2 Vpr, or HIV-2 Vpx was incorporated into mature virions in a type-selective manner. By using chimeric proteins between HIV-1 Vpr and HIV-2 Vpx, we found that the N-terminal side of these proteins was mainly important for type-selective virion incorporation. The C-terminal arginine-rich region of HIV-1 Vpr was also found to transport CAT fusion proteins into virions but without any type selectivity. Furthermore, the corresponding regions of HIV-2 Vpr and HIV-2 Vpx had no such activity. This region of HIV-1 Vpr may interact nonspecifically with viral genomic RNA. Collectively, Vpr and Vpx may provide a means to introduce foreign proteins and other molecules into HIV virions for therapeutic purposes.


Early replication block of human immunodeficiency virus type 1 in monkey cells

The genetic and functional basis of the replication-defective nature of human immunodeficiency virus type 1 (HIV-1) in monkey cells was studied. By the generation and characterization of chimeras between HIV-1 and simian immunodeficiency virus, the sequence encompassing the 3' half of the long terminal repeat, gag and pol genes of HIV-1 was found to be responsible for the growth restriction. Early and late phases of HIV-1 replication in monkey cells were analysed in detail using several assay systems: transfection/coculture, transcomplementation between various proviral clones carrying the CAT gene and effector clones and evaluation of transcription and reverse transcription. All the data were consistent with the notion that HIV-1 replication is blocked at a very early stage(s) such as uncoating and/or reverse transcription in monkey cells.


Function of human immunodeficiency virus type 1 Vpu protein in various cell types

We evaluated the function of human immunodeficiency virus type 1 vpu gene in various cell lines. We established a highly sensitive system consisting of chloramphenicol acetyltransferase and reverse transcriptase assays and used it to monitor the effects of mutation of the vpu gene. In some cell lines, Vpu protein was not required at the early phase of viral replication but was important for efficient virion production. In these cells, the Vpu protein functioned effectively irrespective of the presence of intact env gene products. Likewise, CD4 gene expression had no effects on Vpu function. In the other cell lines tested, Vpu protein was not important for virion release, and the vpu mutant clone generated a normal level of progeny virions upon transfection.


Generation and characterization of a host cell-dependent gag gene mutant of human immunodeficiency virus type 1

An in-frame gag gene mutant of human immunodeficiency virus type 1, which carries two amino acid substitutions in the center of the p24 coding region, was constructed in vitro, and its replication properties in several cell lines were examined. In CD4-negative SW480 cells transfected with the mutant clone, synthesis and processing of viral gag, pol, and env proteins occurred normally, and viral particles were produced. Virions derived from the transfection displayed a severe replication defect when inoculated into some CD4-positive cell lines (H9 and Molt4 clone 8), but in other lines (A3.01 and M8166), the mutant virus grew fairly well. The mutant was demonstrated to be defective at an early infection phase (from adsorption to integration) in Molt4 clone 8 cells but was normal in A3.01 cells. These results indicated that the Gag-p24 protein of human immunodeficiency virus type 1 plays an important role at the early infection phase in a cell-dependent manner.


Rev-dependency of expression of human immunodeficiency virus type 1 gag and env genes

Structural gene expression of human immunodeficiency virus type 1 (HIV-1) requires a viral regulatory protein, Rev transactivator. We investigated Rev-dependency of HIV-1 gene expression by various reporter systems. Expression of unspliced and single-spliced viral mRNAs was demonstrated to be differentially dependent on the Rev function. This difference of Rev-dependency was found not to be determined by cis-elements in gag, pol, and env coding sequences reported so far, and was lost when the reporter constructs containing minimum elements for Rev-responsiveness such as splice signals and rev responsive element were used for experiments. These findings indicated that the fundamental structure of HIV-1 mRNA was critical for the differential regulation of gene expression by Rev transactivator.


Effects of translation initiation factor eIF-5A on the functioning of human T-cell leukemia virus type I Rex and human immunodeficiency virus Rev inhibited trans dominantly by a Rex mutant deficient in RNA binding

The viral transactivator proteins Rex and Rev are necessary for the expression of structural proteins of human T-cell leukemia virus type I and human immunodeficiency virus type 1, respectively. Although the interaction of Rex/Rev with a cellular cofactor(s) has been thought to be required for Rex/Rev action, there is no suitable system to search for the cofactor(s) in mammalian cells. We found that a Rex mutant, TAgRex, which contains a simian virus 40 nuclear localization signal in place of the N-terminal 19 amino acids of Rex, could dominantly inhibit wild-type Rex/Rev functions. The inhibition did not require either Rev response element/Rex response element binding or the oligomerization ability of the mutant, but it did require a region around amino acid 90 of the Rex protein, suggesting that TAgRex sequestered the cellular cofactor. Complementation with the eukaryotic translation initiation factor 5A (eIF-5A) in this system could restore the impaired Rex function. These results indicate that eIF-5A is the cofactor indispensable for Rex function. Additionally, by using a two-hybrid system, the homo-oligomer formation of Rex was found to be mediated by the region around amino acid 90 in addition to Tyr-64 and Trp-65 of Rex protein. Thus, eIF-5A may play a part in the formation of the Rex homo-oligomer.


Functional analysis of human spuma retrovirus genome

Human spuma retrovirus (HSRV) belongs to retroviruses that possess a complex genome organization. HSRV carries at least three extra genes in the region between env and the 3' long terminal repeat, which are not found in simple retroviruses. Via alternative splicing, these HSRV genes can encode several proteins. To genetically study the requirements of these viral proteins for viral replication in tissue cultures, a number of mutant viruses were constructed from an infectious molecular clone of HSRV. All mutants grew normally in the cell lines tested, except for those lacking transcriptional transactivator activity. By reporter-based transient assay systems, no Rev/Rex equivalent was detected in the HSRV proteins.


Functionality of chimeric Rev proteins of HIV/SIV

Studies on functional compatibility of various Rev proteins derived from all known human and simian immunodeficiency virus subgroups have shown that this essential gene product is not always exchangeable among the viruses. In an attempt to map the region of Rev proteins responsible for the observed nonreciprocal complementation, hybrid genomic Rev expression vectors were constructed by exchanging the first and second exons of rev genes, and were examined for their abilities to activate reporter clones by transfection. With one exception, the second coding exon of rev gene determined the functional specificity of Rev proteins.


Human imunodeficiency virus Vpx is required for the early phase of replication in peripheral blood mononuclear cells

Functional importance of Vpx protein of human immunodeficiency virus type 2 was evaluated in various types of cells. In 8 lymphocytic or monocytic cell lines tested, vpx mutant virus grew as well as wild-type virus. Only in primary peripheral blood mononuclear cell cultures, severely retarded growth of mutant virus was observed. No replication of vpx-minus virus was detected in primary macrophage cells. A highly sensitive single-round replication assay system was used to determine the defective replication phase in primary mononuclear cells of vpx mutant virus. In all cell lines examined, vpx mutant displayed no abnormality. In contrast, the vpx mutant was demonstrated to be defective at an early stage of the infection cycle in primary cell cultures. No evidence of a replication-defect at a late phase in primary cells of the vpx mutant was obtained by a transfection-coculture method. These results indicate that the virion-associated Vpx protein is essential for early viral replication process in natural target cells such as primary macrophages.


Growth ability of human immunodeficiency virus type 1 auxiliary gene mutants in primary blood macrophage cultures

A strain of human immunodeficiency virus type 1 that is strictly tropic for primary human blood cell cultures was constructed in vitro. Mutational studies on the vif, vpr, vpu and nef genes of this virus were performed to evaluate their biological functions in natural target cells. For this purpose, replication properties of mutant viruses in peripheral blood mononuclear cells (PBMCs) and macrophages (PBMPs) were determined. Three phenotypes with respect to virus replication were noticed: normal or mildly retarded growth (nef and vpr mutants), impaired growth (vpu mutant), and no growth (vif mutant). These results suggest that the Vif and Vpu proteins are more important than the Nef and Vpr proteins for virus replication in PBMCs and PBMPs.


Persistent infection with SIVmac chimeric virus having tat, rev, vpu, env, and nef of HIV-1 in macaque monkeys

A chimeric human and simian immunodeficiency virus carrying the tat, rev, vpu, env, and nef genes of human immunodeficiency virus type 1 was generated. The chimeric virus, NM-3n, grew competently in peripheral blood mononuclear cells from cynomolgus monkeys like the parental SIVmac. Two cynomolgus monkeys and one rhesus monkey inoculated with NM-3n raised antibodies to SIVmac Gag and HIV-1 Env. The antibodies raised in the cynomolgus monkeys persisted for at least 1.7 years. The antibodies contained virus neutralizing activity not only to the original chimeric virus but also to the parental HIV-1. Infectious viruses were isolated from one of the cynomolgus monkeys 37 and 63 weeks after inoculation and from the rhesus monkey continuously from 6 weeks after infection onward. The recovered virus maintained its chimeric structure but included several clones with mutations in the env V3 region. When the recovered virus was inoculated to another rhesus monkey, no difference in the frequency of virus recovery was seen from the originally infected monkeys. These carrier monkeys have so far shown no sign of the disease.


Inhibition of murine AIDS(MAIDS) development by the transplantation of bone marrow cells carrying the Fv-4 resistant gene to MAIDS virus-infected mice


Feline immunodeficiency virus gene expression: analysis of the RNA splicing pattern and monocistronic rev mRNA

The transcription pattern of the feline immunodeficiency virus (FIV) genome in a feline CD4+ cell line was examined. In addition to the genomic RNA (9.2 kb), at least five FIV-specific transcripts [5.2, 4.4 (doublet), 1.7 and 1.4 kb] were detected by using subgenomic restriction enzyme fragments of an FIV molecular clone or FIV-specific oligonucleotides as probes. Among these transcripts, the 9.2, 5.2 and 4.4 (doublet) kb mRNAs were not expressed in the cytoplasm of cells transfected with a rev- mutant. To determine the location of splice junctions in the FIV genome, we used PCR to amplify and clone cDNAs corresponding to the viral mRNAs from infected cells. The region between pol and env was found to contain at least two splice donor and three splice acceptor sites. Two splice acceptor sites were detected in the 3' region of env. By hybridization analysis and sequencing of cDNA clones, it was revealed that the medium sized mRNAs are derived from a single splice event, with different splice acceptor sites, and that the two smaller transcripts are doubly or triply spliced mRNAs. Our results demonstrate a complex pattern of alternative splicing of FIV mRNAs. Furthermore, we identified monocistronic rev mRNA species that employ a unique splice acceptor site.


The feline immunodeficiency virus ORF-A gene facilitates efficient viral replication in established T-cell lines and peripheral blood lymphocytes

Frameshift mutants corresponding to all of the identified open reading frames of feline immunodeficiency virus, including the ORF-A gene, which has an unknown function, were constructed in vitro. Upon transfection into cells, no significant difference between the phenotypes of ORF-A mutant clones and those of wild-type clones was demonstrated. Although only ORF-A mutant virus among all mutant viruses from transfected cells showed infectivity in established T-cell lines, the replication and propagation of the ORF-A mutant virus were efficiently reduced compared with those of the wild-type virus. Moreover, the loss of the function of the ORF-A gene resulted in a severe defect in productive infection in primary peripheral blood lymphocytes both in the amount of reverse transcriptase activity produced and in core protein expression. These findings demonstrate that the ORF-A gene of feline immunodeficiency virus is required for efficient viral replication and suggest that the ORF-A gene is likely to be important for natural infection.


The AP-1 binding site in the feline immunodeficiency virus long terminal repeat is not required for virus replication in feline T lymphocytes

Sequences of 31 bp containing putative AP-1 and AP-4 binding sequences in the U3 region of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) were deleted and the basal promoter activity of the LTR was measured by the chloramphenicol acetyltransferase (CAT) assay. The activity of the FIV LTR was reduced in Felis catus whole foetus 4 (fcwf-4) cells and Crandell feline kidney cells by this deletion. Cotransfection of murine c-Fos or c-Jun expression plasmids with the FIV LTR-CAT reporter plasmid into fcwf-4 cells revealed that FIV LTR could be activated by c-Fos but not c-Jun in the cells. The mutated LTR was introduced into an infectious molecular clone of FIV and the replication rate and the cytopathogenic activity of the mutant were compared with those of the wild-type in two feline CD4-positive T lymphoblastoid cell lines. It was found that the rate and activity of the mutant were almost the same as those of the wild-type. From these data, we conclude that the 31 bp fragment is important for achieving maximal expression of the FIV genome, but not required for the replication of FIV in feline T lymphocytes.


Compatibility of tat and rev transactivators in the primate lentiviruses

Primate immunodeficiency viruses carry a unique set of transacting regulator genes, which are essential for viral replication. The exchangeability of these Tat and Rev transactivators derived from viruses of the four major subgroups identified to date was assessed in transient transfection and infection assay systems. The human immunodeficiency virus type 1 (HIV-1), a major causative virus of human AIDS, efficiently activated the other viruses. In contrast, the tat and rev gene products of HIV-2, SIVAGM (virus of the African green monkey), and SIVMND (virus of the mandrill) did not fully transactivate the HIV-1. In particular, the rev of HIV-1 was not substantially replaced by those of the other viruses. The result that HIV-1 is distinct from the other immunodeficiency viruses with respect to the compatibility of two transactivators gives a firm functional basis for the unique phylogenetic position of HIV-1.


Cell-dependent requirement of human immunodeficiency virus type 1 Vif protein for maturation of virus particles


Integration is essential for efficient gene expression of human immunodeficiency virus type 1

A mutant of human immunodeficiency virus type 1 which carries a frameshift insertion in the integrase/endonuclease region of pol gene was constructed in vitro. Upon transfection into cells, although this mutant exhibited a normal phenotype with respect to expression of gag, pol, and env genes and to generation of progeny virions, no replication-competent virus in CD4-positive cells emerged. An assay for the single-step replication of a defective viral genome dependent on trans complementation by rev protein was established and used to monitor the early phase of viral infection process. Viral clones with a mutation in the vif, vpr, or vpu gene displayed no abnormality in the early phase. In contrast, the integrase mutant did not direct a marker gene expression after infection. Together with an observation that the mutant lacked the ability to integrate, these results indicated that the integration was required for efficient viral gene expression and productive infection of human immunodeficiency virus type 1.


Sequences responsible for efficient replication of simian immunodeficiency virus SIVmnd in cells of the monocyte/macrophage lineage

We determined the susceptibility of monocytic cell lines to infection with viral strains derived from two infectious clones of simian immunodeficiency virus isolated from a mandrill. One of the strains, which replicates poorly in T cell lines, was found to grow more rapidly than the other in these cells. The viral determinant for this property was genetically mapped within the env gene encoding a surface protein. Six amino acid substitutions identified appeared to be located outside of the domains corresponding to human immunodeficiency virus type 1 env functional domains such as the CD4-binding and V3 loop regions.


Infection of macaque monkeys with a chimeric human and simian immunodeficiency virus

Two macaque monkeys were inoculated with a chimeric human and simian immunodeficiency virus carrying the tat, rev, vpu and env genes of human immunodeficiency virus type 1. Infectious virus was recovered from one of the monkeys at 2 and 6 weeks post-infection. The hybrid nature of the isolated viruses was verified by Southern and Western blotting analyses. Both of the monkeys infected with the chimera elicited a humoral antibody response against the virus.


Identification of a feline immunodeficiency virus gene which is essential for cell-free virus infectivity

Feline immunodeficiency virus (FIV) contains at least three small open reading frames (ORFs) in the genome, in addition to the three structural genes. Two of these ORFs (putative vif and ORF-A) have unknown functions. Northern (RNA) blot analysis of mRNAs from an FIV-infected cell line showed that the putative-vif-specific mRNA was expressed as a 5.2-kb species. To examine the function of the putative vif gene, we constructed mutants carrying a deletion in either the vif-like gene or the rev gene from an infectious molecular clone of FIV. Although the vif mutant produced virion-associated reverse transcriptase at a normal level upon transfection, cell-free virus prepared from the transfected cells could not infect feline CD4+ cells. The infectivity of the vif mutant, however, was demonstrated in a coculture of the transfected cells and feline CD4+ cells. We conclude that FIV contains the vif gene, which is structurally and functionally similar to that of the primate lentiviruses.


Functional classification of simian immunodeficiency virus isolated from a chimpanzee by transactivators

In reporter-based transient expression systems, we characterized simian immunodeficiency virus from a chimpanzee (SIVCPZ), with special reference to the human immunodeficiency virus type 1 (HIV-1). SIVCPZ was not equally activated by tat and rev transactivators derived from representative primate lentiviruses. HIV-1 alone activated SIVCPZ to the full extent in both tat and rev assays. The tat and rev gene products of SIVCPZ, as well as those of HIV-1, efficiently transactivated the other viruses. These results indicate that SIVCPZ is identical to HIV-1 with regard to the compatibility of tat and rev gene activities among four subgroups of primate lentiviruses.


Functional analysis of biologically distinct genetic variants of simian immunodeficiency virus isolated from a mandrill

We examined the biological properties of two infectious clones of a simian immunodeficiency virus, SIVMND, which were designated as pMD121 and pMD122. Upon transfection into CD4-negative cells, pMD122 generated virions much less efficiently than pMD121. Likewise, the growth kinetics in CD4-positive cells of virus derived from pMD122 were remarkably delayed relative to those of virus from pMD121. The cytocidal activity of the MD122 virus was also low. A series of recombinant clones were constructed from pMD121 and pMD122 to determine the sequence responsible for the low virulence of the MD122 virus. The genetic determinant in pMD122 responsible for its properties was mapped to within a region (316 base pairs) encompassing the tat, rev, and env coding sequences. Sequence analysis revealed that the two clones differed by only one nucleotide in this region. A nucleotide substitution G (pMD121) to T (pMD122) altered an arginine codon to a serine codon in the first tat coding exon. Transient transfection experiments showed that the tat activity of pMD122 was about twofold less than that of pMD121. These findings indicate that small differences in tat activity can have a dramatic effect on the biological behavior of SIVMND.


Production of feline immunodeficiency virus in feline and non-feline non-lymphoid cell lines by transfection of an infectious molecular clone

An infectious molecular clone of the TM1 strain of feline immunodeficiency virus (FIV) was transfected into each of one feline (CRFK), two simian (COS and Vero) and two human (SW480 and HeLa) non-lymphoid cell lines, and virus production was assayed on feline T lymphoblastoid MYA-1 cells by monitoring reverse transcriptase activity. Infectious virus was produced in CRFK, Vero and HeLa cells, but not in COS and SW480 cells. When the basal promoter activity of the FIV long terminal repeat (LTR) was examined in these cell lines by using a chloramphenicol acetyltransferase assay, the activity correlated with the virus production in each cell line. Furthermore, when the activity of the FIV LTR was compared with those of three primate lentivirus LTRs, the highest activity in all the cell lines examined was produced by the LTR of simian immunodeficiency virus from African green monkey (SIVAGM), suggesting that it has a wide expression range. In COS and SW480 cells, the activity of the FIV LTR was much lower than that of the SIVAGM LTR. These results indicate that, whereas the primary block to FIV infection of certain cells may occur at the cell surface, the FIV LTR may also participate in controlling virus replication, as an intracellular mechanism.


Genetic characterization of simian immunodeficiency virus isolated from an African mandrill

We constructed an infectious molecular clone of simian immunodeficiency virus from an African mandrill (SIVMND). Upon transfection, this clone directed the production of progeny virus particles infectious to and cytopathic for CD4+ human leukemia cells. Thirteen frameshift proviral mutants with an alteration in the eight open reading frames of SIVMND were generated by recombinant DNA techniques, and were analyzed biologically and biochemically. While mutations in the structural genes gag, pol, and env abolished viral growth and induction of cytopathology, mutants of the vif, vpr, and nef genes were fully biologically active. Of the tat and rev mutants, only one rev mutant grew in CD4+ cells with delayed kinetics. In reporter-based transient expression systems, transactivation potentials of the tat and rev mutants were evaluated. A mutant lacking 2nd coding exon of tat gene exhibited tat activity similar to that of the wild type clone. The infectious rev mutant was partially defective for rev gene activity.