The human immunodeficiency virus type 1 accessory protein Vpu induces apoptosis by suppressing the nuclear factor kappaB-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.

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.

Human immunodeficiency virus type 1 Vif protein is packaged into the nucleoprotein complex through an interaction with viral genomic RNA.

The human immunodeficiency virus type 1 (HIV-1) Vif protein plays a critical role in the production of infectious virions. Previous studies have demonstrated the presence of small amounts of Vif in virus particles. However, Vif packaging was assumed to be nonspecific, and its functional significance has been questioned. We now report that packaging of Vif is dependent on the packaging of viral genomic RNA in both permissive and restrictive HIV-1 target cells. Mutations in the nucleocapsid zinc finger domains that abrogate packaging of viral genomic RNA abolished packaging of Vif. Additionally, an RNA packaging-defective virus exhibited significantly reduced packaging of Vif. Finally, deletion of a putative RNA-interacting domain in Vif abolished packaging of Vif into virions. Virion-associated Vif was resistant to detergent extraction and copurified with components of the viral nucleoprotein complex and functional reverse transcription complexes. Thus, Vif is specifically packaged into virions as a component of the viral nucleoprotein complex. Our data suggest that the specific association of Vif with the viral nucleoprotein complex might be functionally significant and could be a critical requirement for infectivity of viruses produced from restrictive host cells.

The human immunodeficiency virus type 1 Vpu protein inhibits NF-kappa B activation by interfering with beta TrCP-mediated degradation of Ikappa B.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein binds to the CD4 receptor and induces its degradation by cytosolic proteasomes. This process involves the recruitment of human betaTrCP (TrCP), a key member of the SkpI-Cdc53-F-box E3 ubiquitin ligase complex that specifically interacts with phosphorylated Vpu molecules. Interestingly, Vpu itself, unlike other TrCP-interacting proteins, is not targeted for degradation by proteasomes. We now report that, by virtue of its affinity for TrCP and resistance to degradation, Vpu, but not a phosphorylation mutant unable to interact with TrCP, has a dominant negative effect on TrCP function. As a consequence, expression of Vpu in HIV-infected T cells or in HeLa cells inhibited TNF-alpha-induced degradation of IkappaB-alpha. Vpu did not inhibit TNF-alpha-mediated activation of the IkappaB kinase but instead interfered with the subsequent TrCP-dependent degradation of phosphorylated IkappaB-alpha. This resulted in a pronounced reduction of NF-kappaB activity. We also observed that in cells producing Vpu-defective virus, NF-kappaB activity was significantly increased even in the absence of cytokine stimulation. However, in the presence of Vpu, this HIV-mediated NF-kappaB activation was markedly reduced. These results suggest that Vpu modulates both virus- and cytokine-induced activation of NF-kappaB in HIV-1-infected cells.

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.

Peripheral blood extrathymic CD4(+)CD8(+) T cells with high cytotoxic activity are from the same lineage as CD4(+)CD8(-) T cells in cynomolgus monkeys.

We have previously reported that CD4/CD8 double-positive (DP) T cells with the resting memory phenotype are present in the periphery of healthy cynomolgus monkeys. In the present study, we performed functional studies on the T cells. The expression of CD4 and CD8 on DP, CD4 single-positive (SP) or CD8 SP T cells was stable in cultures with either mitogen or anti-CD3 antibody stimulation. In spite of lacking CD28 expression, DP T cells showed similar proliferative ability and apoptosis sensitivity to CD4 SP and CD8 SP T cells. DP T cells showed both helper and cytotoxic activities. Although the helper activity of DP T cells was lower than that of CD4 SP T cells, cytotoxic activity was comparable to that of CD8 SP T cells. Fresh DP T cells killed target cells mainly by the perforin-granzyme pathway. In addition, fresh DP T cells expressed a high level of mRNA for IFN-gamma and produced a high level of IFN-gamma when they were activated by anti-CD3 antibody ligation. On the other hand, several expanded DP T cell clones shared TCR V(beta) with expanded CD4 SP T cell clones, strongly suggesting that those two corresponding clones with DP and CD4 SP phenotypes might be derived from the same ancestor T cell. These results showed that the DP T cells are a novel T cell subset with functions overlapping with those of CD4 SP and CD8 SP T cells, and that they might play protective and regulatory roles in secondary immune response in cynomolgus monkeys.

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.

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.

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.

Effective delivery of a lipophilic 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG) into rat lymphoid tissues.

Lipophilic 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG) was evaluated for its improved lymph node delivery by comparison with the parental nucleoside (ddG) in vitro and in vivo. The in vitro studies with rat plasma, lymph node homogenate and stomach content indicated that 6-Cl-ddG converted to ddG more effectively in the lymph node homogenate and that 6-Cl-ddG was more stable than ddG in the stomach content. In an in vivo study, plasma and lymph nodes were collected from rats after a subcutaneous or oral administration of 6-Cl-ddG or ddG. With the subcutaneous administrations of the drugs, the area under the concentration time-curve (AUC) value in the plasma for converted ddG following a 6-Cl-ddG administration was less than half the value for ddG following a ddG administration but the converted ddG AUC values in the lymph nodes due to 6-Cl-ddG administration were 1.4- to 2.0-fold higher than the ddG AUC values due to ddG administration. Moreover, with the oral administrations, the converted ddG AUC value in plasma after a 6-Cl-ddG administration was 3-fold higher than ddG after a ddG administration, and high levels of converted ddG were detected in the lymph nodes, but no ddG was detected in the lymph node following ddG administration. These results suggest that lipophilic 6-Cl-ddG is a useful prodrug for delivering ddG into the lymph nodes by oral administration.

Induction of apoptosis in Herpesvirus saimiri-immortalized T lymphocytes by blocking interaction of CD28 with CD80/CD86.

We have previously shown that Herpesvirus saimiri (HVS) immortalizes primary macaque monkey T lymphocytes. In this study, we examined the characteristics of the immortalized T cells. The cells showed the phenotype of activated T lymphoblasts (CD3(+) CD25(+) CD69(+) MHC-IIDR(+)) and produced no infectious virus while viral DNA was detected in the Hirt DNA. Interestingly, both a major costimulatory molecule, CD28, and its ligands, CD80/CD86, were coexpressed on the immortalized T cells. The treatment of the cells with a neutralizing monoclonal antibody against CD28, which blocks interaction of CD28 with CD80/CD86, resulted in retarded cell growth and in induction of apoptosis. The effect of the antibody treatment was not overcome by exogenous interleukin-2 treatment. These findings demonstrate the requirement of interaction of CD28 with CD80/CD86 for the optimal growth of HVS-immortalized T cells.

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.

Compatibility of Vpu-like activity in the four groups of primate immunodeficiency viruses.

Env-minus mutants of the viruses of major four human and simian immunodeficiency viruses (HIVs and SIVs) were monitored for their progeny virion production upon transfection into the cells, which are dependent on the HIV-1 Vpu for efficient particle release. Of the env mutants of HIV-1 (one mutant), HIV-2/SIVmac (three mutants), SIVagm (one mutant), and SIVmnd (one mutant) examined, the mutant of SIVmnd generated a very low level of progeny virions similar to that by the HIV-1 Vpu-minus mutant. This effect of the mutation was not observed in the cells which are independent on the Vpu for virion release. The Env of SIVmnd efficiently enhanced virion release of heterologous viruses like the HIV-1 Vpu.

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.

Rev-dependent expression of three species of HIV-1 mRNAs (review).

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.

Exchangeability of accessory Vif and Vpu proteins between various HIV/SIVs (review).

Representative human and simian immunodeficiency viruses (HIV/SIVs) have been monitored for their Vif and Vpu activities in a wide variety of cells. In contrast to the prototype HIV-1, viruses of the other groups do not necessarily have these activities. Only HIV-2 and SIVmnd were clearly demonstrated to show the Vif and Vpu activities, respectively. The exchangeability of these accessory activities between viruses was then assessed to determine the relatedness of the viruses. Quite different from the results for Tat and Rev trans-activators, the activities are almost fully compatible between viruses. These results may facilitate the functional grouping of various HIV/SIVs.

Accumulation of MAC387+ macrophages in paracortical areas of lymph nodes in rhesus monkeys acutely infected with simian immunodeficiency virus.

We investigated the histological features of lymph nodes, focusing on monocytes/macrophages, in rhesus monkeys (Macaca mulatta) acutely infected with simian immunodeficiency virus (SIV). In monkeys infected with a pathogenic SIV, SIVmac239, MAC387(+) newly blood-derived macrophages markedly increased in number at paracortical areas at 11 to 14 days postinoculation, concomitant with the peak of the primary SIV antigenemia. The MAC387(+) macrophages densely gathered around high endothelial venules and formed cell clusters with CD3(+) T lymphocytes, tingible body macrophages, and plasmacytoid monocytes. In the cell clusters, CD3(+) T lymphocytes which closely adhered to the MAC387(+) macrophages enlarged in size, suggesting a histological manifestation of T-lymphocyte activation by macrophages. By 54 days postinoculation, when SIV antigenemia became undetectable, the MAC387(+) macrophages decreased in number and the cell cluster disappeared from paracortical areas. In contrast, the monkeys infected with a nef-deleted mutant of SIVmac239 showed lower levels of SIV antigenemia and lower numbers of MAC387(+) macrophages in paracortical areas than those infected with SIVmac239. These results indicate that MAC387(+) macrophages accumulate in paracortical areas for the period of the intense primary SIV antigenemia and may play an important role in activating naive T lymphocytes.

Functional roles of HIV accessory proteins for viral replication (review).

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.

The non-env tropism of HIV/SIV (Review).

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.