Lymphocyte activation during acute simian/human immunodeficiency virus SHIV(89.6PD) infection in macaques

Host-virus interactions control disease progression in human immunodeficiency virus-infected human beings and in nonhuman primates infected with simian or simian/human immunodeficiency viruses (SHIV). These interactions evolve rapidly during acute infection and are key to the mechanisms of viral persistence and AIDS. SHIV(89.6PD) infection in rhesus macaques can deplete CD4(+) T cells from the peripheral blood, spleen, and lymph nodes within 2 weeks after exposure and is a model for virulent, acute infection. Lymphocytes isolated from blood and tissues during the interval of acute SHIV(89.6PD) infection have lost the capacity to proliferate in response to phytohemagglutinin (PHA). T-cell unresponsiveness to mitogen occurred within 1 week after mucosal inoculation yet prior to massive CD4(+) T-cell depletion and extensive virus dissemination. The lack of mitogen response was due to apoptosis in vitro, and increased activation marker expression on circulating T cells in vivo coincided with the appearance of PHA-induced apoptosis in vitro. Inappropriately high immune stimulation associated with rapid loss of mature CD4(+) T cells suggested that activation-induced cell death is a mechanism for helper T-cell depletion in the brief period before widespread virus dissemination. Elevated levels of lymphocyte activation likely enhance SHIV(89.6PD) replication, thus increasing the loss of CD4(+) T cells and diminishing the levels of virus-specific immunity that remain after acute infection. The level of surviving immunity may dictate the capacity to control virus replication and disease progression. We describe this level of immune competence as the host set point to show its pivotal role in AIDS pathogenesis.     

Construction and in vitro properties of chimeric simian and human immunodeficiency virus with the human TNF-alpha gene

Tumor necrosis factor-alpha (TNF-alpha) has been reported to be involved in the development and progression of acquired immunodeficiency syndrome (AIDS). To study the role of this cytokine in AIDS pathogenesis, we constructed a chimeric simian and human immunodeficiency virus (SHIV) having the human TNF-alpha gene (SHIV-TNF) and characterized its properties in vitro. SHIV-TNF replicated both in M8166, a human T cell line, and in monkey peripheral blood mononuclear cells (PBMCs). Along with SHIV-TNF replication, TNF-alpha was detected in the culture supernatant by ELISA. The maximum expression level of TNF-alpha reached 120 ng/ml in M8166 cells, and 2.5 ng/ml in monkey PBMCs. The expressed TNF was biologically active, as shown by a cytotoxic assay using TNF-sensitive L929 mouse fibroblasts. This activity was detected at least until 10 passages of SHIV-TNF (74 days after the initial infection). In monkey PBMCs, SHIV-TNF replicated much better than the parental SHIV-NI. Flow cytometric analysis showed that the death of monkey PBMCs infected with SHIV-TNF was severer than that caused by the parental SHIV-NI. These results suggest that SHIV-TNF would be useful for inducing the disease in a monkey model, which may contribute to a better understanding of the role of TNF-alpha in AIDS etiology.     

Neuropathogenesis of chimeric simian human immunodeficiency virus infection in rhesus macaques

Comparative studies were performed to determine the neuropathogenesis of infection in macaques with simian human immunodeficiency virus (SHIV)89.6P and SHIV_KU. Both viruses utilize the CD4 receptor and CXCR4 co-receptor. However, in addition, SHIV89.6P uses the CCR5 co-receptor. Both agents are dual tropic for CD4⁺ T cells and blood-derived macrophages of rhesus macaques. Following inoculation into macaques, both caused rapid elimination of CD4⁺ T cells but they varied greatly in mechanisms of neuropathogenesis. Two animals infected with SHIV89.6P developed typical lentiviral encephalitis in which multinucleated giant cell formation, nodular accumulations of microglial cells, activated macrophages and astrocytes, and perivascular accumulations of mononuclear cells were present in the brain. Many of the macrophages in these lesions contained viral RNA. Three macaques infected with SHIV_KU and killed on days 6, 11 and 18, respectively, developed a slowly progressive infection in the CNS but macrophages were not productively infected and there were no pathological changes in the brain. Two other animals infected with this virus and killed several months later showed minimal infection in the brain even though one of the two developed encephalitis of unknown etiology. The basic difference in the mechanisms of neuropathogenesis by the two viruses may be related to co-receptor usage. SHIV89.6P, in utilizing the CCR5 co-receptor, caused neuropathogenic effects that are similar to other neurovirulent primate lentiviruses.     

Neuropathogenesis of chimeric simian human immunodeficiency virus infection in rhesus macaques

Comparative studies were performed to determine the neuropathogenesis of infection in macaques with simian human immunodeficiency virus (SHIV)89.6P and SHIV(KU). Both viruses utilize the CD4 receptor and CXCR4 co-receptor. However, in addition, SHIV89.6P uses the CCR5 co-receptor. Both agents are dual tropic for CD4+ T cells and blood-derived macrophages of rhesus macaques. Following inoculation into macaques, both caused rapid elimination of CD4+ T cells but they varied greatly in mechanisms of neuropathogenesis. Two animals infected with SHIV89.6P developed typical lentiviral encephalitis in which multinucleated giant cell formation, nodular accumulations of microglial cells, activated macrophages and astrocytes, and perivascular accumulations of mononuclear cells were present in the brain. Many of the macrophages in these lesions contained viral RNA. Three macaques infected with SHIV(KU) and killed on days 6, 11 and 18, respectively, developed a slowly progressive infection in the CNS but macrophages were not productively infected and there were no pathological changes in the brain. Two other animals infected with this virus and killed several months later showed minimal infection in the brain even though one of the two developed encephalitis of unknown etiology. The basic difference in the mechanisms of neuropathogenesis by the two viruses may be related to co-receptor usage. SHIV89.6P, in utilizing the CCR5 co-receptor, caused neuropathogenic effects that are similar to other neurovirulent primate lentiviruses.     

Generation of a chimeric human and simian immunodeficiency virus infectious to monkey peripheral blood mononuclear cells.

We constructed five chimeric clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIVMAC) and four SIVMAC mutants by recombinant DNA techniques. Three chimeric clones and all mutants with an alteration in either the vif, vpx, vpr, or nef gene were infectious to human CD4-positive cell lines. The susceptibility of macaque monkey peripheral blood mononuclear cells (PBMC) to infection by these mutants and chimeras was examined in vitro. Macaque PBMC supported the replication of wild-type and vpx, vpr, and nef mutant SIVMAC strains. A chimera carrying the long terminal repeats (LTRs), gag, pol, vif, and vpx of SIVMAC and tat, rev, vpu, and env of HIV-1 was also replication competent in PBMC. In contrast, HIV-1, the vif mutant of SIVMAC, a chimera containing rev and env of SIVMAC, and a chimera containing vpx, vpr, tat, rev, and env of SIVMAC did not grow in PBMC. Western immunoblotting analysis of the replicating chimera in PBMC confirmed the hybrid nature of the virus. These data strongly suggested that the sequence important for macaque cell tropism lies within the LTR, gag, pol, and/or vif sequences of the SIVMAC genome.     

Genetic analysis of the human immunodeficiency virus type 1 integrase protein.

Single-amino-acid changes in a highly conserved central region of the human immunodeficiency virus type 1 (HIV-1) integrase protein were analyzed for their effects on viral protein synthesis, virion morphogenesis, and viral replication. Alteration of two amino acids that are invariant among retroviral integrases, D116 and E152 of HIV-1, as well as a mutation of the highly conserved amino acid S147 blocked viral replication in two CD4+ human T-cell lines. Mutations of four other highly conserved amino acids in the region had no detectable effect on viral replication, whereas mutations at two positions, N117 and Y143, resulted in viruses with a delayed-replication phenotype. Defects in virion precursor polypeptide processing, virion morphology, or viral DNA synthesis were observed for all of the replication-defective mutants, indicating that changes in integrase can have pleiotropic effects on viral replication.     

Construction and in vitro characterization of a chimeric simian and human immunodeficiency virus with the RANTES gene

Chimeric simian-human immunodeficiency virus (SHIV) containing the env gene of HIV-1 infects macaque monkeys and provides basic information that is useful for the development of HIV-1 vaccines. Regulated-on-activation-normal-T-cell-expressed-and-secreted (RANTES), a CC-chemokine, enhances antigen-specific T helper type-1 responses against HIV-1. With the final goal of testing the adjuvant effects of RANTES in SHIV-macaque models, we constructed a SHIV having the RANTES gene (SHIV-RANTES) and characterized its properties in vitro. SHIV-RANTES replicated both in human and monkey T cell lines. Along with SHIV-RANTES replication, RANTES was detected in the supernatant of human and monkey cell cultures, at maximal levels of 98.5 and 4.1 ng/ml, respectively. A flow cytometric analysis showed that the expressed RANTES down-modulated CC-chemokine receptor 5 (CCR5) on PM1 cells, which was restored by adding anti-RANTES antibody. UV-irradiated culture supernatants from the SHIV-RANTES-infected cells suppressed replication of CCR5-tropic HIV-1 BaL in PM-1 cells. Differentiating real-time RT-PCR showed that pre-infection of SHIV-RANTES in C8166 cells expressing CCR5 suppressed the replication of HIV-1 BaL. Biological activity of the expressed RANTES and the inserted RANTES gene in SHIV-RANTES remained stable after 10 passages. These results suggest that SHIV-RANTES is worth testing in macaque models.     

Requirement of active human immunodeficiency virus type 1 integrase enzyme for productive infection of human T-lymphoid cells.

The human immunodeficiency virus type 1 (HIV-1) integrase enzyme exhibits significant amino acid sequence conservation with integrase proteins of other retroviruses. We introduced specific amino acid substitutions at a number of the conserved residue positions of recombinant HIV-1 integrase. Some of these substitutions resulted in proteins which were not able to be purified in the same manner as the wild-type enzyme, and these were not studied further. The remaining mutant enzymes were assessed for their abilities to perform functions characteristic of the integrase protein. These included specific removal of the terminal dinucleotides from oligonucleotide substrates representative of the viral U5-long terminal repeat, nonspecific cleavage of oligonucleotide substrates, and mediation of the strand transfer (integration) reaction. Substitution at position 43, within the protein's zinc finger motif region, resulted in an enzyme with reduced specificity for cleavage of the terminal dinucleotide. In addition, a double substitution of aspartic acid and glutamine for valine and glutamic acid, respectively, at positions 151 and 152 within the D,D(35)E motif region rendered the integrase protein inactive for all of its functions. The introduction of this double substitution into an infectious HIV-1 provirus yielded a mutant virus that was incapable of productively infecting human T-lymphoid cells in culture.     

Elevated frequency of CD1c+ myeloid dendritic cells in the peripheral blood mononuclear cells of simian/human immunodeficiency virus (SHIV) and simian immunodeficiency virus (SIV) repeatedly infected Chinese rhesus macaques

CD1c+ myeloid dendritic cells (mDCs) in the peripheral blood of 30 SHIV-SF162p4 and SIVmac251 sequentially infected Chinese rhesus macaques were examined by flow cytometry to obtain further insight into mDC alterations in HIV/AIDS. The CD1c+ cells were found to be mononuclear leukocytes rather than granulocytes, and most of them expressed CD20. CD1c+mDCs (CD1c+CD20−) consisted of two morphological subsets: the granular and the large CD1c+mDCs. The expression of HLA-DR, CD86, and CD11b, but no CCR7, CD83 and CD123, together with their endocytotic capacity indicated that they were immature mDCs. Their frequency at weeks 10 and 12 post-infection was significantly higher than that of un-infected ones; the large CD1c+mDC level was significantly different between time points and almost absent from un-infected rhesus monkeys; significant correlations between CD1c+mDCs and plasma viral load levels were also observed. These data indicated a possible role for CD1c+mDCs in the pathophysiological process of SIV/HIV infection.     

Mutational analysis of the human immunodeficiency virus type 2 (HIV-2) genome in relation to HIV-1 and simian immunodeficiency virus SIV (AGM).

We constructed an infectious molecular clone of the human immunodeficiency virus type 2 (HIV-2) and generated nine frameshift mutants corresponding to nine open reading frames identified so far. Three structural (gag, pol, env) and two regulative (tat, rev) gene mutants were not infectious, whereas vif, vpx, vpr, and nef genes were dispensable for infectivity. All of the mutants except env and rev were cytopathic in CD4+ human leukemia cells. In transfection assays, the expression of HIV-2 long terminal repeat was activated by infectious clones of HIV-1, HIV-2, and simian immunodeficiency virus from African green monkey but not by the tat mutants. However, an HIV-2 tat mutant could produce small amounts of virus proteins and particles in contrast to a rev mutant, which directed no detectable synthesis of virus proteins and virions.     

DC-SIGN interactions with human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus

Dendritic cells (DCs) efficiently bind and transmit human immunodeficiency virus (HIV) to cocultured T cells and so may play an important role in HIV transmission. DC-SIGN, a novel C-type lectin that is expressed in DCs, has recently been shown to bind R5 HIV type 1 (HIV-1) strains and a laboratory-adapted X4 strain. To characterize the interaction of DC-SIGN with primate lentiviruses, we investigated the structural determinants of DC-SIGN required for virus binding and transmission to permissive cells. We constructed a panel of DC-SIGN mutants and established conditions which allowed comparable cell surface expression of all mutants. We found that R5, X4, and R5X4 HIV-1 isolates as well as simian immunodeficiency and HIV-2 strains bound to DC-SIGN and could be transmitted to CD4/coreceptor-positive cell types. DC-SIGN contains a single N-linked carbohydrate chain that is important for efficient cell surface expression but is not required for DC-SIGN-mediated virus binding and transmission. In contrast, C-terminal deletions removing either the lectin binding domain or the repeat region abrogated DC-SIGN function. Trypsin-EDTA treatment inhibited DC-SIGN mediated infection, indicating that virus was maintained at the surface of the DC-SIGN-expressing cells used in this study. Finally, quantitative fluorescence-activated cell sorting analysis of AU1-tagged DC-SIGN revealed that the efficiency of virus transmission was strongly affected by variations in DC-SIGN expression levels. Thus, variations in DC-SIGN expression levels on DCs could greatly affect the susceptibility of human individuals to HIV infection.     

Characterization of simian immunodeficiency virus SIVSM/human immunodeficiency virus type 2 Vpx function in human myeloid cells

Human immunodeficiency virus type 2 (HIV-2)/simian immunodeficiency virus SIV_SM Vpx is incorporated into virion particles and is thus present during the early steps of infection, when it has been reported to influence the nuclear import of viral DNA. We recently reported that Vpx promoted the accumulation of full-length viral DNA following the infection of human monocyte-derived dendritic cells (DCs). This positive effect was exerted following the infection of DCs with cognate viruses and with retroviruses as divergent as HIV-1, feline immunodeficiency virus, and even murine leukemia virus, leading us to suggest that Vpx counteracted an antiviral restriction present in DCs. Here, we show that Vpx is required, albeit to a different extent, for the infection of all myeloid but not of lymphoid cells, including monocytes, macrophages, and monocytoid THP-1 cells that had been induced to differentiate with phorbol esters. The intracellular localization of Vpx was highly heterogeneous and cell type dependent, since Vpx localized differently in HeLa cells and DCs. Despite these differences, no clear correlation between the functionality of Vpx and its intracellular localization could be drawn. As a first insight into its function, we determined that SIV_SM/HIV-2 and SIV_RCM Vpx proteins interact with the DCAF1 adaptor of the Cul4-based E3 ubiquitin ligase complex recently described to associate with HIV-1 Vpr and HIV-2 Vpx. However, the functionality of Vpx proteins in the infection of DCs did not strictly correlate with DCAF1 binding, and knockdown experiments failed to reveal a functional role for this association in differentiated THP-1 cells. Lastly, when transferred in the context of a replication-competent viral clone, Vpx was required for replication in DCs.     

Importin 7 may be dispensable for human immunodeficiency virus type 1 and simian immunodeficiency virus infection of primary macrophages

In an in vitro assay employing reconstituted nuclei, importin 7 (IPO7) has been implicated in nuclear translocation of human immunodeficiency virus type 1 (HIV-1) cDNA. Using RNA interference technology, we inhibited expression of IPO7 by 80 to 95% in primary macrophages and in HeLa cells and monitored their ability to support HIV-1 and simian immunodeficiency virus (SIV) cDNA synthesis, nuclear translocation, and infection efficiency. Marked IPO7 deficiency did not alter the rate or extent of HIV-1 or SIV cDNA synthesis or nuclear translocation. The infection efficiency of HIV-1 was similarly unaltered. Therefore, in natural, nondividing targets of HIV-1, IPO7 may be dispensable for infection.     

Cross-neutralization of human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus isolates.

In contrast to infrequent and low-titer cross-neutralization of human immunodeficiency virus type 1 (HIV-1) isolates by HIV-2- and simian immunodeficiency virus (SIV)-positive sera, extensive cross-neutralization of HIV-2NIH-Z, SIVMAC251, and SIVAGM208K occurs with high titer, suggesting conservation of epitopes and mechanism(s) of neutralization. The V3 regions of HIV-2 and SIV isolates, minimally related to the HIV-1 homolog, share significant sequence homology and are immunogenic in monkeys as well as in humans. Whereas the crown of the V3 loop is cross-reactive among HIV-1 isolates and elicits neutralizing antibodies of broad specificity, the SIV and especially HIV-2 crown peptides were not well recognized by cross-neutralizing antisera. V3 loop peptides of HIV-2 isolates did not elicit neutralizing antibodies in mice, guinea pigs, or a goat and together with SIV V3 peptides did not inhibit serum neutralization of HIV-2 and SIV. Thus, the V3 loops of HIV-2 and SIV do not appear to constitute simple linear neutralizing epitopes. In view of the immunogenicity of V3 peptides, the failure of conserved crown peptides to react with natural sera implies a significant role of loop conformation in antibody recognition. Our studies suggest that in addition to their grouping by envelope genetic relatedness, HIV-2 and SIV are neutralized similarly to each other but differently from HIV-1. The use of linear peptides of HIV-2 and SIV as immunogens may require greater attention to microconformation, and alternate subunit approaches may be needed in exploiting these viruses as vaccine models. Such approaches may also be applicable to the HIV-1 system in which conformational epitopes, in addition to the V3 loop, participate in virus neutralization.     

Chimeric human immunodeficiency virus type 1 virions that contain the simian immunodeficiency virus nef gene are cyclosporin A resistant

We have previously shown that human immunodeficiency virus type 1 (HIV-1) virions which have their own nef gene deleted and are trans complemented to contain HIV-2 or simian immunodeficiency virus (SIV) Nef become resistant to treatment with cyclosporin A. To expand and confirm these studies, we have tested an HIV-1 isolate in which the HIV-1 nef gene has been replaced by the nef gene from SIV in a multiround infectivity assay using more physiologically relevant cell types. Our results confirm that HIV-1 virions that contain SIV nef can replicate in a cyclophilin-independent fashion.     

An env gene derived from a primary human immunodeficiency virus type 1 isolate confers high in vivo replicative capacity to a chimeric simian/human immunodeficiency virus in rhesus monkeys.

To explore the roles played by specific human immunodeficiency virus type 1 (HIV-1) genes in determining the in vivo replicative capacity of AIDS viruses, we have examined the replication kinetics and virus-specific immune responses in rhesus monkeys following infection with two chimeric simian/human immunodeficiency viruses (SHIVs). These viruses were composed of simian immunodeficiency virus SIVmac239 expressing HIV-1 env and the associated auxiliary HIV-1 genes tat, vpu, and rep. Virus replication was assessed during primary infection of rhesus monkeys by measuring plasma SIVmac p27 levels and by quantifying virus replication in lymph nodes using in situ hybridization. SHIV-HXBc2, which expresses the HIV-1 env of a T-cell-tropic, laboratory-adapted strain of HIV-1 (HXBc2), replicated well in rhesus monkey peripheral blood leukocytes (PBL) in vitro but replicated only to low levels when inoculated in rhesus monkeys. In contrast, SHIV-89.6 was constructed with the HIV-1 env gene of a T-cell- and macrophage-tropic clone of a patient isolate of HIV-1 (89.6). This virus replicated to a lower level in monkey PBL in vitro but replicated to a higher degree in monkeys during primary infection. Moreover, monkeys infected with SHIV-89.6 developed an inversion in the PBL CD4/CD8 ratio coincident with the clearance of primary viremia. The differences in the in vivo consequences of infection by these two SHIVs could not be explained by differences in the immune responses elicited by these viruses, since infected animals had comparable type-specific neutralizing antibody titers, proliferative responses to recombinant HIV-1 gp120, and virus-specific cytolytic effector T-cell responses. With the demonstration that a chimeric SHIV can replicate to high levels during primary infection in rhesus monkeys, this model can now be used to define genetic determinants of HIV-1 pathogenicity.