Mechanisms of immune activation of human immunodeficiency virus in monocytes/macrophages.

Monocytes/macrophages (M/M) are the major host of human immunodeficiency virus (HIV) in solid tissues. However, blood monocytes are nonpermissive for HIV infection, indicating that M/M activation or differentiation is necessary for HIV replication. Since M/M are activated during immune responses, we investigated the effect of T-cell activation on HIV expression in M/M derived from peripheral blood of HIV-infected individuals. Previously, we reported that coculture of monocytes from HIV-infected donors with T cells and mitogens resulted in M/M differentiation and HIV expression. Production of HIV by M/M from infected donors required direct contact between monocytes and T cells (for the first 24 h), and the response to alloantigens, but not mitogens, was restricted to HLA-DR. In this study, we found that HIV was more readily recovered from M/M of asymptomatic HIV seropositive donors (69%) than from M/M of symptomatic donors (57%). Viral antigens (e.g., inactivated herpes simplex virus) could initiate the immune response and HIV expression. The ability of noninfected T cells to activate HIV expression in M/M and observations that treatments of M/M with antibodies to deplete T cells did not reduce HIV expression suggested that the monocytes were endogenously infected. To define the aspects of immune activation specifically involved in initiating HIV expression in M/M, interactions of M/M and T cells and participation of cytokines were investigated. The T cell which activated M/M was CD4+ CD8-. Fixed allogeneic cells are known to induce T-cell activation but were not able to serve as antigen for M/M differentiation, suggesting that M/M may need to function as antigen-presenting cells to receive the signal to differentiate and express HIV. Blocking of M/M-T-cell interaction with antibodies directed against LFA-1 or interleukin-1 prevented HIV expression. However, inhibition of later stages of T-cell activation, such as blocking of interleukin-2 receptors, did not diminish HIV expression in M/M. Consistent with the requirement for cell-cell contact between M/M and T cells, a variety of cytokines were unable to initiate HIV replication in M/M. The ability of T cells to induce cellular differentiation and HIV replication in M/M in vitro suggests that initiation of an immune response to an antigen, such as an opportunistic pathogen, could be a mechanism by which HIV disseminates to tissues in vivo.     

Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts

A number of recent studies have demonstrated the significance of detergent-insoluble, glycolipid-enriched membrane domains or lipid rafts, especially in regard to activation and signaling in T lymphocytes. These domains can be viewed as floating rafts composed of sphingolipids and cholesterol which sequester glycosylphosphatidylinositol (GPI)-linked proteins, such as Thy-1 and CD59. CD45, a 200-kDa transmembrane phosphatase protein, is excluded from these domains. We have found that human immunodeficiency virus type 1 (HIV-1) particles produced by infected T-cell lines acquire the GPI-linked proteins Thy-1 and CD59, as well as the ganglioside GM1, which is known to partition preferentially into lipid rafts. In contrast, despite its high expression on the cell surface, CD45 was poorly incorporated into virus particles. Confocal fluorescence microscopy revealed that HIV-1 proteins colocalized with Thy-1, CD59, GM1, and a lipid raft-specific fluorescent lipid, DiIC(16)(3), in uropods of infected Jurkat cells. CD45 did not colocalize with HIV-1 proteins and was excluded from uropods. Dot immunoassay of Triton X-100-extracted membrane fractions revealed that HIV-1 p17 matrix protein and gp41 were present in the detergent-resistant fractions and that [(3)H]myristic acid-labeled HIV Gag showed a nine-to-one enrichment in lipid rafts. We propose a model for the budding of HIV virions through lipid rafts whereby host cell cholesterol, sphingolipids, and GPI-linked proteins within these domains are incorporated into the viral envelope, perhaps as a result of preferential sorting of HIV Gag to lipid rafts.     

Molecular characterization of feline immunodeficiency virus budding

Infection of domestic cats with feline immunodeficiency virus (FIV) is an important model system for studying human immunodeficiency virus type 1 (HIV-1) infection due to numerous similarities in pathogenesis induced by these two lentiviruses. However, many molecular aspects of FIV replication remain poorly understood. It is well established that retroviruses use short peptide motifs in Gag, known as late domains, to usurp cellular endosomal sorting machinery and promote virus release from infected cells. For example, the Pro-Thr/Ser-Ala-Pro [P(T/S)AP] motif of HIV-1 Gag interacts directly with Tsg101, a component of the endosomal sorting complex required for transport I (ESCRT-I). A Tyr-Pro-Asp-Leu (YPDL) motif in equine infectious anemia virus (EIAV), and a related sequence in HIV-1, bind the endosomal sorting factor Alix. In this study we sought to identify and characterize FIV late domain(s) and elucidate cellular machinery involved in FIV release. We determined that mutagenesis of a PSAP motif in FIV Gag, small interfering RNA-mediated knockdown of Tsg101 expression, and overexpression of a P(T/S)AP-binding fragment of Tsg101 (TSG-5′) each inhibited FIV release. We also observed direct binding of FIV Gag peptides to Tsg101. In contrast, mutagenesis of a potential Alix-binding motif in FIV Gag did not affect FIV release. Similarly, expression of the HIV-1/EIAV Gag-binding domain of Alix (Alix-V) did not disrupt FIV budding, and FIV Gag peptides showed no affinity for Alix-V. Our data demonstrate that FIV relies predominantly on a Tsg101-binding PSAP motif in the C terminus of Gag to promote virus release in HeLa cells, and this budding mechanism is highly conserved in feline cells.     

Positionally independent and exchangeable late budding functions of the Rous sarcoma virus and human immunodeficiency virus Gag proteins.

The Gag proteins of Rous sarcoma virus and human immunodeficiency virus (HIV) each contain a function involved in a late step in budding, defects in which result in the accumulation of these molecules at the plasma membrane. In the Rous sarcoma virus Gag protein (Pr76gag), this assembly domain is associated with a PPPY motif, which is located at an internal position between the MA and CA sequences. This motif is not contained anywhere within the HIV Gag protein (Pr55gag), and the MA sequence is linked directly to CA. Instead, a late assembly function of HIV has been associated with the p6 sequence situated at the C terminus of Gag. Here we demonstrate the remarkable finding that the late assembly domains from these two unrelated Gag proteins are exchangeable between retroviruses and can function in a positionally independent manner.     

Defects in human immunodeficiency virus budding and endosomal sorting induced by TSG101 overexpression

Retrovirus budding is greatly stimulated by the presence of Gag sequences known as late or L domains. The L domain of human immunodeficiency virus type 1 (HIV-1) maps to a highly conserved Pro-Thr-Ala-Pro (PTAP) sequence in the p6 domain of Gag. We and others recently observed that the p6 PTAP motif interacts with the cellular endosomal sorting protein TSG101. Consistent with a role for TSG101 in virus release, we demonstrated that overexpressing the N-terminal, Gag-binding domain of TSG101 (TSG-5') suppresses HIV-1 budding by blocking L domain function. To elucidate the role of TSG101 in HIV-1 budding, we evaluated the significance of the binding between Gag and TSG-5' on the inhibition of HIV-1 release. We observed that a mutation in TSG-5' that disrupts the Gag/TSG101 interaction suppresses the ability of TSG-5' to inhibit HIV-1 release. We also determined the effect of overexpressing a panel of truncated TSG101 derivatives and full-length TSG101 (TSG-F) on virus budding. Overexpressing TSG-F inhibits HIV-1 budding; however, the effect of TSG-F on virus release does not require Gag binding. Furthermore, overexpression of the C-terminal portion of TSG101 (TSG-3') potently inhibits budding of not only HIV-1 but also murine leukemia virus. Confocal microscopy data indicate that TSG-F and TSG-3' overexpression induces an aberrant endosome phenotype; this defect is dependent upon the C-terminal, Vps-28-binding domain of TSG101. We propose that TSG-5' suppresses HIV-1 release by binding PTAP and blocking HIV-1 L domain function, whereas overexpressing TSG-F or TSG-3' globally inhibits virus release by disrupting the cellular endosomal sorting machinery. These results highlight the importance of TSG101 and the endosomal sorting pathway in virus budding and suggest that inhibitors can be developed that, like TSG-5', target HIV-1 without disrupting endosomal sorting.     

Heterologous late-domain sequences have various abilities to promote budding of human immunodeficiency virus type 1

Retroviral late (L) domains present within Gag act in conjunction with cellular proteins to efficiently release virions from the surface of the cell. Three different critical core sequences have been identified as required elements for L-domain function: PPPY, PTAP (also PSAP), and YPDL, with different retroviruses utilizing one or two of these core sequences. The human immunodeficiency virus type 1 (HIV-1) L domain is centered around a PTAP sequence in the p6 region of Gag. To assess the ability of heterologous L-domain sequences to be functionally interchanged for those in full-length HIV-1, we produced a series of constructs that replaced PTAP-containing p6(Gag) sequences with those of PPPY- or YPDL-based L domains. While previous studies had found that L domains are interchangeable in other retroviruses, most of the sequences introduced into p6(Gag) failed to substitute for PTAP-mediated L-domain function. One exception was the 11-amino-acid p2b sequence of Rous sarcoma virus (RSV) Gag, which could fully restore HIV-1 budding, while a PPPPY sequence exchange alone did not. This suggests that the RSV L domain consists of more than simply its core L-domain sequence. The HIV-p2b chimera was as infectious as the wild type, produced normal virions, and was sensitive to proteasome inhibitors. These results show that L-domain sequences are not necessarily interchangeable. Thus, HIV-1 Gag might have a more stringent requirement for L-domain function than the other retroviruses previously studied.     

Protection of human immunodeficiency virus type 2-exposed seronegative macaques from mucosal simian immunodeficiency virus transmission.

At present it is not known which form of immunity would be most effective against infection with human immunodeficiency virus (HIV). To evaluate the possible role of cellular immunity, we examined whether four HIV type 2-exposed but seronegative macaques developed cellular immune responses and determined whether these exposed macaques were resistant to mucosal transmission of simian immunodeficiency virus (SIV). Following intrarectal challenge with SIV, 2 monkeys were protected against detectable SIV replication and another showed suppressed viral replication compared to 14 persistently infected controls. The two protected monkeys demonstrated SIV-specific cytotoxic T lymphocytes before as well as after SIV challenge. Here we provide evidence that activation of the cell-mediated arm of the immune system only, without antibody formation, can control SIV replication in macaques. The results imply that vaccines that stimulate a strong and broad cellular immune response could prevent mucosal HIV transmission.     

Fusion from without directed by human immunodeficiency virus particles.

Fusion from without is the process through which particles of some enveloped viruses can direct fusion of target cells in the absence of viral replication. We demonstrate here that human immunodeficiency virus (HIV) particles can efficiently promote fusion from without. Using HeLa-CD4 cells carrying a Tat-inducible lacZ gene, we observed syncytia as early as 6 h after exposure to HIV particles, before HIV gene expression could be detected. Efficient syncytium formation could be obtained when cells were treated with zidovudine, which prevented HIV replication and expression but not cell-cell fusion. Fusion was also observed when cells were exposed to particles of a replication-defective HIV integrase mutant. Fusion from without by HIV particles could be blocked by a monoclonal antibody specific for the V3 loop of the HIV-1 envelope glycoprotein and by soluble CD4. This mechanism of cytopathicity, which can involve cells that do not actively replicate HIV and can be directed by replication-defective particles, could participate in the pathogenicity of the CD4 cell depletion that characterizes HIV infection.     

The intracytoplasmic domain of gp41 mediates polarized budding of human immunodeficiency virus type 1 in MDCK cells.

Human immunodeficiency virus type 1 (HIV-1) has been shown to exhibit a specific basolateral release in polarized epithelial cells. Previous investigators have used vaccinia virus recombinants expressing HIV proteins to demonstrate that virus release is nonpolarized in the absence of viral envelope glycoproteins. In this study, we developed a transient expression system which allows the use of Madin-Darby canine kidney polarized epithelial cells directly grown on semipermeable membranes. This procedure allowed us to investigate polarized HIV viral budding following introduction of proviral DNA constructs. Expression of env gene products in trans demonstrated the ability to polarize env-negative viruses in a dose-dependent manner. The targeting signal for polarized virus release was shown to be present in the envelope gp41 transmembrane protein and absent from the gp120 portion of env. At least part of this signal is within the gp41 intracytoplasmic domain. Mutants of the p17gag matrix protein were shown to be nonpolarized only when unable to interact with the envelope glycoproteins. Together, these data are consistent with a model of polarized virus budding in which capsid proteins, lacking a targeting signal, are targeted for specific basolateral release via an interaction of p17 with the envelope glycoprotein containing the polarization signal in its intracytoplasmic domain.     

Genetic recombination of human immunodeficiency virus.

We investigated genetic recombination of the human immunodeficiency virus (HIV) in a tissue culture system. A clonal cell line expressing a single integrated HIV provirus with a termination codon affecting pol gene expression was transfected with different defective mutants derived from an infectious molecular clone of HIV. Replication-competent viral particles were recovered, passaged, and plaque purified. Restriction analyses of the proviral DNA corresponding to several of these viruses indicated that their emergence was the result of genetic recombination.     

Functional comparison of transactivation by simian immunodeficiency virus from rhesus macaques and human immunodeficiency virus type 1.

Simian immunodeficiency virus from rhesus macaques (SIVmac), like human immunodeficiency virus type 1 (HIV-1), encodes a transactivator (tat) which stimulates long terminal repeat (LTR)-directed gene expression. We performed cotransfection assays of SIVmac and HIV-1 tat constructs with LTR-CAT reporter plasmids. The primary effect of transactivation for both SIVmac and HIV-1 is an increase in LTR-directed mRNA accumulation. The SIVmac tat gene product partially transactivates an HIV-1 LTR, whereas the HIV-1 tat gene product fully transactivates an SIVmac LTR. Significant transactivation is achieved by the product of coding exon 1 of the HIV-1 tat gene; however, inclusion of coding exon 2 results in a further increase in mRNA accumulation. In contrast, coding exon 2 of the SIVmac tat gene is required for significant transactivation. These results imply that the tat proteins of SIVmac and HIV-1 are functionally similar but not interchangeable. In addition, an in vitro-generated mutation in SIVmac tat disrupts splicing at the normal splice acceptor site at the beginning of coding exon 2 and activates a site approximately 15 nucleotides downstream. The product of this splice variant stimulates LTR-directed gene expression. This alternative splice acceptor site is also used by a biologically active provirus with an efficiency of approximately 5% compared with the upstream site. These data suggest that a novel tat protein is encoded during the course of viral infection.     

A critical role of nitric oxide in human immunodeficiency virus type 1-induced hyperresponsiveness of cultured monocytes.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) infection leads to a general exhaustion of the immune system. Prior to this widespread decline of immune functions, however, there is an evident hyperactivation of the monocyte/macrophage arm. Increased levels of cytokines and other biologically active molecules produced by activated monocytes may contribute to the pathogenesis of HIV disease both by activating expression of HIV-1 provirus and by direct effects on cytokine-sensitive tissues, such as lung or brain. In this article, we investigate mechanisms of hyperresponsiveness of HIV-infected monocytes. MATERIALS AND METHODS: The study was performed on monocyte cultures infected in vitro with a monocytetropic strain HIV-1ADA. Cytokine production was induced by stimulation of cultures with lipopolysaccharides (LPS) and measured by ELISA. To study involvement of nitric oxide (NO) in the regulation of cytokine expression, inhibitors of nitric oxide synthase (NOS) or chemical donors of NO were used. RESULTS: We demonstrate that infection with HIV-1 in vitro primes human monocytes for subsequent activation with LPS, resulting in increased production of pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin 6 (IL-6). This priming effect can be blocked by Ca(2+)-chelating agents and by the NOS inhibitor L-NMMA, but not by hemoglobin. It could be reproduced on uninfected monocyte cultures by using donors of NO, but not cGMP, together with LPS. CONCLUSIONS: NO, which is expressed in HIV-1-infected monocyte cultures, induces hyperresponsiveness of monocytes by synergizing with calcium signals activated in response to LPS stimulation. This activation is cGMP independent. Our findings demonstrate the critical role of NO in HIV-1-specific hyperactivation of monocytes.     

Pathogenesis of human immunodeficiency virus infection.

The lentivirus human immunodeficiency virus (HIV) causes AIDS by interacting with a large number of different cells in the body and escaping the host immune response against it. HIV is transmitted primarily through blood and genital fluids and to newborn infants from infected mothers. The steps occurring in infection involve an interaction of HIV not only with the CD4 molecule on cells but also with other cellular receptors recently identified. Virus-cell fusion and HIV entry subsequently take place. Following virus infection, a variety of intracellular mechanisms determine the relative expression of viral regulatory and accessory genes leading to productive or latent infection. With CD4+ lymphocytes, HIV replication can cause syncytium formation and cell death; with other cells, such as macrophages, persistent infection can occur, creating reservoirs for the virus in many cells and tissues. HIV strains are highly heterogeneous, and certain biologic and serologic properties determined by specific genetic sequences can be linked to pathogenic pathways and resistance to the immune response. The host reaction against HIV, through neutralizing antibodies and particularly through strong cellular immune responses, can keep the virus suppressed for many years. Long-term survival appears to involve infection with a relatively low-virulence strain that remains sensitive to the immune response, particularly to control by CD8+ cell antiviral activity. Several therapeutic approaches have been attempted, and others are under investigation. Vaccine development has provided some encouraging results, but the observations indicate the major challenge of preventing infection by HIV. Ongoing research is necessary to find a solution to this devastating worldwide epidemic.     

Polarized human immunodeficiency virus budding in lymphocytes involves a tyrosine-based signal and favors cell-to-cell viral transmission

Maturation and release of human immunodeficiency virus type 1 (HIV-1) is targeted at the pseudopod of infected mononuclear cells. However, the intracellular mechanism or targeting signals leading to this polarized viral maturation are yet to be identified. We have recently demonstrated the presence of a functional YXXL motif for specific targeting of HIV-1 virions to the basolateral membrane surface in polarized epithelial Madin-Darby canine kidney cells (MDCK). Site-directed mutagenesis was used to demonstrate that the membrane-proximal tyrosine in the intracytoplasmic tail of the HIV-1 transmembrane glycoprotein (gp41) is an essential component of this signal. In the present study, immunolocalization of viral budding allowed us to establish that this tyrosine-based signal is involved in determining the exact site of viral release at the surface of infected mononuclear cells. Substitution of the critical tyrosine residue was also shown to increase the amount of envelope glycoprotein at the cell surface, supporting previous suggestions that the tyrosine-based motif can promote endocytosis. Although alteration of the dual polarization-endocytosis motif did not affect the infectivity of cell-free virus, it could play a key role in cell-to-cell viral transmission. Accordingly, chronically infected lymphocytes showed a reduced ability to transmit the mutant virus to a cocultivated cell line. Overall, our data indicate that the YXXL targeting motif of HIV is active in various cell types and could play an important role in viral propagation; this may constitute an alternative target for HIV therapeutics and vaccine development.     

Differential regulation of the human immunodeficiency virus type 2 enhancer in monocytes at various stages of differentiation.

We have demonstrated that stimulation of the human immunodeficiency virus type 2 (HIV-2) enhancer in T cells is dependent upon at least four cis-acting elements, including two purine-rich binding sites, PuB1 and PuB2, which are capable of binding members of the ets family of proto-oncogenes, the pets (peri-ets) site, which lies just upstream of the PuB2 site, and a single kappa B site (D. M. Markovitz, M. Smith, J. M. Hilfinger, M. C. Hannibal, B. Petryniak, and G. J. Nabel, J. Virol. 66:5479-5484, 1992). In this study, we examined the regulation of the HIV-2 enhancer in cells of monocytic lineage. We found that in immature monocytic cell lines, the HIV-2 enhancer is markedly induced by phorbol esters and that all four cis-acting elements are required for activation. In mature monocytic cells, constitutive activity is high, with only modest stimulation following phorbol ester treatment. Mutation of any of the four cis-acting elements resulted in greatly reduced basal expression in mature monocytes. This is in contrast to HIV-1, in which developmentally controlled expression of the enhancer in monocytes is mediated largely through the kappa B sites alone [G. E. Griffin, K. Leung, T. M. Folks, S. Kunkel, and G. J. Nabel, Nature (London) 339:70-73, 1989]. Further, we demonstrated that although both Elf-1, an ets family member with significant similarity to the drosophila developmental regulatory protein E74, and Pu.1, a monocyte- and B-cell-specific member of the ets family, bind the purine-rich enhancer region, Elf-1 is the protein which binds predominantly in vivo. A nuclear factor(s) which binds the pets site, an element which has been described only in HIV-2, was detected in extracts of all of the monocytic cells tested. These findings indicate that the mechanism by which cellular factors regulate HIV-2 enhancer function in monocytic cells differs significantly from that of HIV-1 and may offer a partial explanation for the differences in the biological and clinical characteristics of the two viruses.