Disruption of the putative splice acceptor site for SIV(mac239)Vif reveals tight control of SIV splicing and impaired replication in Vif non-permissive cells

Vif is dispensable for simian immunodeficiency virus (SIV) replication in some cells, termed permissive (i.e., CEM-SS), but not in others, termed non-permissive (i.e., H9, CEMx174, and peripheral blood lymphocytes). Non-permissive cells express the RNA editing enzyme, APOBEC3G. To determine whether vif mRNA could be alternatively spliced, a mutation altering the putative vif splice acceptor site (SA1) was introduced into SIV(mac239) (SIV(Deltavif-SA)). Despite three consensus splice acceptor sites nearby SA1, SIV(Deltavif-SA) did not efficiently generate alternatively spliced vif mRNA. SIV(Deltavif-SA) was growth attenuated in CEMx174 and H9 cells but not in CEM-SS cells. Following SIV(Deltavif-SA), but not SIV(mac239), infection in either H9 or CEMx174 cells viral cDNA contained numerous G to A mutations; no such differences were observed in CEM-SS cells. This pattern is consistent with mutations generated by APOBEC3G in the absence of Vif. Therefore, efficient splicing of SIV vif mRNA is tightly controlled and requires the SA1 site.     

Analysis of the viral determinants underlying replication kinetics and cellular tropism of human immunodeficiency virus.

Human immunodeficiency viruses (HIVs) isolated from infected individuals show genetic and biological diversity. To delineate the genetic determinants underlying specific biological characteristics such as rate of replication and cellular tropism, generation of hybrid HIV using viruses which exhibit distinct biological feature is essential. We have used three different infectious HIV proviral DNAs, designated pZ6, pHXB2 and pARV, derived from HIVZr6, HIVHTLV-IIIB and HIVSF-2 strains, respectively, to construct hybrid HIV. Proviral DNAs differed in their ability to direct the synthesis of viral particles upon transfection into cells and the viruses derived from the molecular clones exhibited different cellular tropism. Three different methods were utilized to generate hybrid HIV, including construction of hybrid proviral DNA using molecular techniques, intracellular ligation of viral DNA fragments and the homologous recombination approach. The chimeric proviral DNAs with exchanges involving only the long terminal repeat (LTR) region indicated that LTR does not exert influence on the overall level of virus production despite extensive differences in the U3 region of the LTR. Regarding the cellular tropism of HIV, the virus derived from pHXB2 productively infected CEMx174 cells. On the other hand, pARV-derived virus did not show productive infection of CEMx174 cells. The hybrid HIV containing the 3'-end of the genome from pARV and the 5'-end of the genome from pHXB2 was effective in infecting CEMx174 cells. However, the converse hybrid containing the 5'-pARV and the 3'-pHXB2 was not effective in infecting CEMx174 cells. These results suggest that differences in the genes outside of env and nef may play a role in the ability of virus to infect a certain cell type.     

Rhesus monkey simian immunodeficiency virus infection as a model for assessing the role of selenium in AIDS

The objective of this study was to determine whether simian immunodeficiency virus (SIV) infection of macaques could be used as a model system to assess the role of selenium in AIDS. Plasma and serum selenium levels were determined by standard assays in monkeys before and after inoculation of SIV. SIV-infected cells or cells expressing the HIV Tat protein were labeled with 75Se, and protein extracts were prepared and electrophoresed to analyze selenoprotein expression. Total tRNA was isolated from CEMx174 cells infected with SIV or from KK1 cells infected with HIV, and selenocysteine tRNA isoforms were characterized by reverse phase chromatography. SIV-infected monkeys show a decrease in blood selenium levels similar to that observed in AIDS with development of SAIDS. Cells infected with SIV in vitro exhibit reduced selenoprotein levels and an accumulation of small molecular weight selenium compounds relative to uninfected cells. Examination of the selenocysteine tRNA isoforms in HIV-infected KK1 cells or SIV-infected CEMx174 cells reveals an isoform distribution characteristic of selenium-deficient cells. Furthermore, transfection of Jurkat E6 cells with the Tat gene selectively altered selenoprotein synthesis, with GPX4 and Sep15 being the most inhibited and TR1 the most enhanced. Taken together, the data show that monkeys infected with SIV in vivo and cells infected with SIV in vitro will provide appropriate models for investigating the mechanism(s) responsible for reduced selenium levels that accompany the progression of AIDS in HIV disease.     

Viral and cellular gene expression in CD4+ human lymphoid cell lines infected by the simian immunodeficiency virus, SIV/Mne.

Our laboratory has undertaken an analysis of cellular and viral gene expression in CD4+ human lymphoid cell lines infected by the human and simian immunodeficiency viruses, HIV-1 and SIV/Mne, respectively. The purpose of the current study was to: (i) examine the effects of SIV/Mne infection on host macromolecular synthesis and compare the results to those in the HIV-1 system; and (ii) investigate the mechanisms responsible for the restriction of SIV/Mne infection in CD4 positive lymphoid cells which are readily infected by HIV-1. First we determined that SIV does not impose selective blocks on host macromolecular synthesis, unlike HIV-1, which induces both the selective inhibition of cellular protein synthesis and the degradation of cellular mRNAs (Agy, M., Wambach, M., Foy, K., and Katze, M. G., 1990, Virology 177, 251-258). No such selective reduction in cellular mRNA stability or protein synthesis was observed in cells infected by SIV/Mne. Additional differences between SIV and HIV-1 were observed using a panel of CD4+ human cell lines. While HIV-1-infected all cell lines. SIV/Mne efficiently infected only the MT-4, C8166, and 174 x CEM cell lines. Repeated efforts to infect CEM or Jurkat cells were unsuccessful as determined by PCR analysis of viral DNA. HUT 78 cells supported a limited infection detectable only by PCR analysis. These data suggest the block in viral replication in the nonsusceptible cell lines is at an early step. Interestingly, all the SIV susceptible cells were virally transformed, C8166 and MT-4 by HTLV-1, and 174 x CEM by Epstein-Barr virus. Furthermore FACS analysis revealed that all susceptible cells expressed two B cell associated markers, B7/BB1 and CD40. These observations taken together highlight differences between the HIV and SIV viruses, and suggest that for efficient replication, SIV/Mne may require an additional cell surface molecule, cofactors provided by transforming viruses, or a complex interplay between the two.     

Recombinant vesicular stomatitis viruses encoding simian immunodeficiency virus receptors target infected cells and control infection

We have constructed VSV recombinants lacking the viral glycoprotein gene and instead expressing rhesus macaque SIV receptors CD4 and CCR5 with or without the receptor DC-SIGN. The recombinant expressing CD4 and CCR5 specifically infected SIV envelope protein-expressing cells. Incorporation of DC-SIGN into the particles required deletion of the cytoplasmic domain. Inclusion of DC-SIGN in the particles definitely enhanced infection, indicating that the enhancement by coexpression of DC-SIGN with CD4 and CCR5 does not require internalization of the virus into cells. The recombinants also specifically infected, killed, and propagated in CEMx174 cells that were first infected with an SIV expressing EGFP. If cells were superinfected with either of the recombinants after the primary SIV infection, the numbers of SIV-infected cells and titers of infectious SIV in the cultures were significantly reduced. Such antivirals can now be tested in the SIV/non-human primate model for AIDS to determine their therapeutic value in vivo.     

Structural constraints on viral escape from HIV- and SIV-specific cytotoxic T-lymphocytes

Cytotoxic T lymphocytes (CTL) play a central role in controlling lentiviral infections in both humans and monkeys. While they contain the spread of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), CTL are not capable of fully eradicating virus following infection. Ongoing viral replication can therefore lead to the accumulation of viral mutations within CTL epitopes that can undermine cellular immune control of virus. Here we review the importance of CTL in controlling HIV/SIV infection and how immunologic pressure exerted by effector T cells selects for viral variants that escape CTL recognition. We review two examples of viral escape from CTL at highly conserved epitopes that illustrate the extraordinary capacity of lentiviruses to adapt to their immunologic environment despite structural constraints on the ability of the virus to accommodate mutations.     

Recursion-based depletion of human immunodeficiency virus-specific naive CD4+ T cells may facilitate persistent viral replication and chronic viraemia leading to acquired immunodeficiency syndrome

Although antiretroviral therapy has made human immunodeficiency virus (HIV) infection a controllable disease, it is still unclear how viral replication persists in untreated patients and causes CD4⁺ T-cell depletion leading to acquired immunodeficiency syndrome (AIDS) in several years. Theorists tried to explain it with the diversity threshold theory in which accumulated mutations in the HIV genome make the virus so diverse that the immune system will no longer be able to recognize all the variants and fail to control the viraemia. Although the theory could apply to a number of cases, macaque AIDS models using simian immunodeficiency virus (SIV) have shown that failed viral control at the set point is not always associated with T-cell escape mutations. Moreover, even monkeys without a protective major histocompatibility complex (MHC) allele can contain replication of a super infected SIV following immunization with a live-attenuated SIV vaccine, while those animals are not capable of fighting primary SIV infection. Here we propose a recursion-based virus-specific naive CD4⁺ T-cell depletion hypothesis through thinking on what may happen in individuals experiencing primary immunodeficiency virus infection. This could explain the mechanism for impairment of virus-specific immune response in the course of HIV infection.     

Transactivation is a conserved function among primate lentivirus Vpr proteins but is not shared by Vpx.

OBJECTIVE: To investigate the transactivating activity of Vpr proteins from human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency viruses (SIVs) on various primate lentivirus long terminal repeats (LTRs), and to determine whether the Vpx proteins shared by HIV-2 and SIV are able to transactivate any HIV or SIV promoter. STUDY DESIGN/METHODS: The vpr and vpx genes of the HIVs and SIVs encode virion-associated proteins, which are implicated in viral replication and pathogenesis. HIV-1 Vpr is involved in the transport of the preintegration complex (PIC) to the nucleus, transactivates the viral LTR, and induces cell cycle arrest. HIV-2 and SIV Vpx proteins share amino acid sequence similarities with Vpr and are involved in PIC translocation into the nucleus but are unable to induce cell cycle arrest. We cloned and expressed the vpr and vpx genes from several primate lentiviruses and tested their transactivating ability on HIV-1, HIV-2, SIVmac and SIVagm LTRs cloned upstream of the CAT reporter gene. RESULTS: All Vpr tested had a transactivating effect on several viral LTRs; however, none of the Vpx proteins showed a detectable transactivating effect. CONCLUSIONS: These results indicate that the transactivating properties of Vpr proteins were conserved throughout evolution in primate lentiviruses, which suggests that they have an important role in virus replication.     

Live and let die: Nef functions beyond HIV replication

The viral Nef protein is important for the progression of the human and simian immunodeficiency virus (HIV/SIV) infection. So far, experimental evidence has suggested that Nef enhances viral replication and infectivity through a combination of different effects. Recent insights, however, indicate that its functions are more complex than previously anticipated. By targeting the T cell receptor, Nef may not only prime viral replication but, more importantly, ensure viral survival through distinct mechanisms of immune evasion and antiapoptosis.     

Into the wild: simian immunodeficiency virus (SIV) infection in natural hosts

Identifying distinctions between pathogenic HIV and simian immunodeficiency virus (SIV) infections and nonprogressive SIV in natural African primate hosts might provide key insights into HIV pathogenesis. Similar to pathogenic HIV infection in humans, natural SIV infections result in high viral replication and massive acute depletion of mucosal CD4(+) T cells. A key distinction of natural SIV infections is a rapidly developing anti-inflammatory milieu that prevents chronic activation, apoptosis and proliferation of T cells and preserves the function of other immune cell subsets, thus contributing to the integrity of the mucosal barrier and the lack of microbial translocation from the gut to the peritoneum. Immunologic features observed during natural SIV infections suggest approaches for designing new strategies for producing novel second-generation vaccines and therapeutic approaches to inhibit disease progression in HIV-infected humans.     

RANTES, IFN-gamma, CCR1, and CCR5 mRNA expression in peripheral blood, lymph node, and bronchoalveolar lavage mononuclear cells during primary simian immunodeficiency virus infection of macaques

Primary infection of macaques with pathogenic isolates of simian immunodeficiency virus (SIV) (as a model of HIV infection in humans) represents a unique opportunity to study early lentivirus/host interactions. We sought to determine whether there is a temporal relationship linking SIV replication and dissemination and the expression of the chemokine RANTES (regulated upon activation normal T cell expressed and secreted) and the SIV/HIV coreceptor CCR5 in different tissues during acute SIV infection of macaques. Four cynomolgus macaques were inoculated intravenously with a pathogenic primary isolate of SIVmac251. RT-PCR was used to monitor the expression of RANTES and CCR5 mRNA in fresh isolated mononuclear cells from blood, lymph node, and bronchoalveolar lavages. These expressions were compared to those of IFN-gamma as an indicator of the development of the immune response and to another receptor for RANTES, CCR1, which is not described as a coreceptor for SIV/HIV-1 entry. An enhancement of CCR1/CCR5 mRNA expression was noticed during primary SIVmac251 infection of macaques, mainly in tissue. In the three different compartments investigated, IFN-gamma and RANTES overexpression was noticed by the time of systemic viral replication containment. Our results put CCR5 and RANTES mRNA expression back in the context of inflammatory and immune responses to SIV primary infection.     

The role of the alternative coreceptor GPR15 in SIV tropism for human cells

Many SIV isolates can employ the orphan receptor GPR15 as coreceptor for efficient entry into transfected cell lines, but the role of endogenously expressed GPR15 in SIV cell tropism is largely unclear. Here, we show that several human B and T cell lines express GPR15 on the cell surface, including the T/B cell hybrid cell line CEMx174, and that GPR15 expression is essential for SIV infection of CEMx174 cells. In addition, GPR15 expression was detected on subsets of primary human CD4⁺, CD8⁺ and CD19⁺ peripheral blood mononuclear cells (PBMCs), respectively. However, GPR15⁺ PBMCs were not efficiently infected by HIV and SIV, including cells from individuals homozygous for the defective Δ32 ccr5 allele. These results suggest that GPR15 is coexpressed with CD4 on PBMCs but that infection of CD4⁺, GPR15⁺ cells is not responsible for the well documented ability of SIV to infect CCR5⁻ blood cells.     

Phosphorylation of HIV Nef by cAMP-dependent protein kinase

Nef, a multifunctional accessory protein of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), is important for disease progression. Nef downmodulates CD4 and MHC class I expression, alters host-cell signal transduction pathways, and enhances viral replication. We have identified a novel interaction between Nef and cAMP-dependent kinase (PKA). N-terminal serine residues Ser6,9 of HIVNL4-3 Nef and Ser10 of SIVmac239 Nef were phosphorylated by PKA in a cell-free system; intracellularly, only Ser9 of HIVNL4-3 Nef was phosphorylated by PKA. Mutation of Ser9 to alanine in the context of full-length HIVNL4-3 lowered HIV replication in resting peripheral blood mononuclear cells (PBMC) compared to parental virus. As this mutation played a major role in abrogating the Nef effect on HIV replication in unstimulated primary cells, we postulate that Nef phosphorylation by PKA is an important step in the viral life cycle in resting cells.     

Determination of HIV-1 infectivity by lymphocytic cell lines with integrated luciferase gene

We have established lymphocytic cell lines H9 and M8166 that contain integrated copy of luciferase gene under the control of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). While H9 is known to be non-permissive for or insensitive to some particular mutant strains of HIV/simian immunodeficiency virus (SIV), M8166 is one of the most susceptible lines to various HIV/SIVs. The luciferase gene driven by HIV-1 LTR was transfected into H9 and M8166 cells with the neo gene, and cell lines were selected by G418. The indicator cell lines thus obtained were designated H9/H1luc and M8166/H1luc, and monitored for their susceptibility to various HIV clones including in vitro-constructed mutants. Both cell lines, particularly M8166/H1luc, were found to be exquisitely sensitive to HIV-1 and HIV-2. Furthermore, they exhibited the response to infections by various viral clones exactly as expected from the characteristics of the original cell lines. These results indicated that our new indicator cell lines H9/H1luc and M8166/H1luc are eminently useful for a variety of molecular virological studies on HIV/SIV.     

ICAM-3 influences human immunodeficiency virus type 1 replication in CD4(+) T cells independent of DC-SIGN-mediated transmission

We investigated the role of ICAM-3 in DC-SIGN-mediated human immunodeficiency virus (HIV) infection of CD4(+) T cells. Our results demonstrate that ICAM-3 does not appear to play a role in DC-SIGN-mediated infection of CD4(+) T cells as virus is transmitted equally to ICAM-3(+) or ICAM-3(-) Jurkat T cells. However, HIV-1 replication is enhanced in ICAM-3(-) cells, suggesting that ICAM-3 may limit HIV-1 replication. Similar results were obtained when SIV replication was examined in ICAM-3(+) and ICAM-3(-) CEMx174 cells. Furthermore, while ICAM-3 has been proposed to play a co-stimulatory role in T cell activation, DC-SIGN expression on antigen presenting cells did not enhance antigen-dependent activation of T cells. Together, these data indicate that while ICAM-3 may influence HIV-1 replication, it does so independent of DC-SIGN-mediated virus transmission or activation of CD4(+) T cells.