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.

Dual phasic suppression of viral replication following de novo human immunodeficiency virus type 1 (HIV-1) infection in lymphocytes of asymptomatic HIV-1 carriers.

Replication of human immunodeficiency virus type 1 (HIV-1) is suppressed in asymptomatic HIV-1 carriers (ACs). By using an in vitro experimental system, the mechanism of this suppression was investigated. Following in vitro infection of a laboratory HIV-1 strain, the peripheral blood mononuclear cells (PBMC) of ACs transiently supported a low level of viral replication, then the virus production rapidly decreased. PCR analysis revealed that HIV-1 proviral DNA integrated in the PBMC of ACs following infection gradually decreased. Such tapering consequences of in vitro HIV-1 infection in the PBMC of ACs were abrogated by depletion of CD8+ T cells from the culture. Furthermore, the viruses subsequently produced by the PBMC of an AC were less able to replicate than the virus produced by CD8+ cell-depleted PBMC of the same donor. These observations suggested that the CD8+ T cell-mediated suppression of HIV-1 replication in ACs may involve both cytocidal and cytostatic mechanisms: the former kills the cells producing viruses, and the latter inhibits viral spread by reducing viral infectivity.

Establishment of a phylogenetic survey system for AIDS-related lentiviruses and demonstration of a new HIV-2 subgroup.

We designed a universal primer (UNIPOL) for DNA amplification of AIDS-related viruses. The phylogenetic tree constructed from the presumed sequences amplified with UNIPOL was representative of the tree calculated from whole pol gene sequences so far reported. UNIPOL was able to amplify the sequences of all four major groups of primate lentiviruses and also that of a distinct virus from a Ghanaian patient with an AIDS-related complex, designated GH-2. This strain scarcely hybridizes with known HIV/simian immunodeficiency virus (SIV) DNA probes. Sequence analysis of the only amplified fragment revealed rapidly that GH-2 was quite similar to the recently reported HIV-2ALT(D205) and that these two viruses form a new subgroup distint from known HIV-2 and SIVmac/SIVsm in the large HIV-2 group. This system will be useful for further phylogenetic study of various primate lentiviruses.

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.

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.

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.

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.

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.

Age-dependent remodeling of peripheral blood CD4+ CD8+ T lymphocytes in cynomolgus monkeys.

Recently, we have found in adult cynomolgus monkeys that substantial peripheral blood CD4+ CD8+ double-positive (DP) T lymphocytes exhibit a resting memory phenotype and increase in proportion with age. In this study, we investigated whether phenotypic changes occur in the course of the increase in proportion of the DP T cells. The results obtained from 195 clinically healthy monkeys aged from 1 month to 31 years showed that the CD29hi and CD28 subpopulation in the DP T subset increased in proportion with age and that the increase reached a plateau at six years old for the CD29hi subpopulation and at eleven years old for the CD28 one, respectively. The phenotypic alteration preceded the abrupt increase in proportion of the DP T cells and was able to be classified into four phases on the basis of the qualitative and quantitative alteration.

Induction of MHC-IIDR 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.

Phenotypic changes in peripheral blood monocytes of cynomolgus monkeys acutely infected with simian immunodeficiency virus.

The quantitative and phenotypic changes of peripheral blood monocytes during the acute stage of simian immunodeficiency virus infection were investigated. We inoculated intravenously three cynomolgus monkeys (Macaca fascicularis) with 100 TCID50 of SIVmac239 and collected whole blood twice a week until 35 days postinoculation. We found that the relative number of monocytes in peripheral blood leukocytes significantly increased at 7-17 days postinoculation. This increase was concomitant with the peak of primary SIV antigenemia. To determine if the monocytes observed during the acute stage were phenotypically altered, they were periodically examined for the expression of surface markers (i.e., CD11b, CD14, CD16, CD29, D32, CD56, CD62L, CD64, CD80, and MHC-II-DR) by flow cytometry. The results showed that the expression levels of CD14 and CD56 on most of the monocytes were remarkably reduced at 7-17 days postinoculation, and a new subpopulation, CD14lowCD16+CD80+ monocytes, was clearly detected at 10 days postinoculation. These results indicate that the phenotypic alteration of peripheral blood monocytes occurs during the primary SIV infection.

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.

Antiviral effects of 6-chloro-2',3'-dideoxyguanosine in rhesus monkeys acutely infected with simian immunodeficiency virus.

A lipophilic dideoxynucleoside analogue, 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG), was expected to be effective against AIDS-related dementia. In this study, we tested the effect of 6-Cl-ddG on simian immunodeficiency virus (SIVmac239) replication in vitro and on acute infection of six rhesus monkeys (Macaca mulatta) with SIVmac239. This compound inhibited SIV-induced cytopathic effect in CEM x 174 cells and SIV replication in vitro with an ED50 value of 2.5 microM. A dose of 25 mg/kg 6-Cl-ddG was administered to three monkeys every 8 h for 10 days and an untreated group of three monkeys was injected with the solvent without drug. Although 6-Cl-ddG was not detected in the plasma, the metabolite ddG was maintained at a concentration of more than 3 microM for 8 h after administration. In the cerebrospinal fluid, the ddG concentration was 2 microM at 2 h after administration. SIV antigen (p27) and antibody appearance in the plasma were delayed for 5-8 days compared with the mock-treated group. The occurrence of lymphadenopathy in treated monkeys was delayed for 6 days compared with the mock-treated group. Signs of 6-Cl-ddG toxicity were minimal after the treatment. The results of this study provide further evidence that 6-Cl-ddG may act as a potent anti-human immunodeficiency virus agent in vivo.

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.

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.

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.

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.

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.

Role of virus-induced apoptosis in a host defense mechanism against virus infection.

Many animal viruses are known to induce apoptosis in infected cells. This virus-induced apoptosis has been often described as a mechanism of host defense against virus infection, based on the finding that mutants of an insect virus with the ability to induce extensive apoptosis in some cells cannot grow in the same cells. In animal virus infection, we have shown that (1) viruses can somehow overcome this defense mechanism and that (2) virus multiplication in the apoptotic cells is not as completely suppressed as in the insect virus infection. These results suggest that, in the case of animal viruses, the virus-induced apoptosis does not play the same role in the host defense system as in insect cells. However, by examining the virus infection under the conditions comparable to the infection in vivo, we demonstrated the defensive role of apoptosis in animal virus infection.

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.