Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Cell-Cell Spread of Human Immunodeficiency Virus Type 1 Overcomes Tetherin/BST-2-Mediated Restriction in T cells

Direct cell-to-cell spread of human immunodeficiency virus type 1 (HIV-1) between T cells at the virological synapse (VS) is an efficient mechanism of viral dissemination. Tetherin (BST-2/CD317) is an interferon-induced, antiretroviral restriction factor that inhibits nascent cell-free particle release. The HIV-1 Vpu protein antagonizes tetherin activity; however, whether tetherin also restricts cell-cell spread is unclear. We performed quantitative cell-to-cell transfer analysis of wild-type (WT) or Vpu-defective HIV-1 in Jurkat and primary CD4⁺ T cells, both of which express endogenous levels of tetherin. We found that Vpu-defective HIV-1 appeared to disseminate more efficiently by cell-to-cell contact between Jurkat cells under conditions where tetherin restricted cell-free virion release. In T cells infected with Vpu-defective HIV-1, tetherin was enriched at the VS, and VS formation was increased compared to the WT, correlating with an accumulation of virus envelope proteins on the cell surface. Increasing tetherin expression with type I interferon had only minor effects on cell-to-cell transmission. Furthermore, small interfering RNA (siRNA)-mediated depletion of tetherin decreased VS formation and cell-to-cell transmission of both Vpu-defective and WT HIV-1. Taken together, these data demonstrate that tetherin does not restrict VS-mediated T cell-to-T cell transfer of Vpu-defective HIV-1 and suggest that under some circumstances tetherin might promote cell-to-cell transfer, either by mediating the accumulation of virions on the cell surface or by regulating integrity of the VS. If so, inhibition of tetherin activity by Vpu may balance requirements for efficient cell-free virion production and cell-to-cell transfer of HIV-1 in the face of antiviral immune responses.Human immunodeficiency virus type 1 can disseminate between and within hosts by cell-free infection or by direct cell-cell spread. Cell-cell spread of HIV-1 between CD4⁺ T cells is an efficient means of viral dissemination (65) and has been estimated to be several orders of magnitude more rapid than cell-free virus infection (6, 8, 41, 64, 74). Cell-cell transmission of HIV-1 takes place at the virological synapse (VS), a multimolecular structure that forms at the interface between an HIV-1-infected T cell and an uninfected target T cell during intercellular contact (27). Related structures that facilitate cell-cell spread of HIV-1 between dendritic cells and T cells (42) and between macrophages and T cells (16, 17) and for cell-cell spread of the related retrovirus human T-cell leukemia virus type 1 (HTLV-1) (24) have also been described. Moreover, more long-range cell-cell transfer can occur via cellular projections, including filopodia (71) and membrane nanotubes (75). The VS is initiated by binding of the HIV-1 envelope glycoprotein (Env), which is expressed on the surfaces of infected T cells, to HIV-1 entry receptors (CD4 and either CXCR4 or CCR5) present on the target cell membrane (6, 22, 27, 41, 61, 73). Interactions between LFA-1 and ICAM-1 and ICAM-3 further stabilize the conjugate interface and, together with Env receptor binding, help trigger the recruitment of viral proteins, CD4/coreceptor, and integrins to the contact site (27, 28, 61). The enrichment of viral and cellular proteins at the VS is an active process, dependent on cytoskeletal remodeling, and in the infected T cell both the actin and tubulin network regulate polarization of HIV-1 proteins at the cell-cell interface, thus directing HIV-1 assembly and egress toward the engaged target cell (27, 29). Virus is transferred by budding into the synaptic cleft, and virions subsequently attach to the target cell membrane to mediate entry, either by fusion at the plasma membrane or possibly following endocytic uptake (2, 22). In this way, the VS promotes more rapid infection kinetics and may enhance HIV-1 pathogenesis in vivo.Cells have evolved a number of barriers to resist invading microorganisms. One mechanism that appears to be particularly important in counteracting HIV-1 infection is a group of interferon-inducible, innate restriction factors that includes TRIM5α, APOBEC3G, and tetherin (38, 49, 69, 79). Tetherin (BST-2/CD317) is a host protein expressed by many cell types, including CD4⁺ T cells, that acts at a late stage of the HIV-1 life cycle to trap (or “tether”) mature virions at the plasma membranes of virus-producing cells, thereby inhibiting cell-free virus release (49, 56, 81). This antiviral activity of tetherin is not restricted to HIV-1, and tetherin can also inhibit the release of other enveloped viruses from infected cells (31, 40, 54, 62). What the cellular function of tetherin is besides its antiviral activity is unclear, but because expression is upregulated following alpha/beta interferon (IFN-α/β) treatment (1) and tetherin can restrict a range of enveloped viruses, tetherin has been postulated to be a broad-acting mediator of the innate immune defense against enveloped viruses.To circumvent restriction of particle release, HIV-1 encodes the 16-kDa accessory protein Vpu, which antagonizes tetherin and restores normal virus budding (47, 78). The molecular mechanisms by which Vpu does this are not entirely clear, but evidence suggests that Vpu may exert its antagonistic function by downregulating tetherin from the cell surface, trapping it in the trans-Golgi network (10) and targeting it for degradation by the proteasome (12, 39, 81) or lysosome (9, 25, 44); however, degradation of tetherin may be dispensable for Vpu activity (13), and in HIV-1-infected T cells, surface downregulation of tetherin has been reported to be minor (45), suggesting that global removal of tetherin from the plasma membrane may not be necessary to antagonize its function.Tetherin-mediated restriction of HIV-1 and antagonism by Vpu have been the focus of much research, and inhibition of cell-free virus infection has been well documented (33, 47-49, 77, 81, 82). In contrast, less studied is the impact of tetherin on direct cell-cell dissemination. For example, it is not clear if tetherin-mediated restriction inhibits T cell-T cell spread as efficiently as cell-free release or whether tetherin affects VS formation. To address these questions, we analyzed Vpu⁺ and Vpu⁻ viruses for their ability to spread directly between Jurkat T cells and primary CD4⁺ T cells in the presence or absence of endogenous tetherin. Our data suggest that tetherin does not restrict HIV-1 in the context of cell-to-cell transmission of virus between T cells expressing endogenous tetherin. Interestingly, we also that observed that Vpu-defective virus may disseminate more efficiently by cell-cell spread at the VS. We postulate that cell-cell spread may favor viral pathogenesis by allowing HIV-1 to disseminate in the presence of tetherin during an interferon-producing innate response.     

Antagonism to and Intracellular Sequestration of Human Tetherin by the Human Immunodeficiency Virus Type 2 Envelope Glycoprotein

Tetherin (CD317/BST-2), an interferon-induced membrane protein, restricts the release of nascent retroviral particles from infected cell surfaces. While human immunodeficiency virus type 1 (HIV-1) encodes the accessory gene vpu to overcome the action of tetherin, the lineage of primate lentiviruses that gave rise to HIV-2 does not. It has been previously reported that the HIV-2 envelope glycoprotein has a Vpu-like function in promoting virus release. Here we demonstrate that the HIV-2 Rod envelope glycoprotein (HIV-2 Rod Env) is a tetherin antagonist. Expression of HIV-2 Rod Env, but not that of HIV-1 or the closely related simian immunodeficiency virus (SIV) SIVmac1A11, counteracts tetherin-mediated restriction of Vpu-defective HIV-1 in a cell-type-specific manner. This correlates with the ability of the HIV-2 Rod Env to mediate cell surface downregulation of tetherin. Antagonism requires an endocytic motif conserved across HIV/SIV lineages in the gp41 cytoplasmic tail, but specificity for tetherin is governed by extracellular determinants in the mature Env protein. Coimmunoprecipitation studies suggest an interaction between HIV-2 Rod Env and tetherin, but unlike studies with Vpu, we found no evidence of tetherin degradation. In the presence of HIV-2 Rod Env, tetherin localization is restricted to the trans-Golgi network, suggesting Env-mediated effects on tetherin trafficking sequester it from virus assembly sites on the plasma membrane. Finally, we recapitulated these observations in HIV-2-infected CD4⁺ T-cell lines, demonstrating that tetherin antagonism and sequestration occur at physiological levels of Env expression during virus replication.Various stages of the replication cycle of primate lentiviruses can be targeted by host antiviral restriction factors (reviewed in reference 49). In addition to the well-characterized antiviral effects of members of the APOBEC3 family of cytidine deaminases, particularly APOBEC3G and -3F, and species-specific variants of tripartite motif family 5α, the release of nascent retroviral particles has recently been shown to be a target for a novel restriction factor, tetherin (CD317/bone marrow stromal cell antigen 2 [BST-2]) (31, 46). Tetherin is an interferon-inducible gene that was originally shown to impart a restriction on the release of mutants of human immunodeficiency virus type 1 (HIV-1) that lack a vpu gene (31, 46). In tetherin-positive cells, mature Vpu-defective HIV-1 particles are retained on the cell surface, linked to the plasma membrane (PM) and each other via protease-sensitive tethers, and can be subsequently endocytosed and accumulate in late endosomes (30, 31). Tetherin is not HIV specific and restricts the release of virus-like particles derived from all retroviruses tested (18), as well as those of filoviruses and arenaviruses (18, 19, 39).Tetherin is a small (181-amino-acid) type II membrane protein with an unusual topology that exists mainly as a disulfide-linked dimer (34). It consists of an N-terminal cytoplasmic tail, a transmembrane anchor, an extracellular domain that includes three cysteine residues important for dimerization, a putative coiled-coil, and finally a glycophosphatidyinosityl-linked lipid anchor (22) that is essential for restriction (31). Tetherin localizes to retroviral assembly sites on the PM (18, 31), and this unusual structure is highly suggestive that tetherin restricts virion release by incorporation into the viral membrane and cross-linking virions to cells. Such a mechanism would make tetherin a powerful antiviral effector that can target an obligate part of most, if not all, enveloped virus assembly strategies. Moreover, since tetherin restriction has no specific requirement for virus protein sequences, to avoid its action, mammalian viruses have evolved to encode several distinct countermeasures that specifically inhibit tetherin''s antiviral function.The Vpu accessory protein antagonizes tetherin-mediated restriction of HIV-1 (31, 46). In the presence of Vpu, tetherin is downregulated from the cell surface (2, 46) and is targeted for degradation (10, 13, 14), although whether these processes are required for antagonism of tetherin function is unclear (27). HIV-1 Vpu displays a distinct species specificity in that it is unable to target tetherin orthologues from rhesus macaques or African green monkeys (14, 25). This differential sensitivity maps to the tetherin transmembrane domain, particularly residues that are predicted to have been under high positive selection pressure during primate evolution (14, 16, 25). This suggests that tetherin evolution may have been driven in part by viral countermeasures like Vpu. Vpu, however, is only encoded by HIV-1 and its direct simian immunodeficiency virus (SIV) lineage precursors. The majority of SIVs, including the SIVsm, the progenitor of both HIV-2 and SIVmac, do not encode a Vpu protein (21). In some of these SIVs, tetherin antagonism was recently shown to map to the nef gene (16, 51). SIV Nef proteins, however, are generally ineffective against human tetherin because they target a (G/D)DIWK motif that was deleted from the human tetherin cytoplasmic tail sometime after the divergence of humans and chimpanzees (51). This raises the question of how HIV-2 is able to overcome human tetherin, as recent data show chronically HIV-2-infected CEM T cells have reduced tetherin levels on their surface (10).Interestingly, it has long been known that the envelope glycoprotein of certain HIV-2 isolates can stimulate the release of Vpu-defective HIV-1 virions from cells we now know to be tetherin positive (5, 6, 43). HIV and SIV Envs form trimeric spikes of dimers of the surface subunit (SU-gp105 in HIV-2/SIVmac and gp120 in HIV-1) that bind CD4 and the chemokine coreceptor and gp41 (the transmembrane [TM] subunit that facilitates fusion with and entry into the target cell). Envelope precursors (gp140 or gp160) are synthesized in the endoplasmic reticulum, where they become glycosylated and are exported to the surface via the secretory pathway (8). During transit through the Golgi apparatus and possibly in endosomal compartments, the immature precursors are cleaved by furin-like proteases to form mature spikes (15, 29). Multiple endocytosis motifs in the gp41 cytoplasmic tail lead to only minor quantities of Env being exposed at the cell surface at any given time (7, 40). Recent data demonstrated that the conserved GYxxθ motif, a binding site for the clathrin adaptor protein AP-2 (3), in the membrane-proximal region of HIV-2 gp41 is required to promote Vpu-defective HIV-1 release from HeLa cells (1, 32). Based on experiments with HIV-1/HIV-2 chimeric envelopes, an additional requirement in the extracellular component was suggested (1). In this study we set out to examine the Vpu-like activity of HIV-2 envelope in light of the discovery of tetherin. We demonstrate that the HIV-2 Env is a tetherin antagonist, and we provide mechanistic insight into the basis of this antagonism.     

Prime-Boost Vaccination with Recombinant Mumps Virus and Recombinant Vesicular Stomatitis Virus Vectors Elicits an Enhanced Human Immunodeficiency Virus Type 1 Gag-Specific Cellular Immune Response in Rhesus Macaques

Intramuscular inoculation of rhesus macaques with one or more doses of recombinant vesicular stomatitis virus (rVSV) expressing human immunodeficiency virus type 1 (HIV-1) Gag (rVSVgag) typically elicits peak cellular immune responses of 500 to 1,000 gamma interferon (IFN-γ) enzyme-linked immunospots (ELISPOTS)/10⁶ peripheral blood lymphocytes (PBL). Here, we describe the generation of a novel recombinant mumps virus (rMuV) expressing HIV-1 Gag (rMuVgag) and measure the Gag-specific cellular immune responses detected in rhesus macaques following vaccination with a highly attenuated form of rVSV expressing HIV-1 Gag (rVSVN4CT1gag1) and rMuVgag in various prime-boost combinations. Notably, peak Gag-specific cellular immune responses of 3,000 to 3,500 ELISPOTS/10⁶ PBL were detected in macaques that were primed with rMuVgag and boosted with rVSVN4CT1gag1. Lower peak cellular immune responses were detected in macaques that were primed with rVSVN4CT1gag1 and boosted with rMuVgag, although longer-term gag-specific responses appeared to remain higher in this group of macaques. These findings indicate that rMuVgag may significantly enhance Gag-specific cellular immune responses when administered with rVSVN4CT1gag1 in heterologous prime-boost regimens.The ability to recover infectious virus from genomic cDNA has enabled the development of nonsegmented negative-strand RNA viruses as candidate vaccine vectors (8, 20); vesicular stomatitis virus (VSV), which predominantly infects insects and livestock in nature (29, 51, 52), is one of the most extensively studied in this group of RNA viruses. Recombinant forms of VSV (rVSVs) have been tested in preclinical studies as potential vaccine vectors to combat a wide range of human diseases including human immunodeficiency virus (HIV)/AIDS (16, 28, 30, 31, 40-43). In one of these studies, nonhuman primates (NHPs) vaccinated with rVSV vaccine vectors expressing simian immunodeficiency virus (SIV) Gag and HIV Env proteins were protected from disease following challenge with a pathogenic SIV/HIV-1 recombinant (SHIV) (46). Although these prototypic rVSV vaccine vectors elicited robust SHIV-specific immune responses in NHPs and demonstrated protective efficacy in the SHIV challenge model, they were found to be insufficiently attenuated for human trials when tested in a stringent NHP neurovirulence model (27). This finding was addressed by the development of a highly attenuated rVSV vector (rVSVN4CT1gag1). This vector was attenuated by combination of a specific N gene translocation and G gene truncation (9), with the N gene in position 4 (N4), the G gene expressing a G protein with a single amino acid in the cytoplasmic tail (CT1), and the HIV-1 Gag gene added in the first position of the genome (gag1). The rVSVN4CT1gag1 vector caused no obvious signs of neurological disease in young mice following intracranial inoculation with >10⁷ PFU of virus (12) and produced only very minimal, predominantly inflammatory lesions following intrathalamic inoculation of NHPs with 10⁷ PFU of virus (unpublished data). Although rVSVN4CT1gag1 demonstrated reduced in vitro replication efficiency and in vivo virulence, it was as immunogenic in mice (12) and NHPs (unpublished data) as the much more virulent prototypic rVSV vectors that provided protection from disease in the SHIV challenge model.Mumps virus (MuV), the agent of mumps in humans, is a nonsegmented negative-strand RNA virus in the family Paramyxoviridae. The incidence of mumps has been greatly reduced in the developed world by the introduction of live attenuated MuV vaccine strains over the past 30 to 35 years. The most commonly used MuV vaccine in the United States and Western Europe is the Jeryl Lynn strain, which has demonstrated excellent efficacy and an outstanding safety record for the >100 million doses administered to the pediatric population. A system for the recovery of the Jeryl Lynn strain of MuV from genomic cDNA has been described previously (10). This methodology has enabled targeted alteration of the MuV genome to study virus-associated neurovirulence and neuroattenuation (33) as well as the possibility of developing MuV as a vaccine vector for other pathogens.There is currently no proven method of inducing broadly neutralizing antibodies in HIV type 1 (HIV-1) vaccinees. It has been postulated, however, that robust vaccine-induced cellular immune responses directed against one or more HIV-1 proteins may be sufficient to prevent HIV-1-infected humans from developing AIDS in the absence of broadly neutralizing antibodies (34). Although this hypothesis has been called into question recently following the results of an HIV-1 phase II clinical trial (the STEP trial), there is still reason to believe that a robust cellular immune response against specific cytotoxic T-lymphocyte epitopes within highly conserved regions of the viral proteome could result in a significantly reduced viral load following HIV-1 infection (45). One rational approach for maximizing vaccine-induced HIV-1-specific peak cellular immune responses is the administration of completely heterologous vaccine vectors in prime-boost regimens (2, 15, 24, 40), unlike the serotype switch used previously in rVSV prime-boost vaccination regimens (12, 46, 47). In general, the magnitudes of the resulting cellular immune responses were higher than those detected for comparable prime-boost regimens with homologous vectors (14, 46) although any associated enhancement of protective efficacy in challenge models remains unclear (3, 48). Here, we describe the generation of a novel rMuV vector expressing HIV-1 Gag (rMuVgag) and the immune responses elicited in rhesus macaques when this vector was administered with a highly attenuated rVSVN4CT1gag1 vector in heterologous prime-boost regimens.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Analysis of Human Immunodeficiency Virus Type 1 Matrix Binding to Membranes and Nucleic Acids

The human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein targets HIV-1 precursor Gag (PrGag) proteins to assembly sites at plasma membrane (PM) sites that are enriched in cholesterol and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂]. MA is myristoylated, which enhances membrane binding, and specifically binds PI(4,5)P₂ through headgroup and 2′ acyl chain contacts. MA also binds nucleic acids, although the significance of this association with regard to the viral life cycle is unclear. We have devised a novel MA binding assay and used it to examine MA interactions with membranes and nucleic acids. Our results indicate that cholesterol increases the selectivity of MA for PI(4,5)P₂-containing membranes, that PI(4,5)P₂ binding tolerates 2′ acyl chain variation, and that the MA myristate enhances membrane binding efficiency but not selectivity. We also observed that soluble PI(4,5)P₂ analogues do not compete effectively with PI(4,5)P₂-containing liposomes for MA binding but surprisingly do increase nonspecific binding to liposomes. Finally, we have demonstrated that PI(4,5)P₂-containing liposomes successfully outcompete nucleic acids for MA binding, whereas other liposomes do not. These results support a model in which RNA binding protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to PM assembly sites.The matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) precursor Gag (PrGag) protein serves several functions in the viral replication cycle. One essential function is to target PrGag proteins to their assembly sites at the plasma membranes (PMs) of infected cells (4, 5, 11, 16, 25, 29, 30, 33, 35, 39, 43-45, 47, 50, 54, 56, 57). A second function is the recruitment of the viral surface/transmembrane (SU/TM; also referred to as gp120/gp41) envelope (Env) protein complex into virions (14, 15, 18, 19, 27, 51-53). In addition to these activities, numerous reports have attributed nucleic acid binding properties to retroviral MAs (24, 38, 47), and with some viruses MA appears to serve in an encapsidation capacity (24). While no encapsidation role has been assigned for HIV-1 MA, experiments have shown that MA can substitute for the HIV-1 nucleocapsid (NC) protein assembly function (38) under some circumstances, presumably by virtue of its facility to concentrate PrGag proteins by binding them to RNAs (38).A number of structural studies have been conducted on HIV-1 MA (1, 22, 41, 42, 49). The protein is N terminally myristoylated and composed of six α helices, capped by a three-strand β sheet (7, 22, 41, 42, 49). The protein trimerizes in solution and in crystals (22, 28, 49) and recently has been shown to organize as hexamers of trimers on lipid membranes (1). The membrane binding face of HIV-1 MA is basic, fostering its ability to associate with negatively charged phospholipid headgroups (1, 22, 30, 41, 42, 49). The importance of such an interaction has been underscored in molecular genetic experiments which demonstrated that depletion of PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] reduced the assembly efficiency of HIV-1 (9, 36). Consistent with these observations, HIV-1 MA preferentially binds to soluble PI(4,5)P₂ mimics through contacts with the headgroup and 2′ acyl chain, and binding promotes exposure of the MA myristate group and protein oligomerization (17, 21, 40-43, 46). However, PI(4,5)P₂ is not the only lipid to demonstrate an association with HIV-1. In particular, HIV-1 appears to assemble at cholesterol-rich PM sites, cholesterol is highly enriched in HIV-1 virions, and cholesterol depletion reduces viral infectivity (2, 6, 8, 20, 23, 26, 31, 34, 37). The HIV-1 lipidome shows additional differences from the PM lipids of infected cells (2, 5, 8), suggesting that other lipids could affect PrGag-membrane binding or virus assembly site selection.To gain a better understanding of the functions and interactions of HIV-1 MA, we have examined the liposome and nucleic acid binding properties of purified myristoylated MA. Using liposome flotation assays and a novel liposome bead binding assay, we have demonstrated that the PI(4,5)P₂ binding specificity of MA is enhanced by cholesterol, that protein myristoylation increases membrane binding efficiency but not specificity, and that 2′ acyl chain variation is compatible with PI(4,5)P₂ binding. We also examined whether soluble PI(4,5)P₂ mimics could compete with liposomes for MA binding. Surprisingly, we found that soluble mimics not only failed to compete with PI(4,5)P₂ liposomes but also increased MA binding to membranes that do not contain acidic phospholipids. Finally, we have observed that while MA does bind nucleic acids, nucleic acid binding is outcompeted by PI(4,5)P₂-containing liposomes. Our results suggest models for PrGag-membrane and RNA association and the HIV-1 assembly pathway.     

Safety and Immunogenicity of Novel Recombinant BCG and Modified Vaccinia Virus Ankara Vaccines in Neonate Rhesus Macaques

Although major inroads into making antiretroviral therapy available in resource-poor countries have been made, there is an urgent need for an effective vaccine administered shortly after birth, which would protect infants from acquiring human immunodeficiency virus type 1 (HIV-1) through breast-feeding. Bacillus Calmette-Guérin (BCG) is given to most infants at birth, and its recombinant form could be used to prime HIV-1-specific responses for a later boost by heterologous vectors delivering the same HIV-1-derived immunogen. Here, two groups of neonate Indian rhesus macaques were immunized with either novel candidate vaccine BCG.HIVA⁴⁰¹ or its parental strain AERAS-401, followed by two doses of recombinant modified vaccinia virus Ankara MVA.HIVA. The HIVA immunogen is derived from African clade A HIV-1. All vaccines were safe, giving local reactions consistent with the expected response at the injection site. No systemic adverse events or gross abnormality was seen at necropsy. Both AERAS-401 and BCG.HIVA⁴⁰¹ induced high frequencies of BCG-specific IFN-γ-secreting lymphocytes that declined over 23 weeks, but the latter failed to induce detectable HIV-1-specific IFN-γ responses. MVA.HIVA elicited HIV-1-specific IFN-γ responses in all eight animals, but, except for one animal, these responses were weak. The HIV-1-specific responses induced in infants were lower compared to historic data generated by the two HIVA vaccines in adult animals but similar to other recombinant poxviruses tested in this model. This is the first time these vaccines were tested in newborn monkeys. These results inform further infant vaccine development and provide comparative data for two human infant vaccine trials of MVA.HIVA.Close to 2.3 million of children globally are infected with human immunodeficiency virus type 1 (HIV-1). The majority of neonatal infections occur in utero or intrapartum and, in the absence of preventative interventions, up to 29% of infants breast-fed by infected mothers acquire HIV-1 (6). Furthermore, HIV-1-infected children face a worse prognosis than adults in that, without antiretroviral treatment (ART), 25% of perinatally infected children progress to AIDS within 1 year (10), and the median time to AIDS for the remaining children is less than 7 years (2). It is now clearly established that maternal and extended infant ART can substantially reduce transmission of HIV-1 through breast-feeding (23). However, in a resource-poor setting, many logistical barriers to implementation of the ART-based prevention of mother-to-child-transmission (PMTCT) remain (23). Because nutrition and hygiene makes breast milk an important determinant of infant survival (22, 28), formula feeding as a protective measure against HIV-1 acquisition is recommended only if it is AFASS (acceptable, feasible, affordable, sustainable, and safe). Unfortunately, AFASS it is still not for majority of infected mothers in sub-Saharan Africa. Also, mixed bottle and breast feeding is associated with a 10-fold increase in HIV-1 transmission relative to exclusive breast-feeding (4). Thus, an effective infant vaccine against HIV-1 infection is the best and safest solution for PMTCT of HIV-1 with the added practical option of prolonging breast-feeding.Neonatal immunity is immature compared to the adult immune system (25). The differences include naivety of the immune cells, a tendency to develop Th2 responses (5) and antigen-presenting cells with inefficient cytokine production (35). For example, human cord blood T cells proliferated poorly and produced low levels of interleukin-2 (IL-2) and gamma interferon (IFN-γ) when endogenous antigen-presenting cells presented the antigen (35, 44). Also, infant myeloid dendritic cells are less efficient in priming Th1 responses because of their decreased responsiveness to Toll-like receptor stimulation, lower levels of surface costimulatory molecules, and lower production of IL-12 (8, 27). In several infections, qualitative and quantitative differences between human newborn and adult responses were detected (1, 9, 26, 37). In contrast, other studies of infants reported proliferation as well as IL-2 and IFN-γ production by T cells equal to that of adults following T-cell receptor-independent activation (21, 46). These latter observations indicate that neonate T cells are not intrinsically “locked” into an immature phenotype but, given the correct stimuli, they can develop mature immune responses (25). The requirement for specific stimuli will likely differ for different pathogens and vaccine vectors.Mycobacterium bovis bacillus Calmette-Guérin (BCG) is commonly delivered at birth as an antituberculosis vaccine as a part of the WHO Expanded Programme on Immunization (EPI). It has been reported by several studies to promote an adultlike Th1 response in newborns (16, 24, 34, 43), although it was also suggested that delaying the BCG delivery to 10 weeks of age benefits the quantity and quality of BCG-induced CD4 T-cell responses (20). BCG and related mycobacterial vectors have been explored as vaccines against other infectious agents, including human and simian immunodeficiency viruses (19), and in adult animals showed immunogenicity and protection (3, 36, 39, 47, 48). The only clinical study of recombinant BCG (rBCG) in adults failed to provide consistent efficacy (7). We have suggested the use of rBCG expressing an HIV-1-derived immunogen as the priming component of a heterologous vaccine platform for PMTCT of HIV-1 through infected breast milk (18), where it is critical to prime HIV-1-specific responses as soon as possible after birth. These responses could be boosted a few weeks later or shortly after the already busy EPI by heterologous vaccines delivering the same HIV-1-derived immunogen. To this extent, we constructed the novel candidate vaccine BCG.HIVA⁴⁰¹ (36) by inserting a gene coding for the HIV-1 clade A-derived immunogen HIVA (14) into recombinant BCG strain AREAS-401 (40). AERAS-401 is a newly developed strain that displayed enhanced safety (40) and immunogenicity (11, 15) in murine models relative to its parental BCG vaccine strain Danish SSI-1331. Increased safety represents an important feature should the BCG.HIVA⁴⁰¹ vaccine be deployed in babies born to HIV-1-infected mothers. We showed that BCG.HIVA⁴⁰¹ in a heterologous combination with recombinant modified vaccinia virus Ankara MVA.HIVA and recombinant ovine atadenovirus OAdV.HIVA induced robust polyfunctional HIV-1-specific T-cell responses in adult macaques (36). Here, we assess the safety and immunogenicity of the BCG.HIVA prime-MVA.HIVA boost regimen in newborn rhesus macaques.     

Virological Synapse-Mediated Spread of Human Immunodeficiency Virus Type 1 between T Cells Is Sensitive to Entry Inhibition

Human immunodeficiency virus type 1 (HIV-1) can disseminate between CD4⁺ T cells via diffusion-limited cell-free viral spread or by directed cell-cell transfer using virally induced structures termed virological synapses. Although T-cell virological synapses have been well characterized, it is unclear whether this mode of viral spread is susceptible to inhibition by neutralizing antibodies and entry inhibitors. We show here that both cell-cell and cell-free viral spread are equivalently sensitive to entry inhibition. Fluorescence imaging analysis measuring virological synapse lifetimes and inhibitor time-of-addition studies implied that inhibitors can access preformed virological synapses and interfere with HIV-1 cell-cell infection. This concept was supported by electron tomography that revealed the T-cell virological synapse to be a relatively permeable structure. Virological synapse-mediated HIV-1 spread is thus efficient but is not an immune or entry inhibitor evasion mechanism, a result that is encouraging for vaccine and drug design.As with enveloped viruses from several viral families, the human immunodeficiency virus type 1 (HIV-1) can disseminate both by fluid-phase diffusion of viral particles and by directed cell-cell transfer (39). The primary target cell for HIV-1 replication in vivo is the CD4⁺ T-cell (13), which is infectible by CCR5-tropic (R5) and CXCR4-tropic (X4) viral variants (29). R5 HIV-1 is the major transmitted viral phenotype and dominates the global pandemic, whereas X4 virus is found later in infection in ca. 50% of infected individuals, and its presence indicates a poor disease progression prognosis (23). Cell-cell HIV-1 transfer between T cells is more efficient than diffusion-limited spread (8, 16, 32, 38), although recent estimates for the differential range from approximately 1 (42) to 4 (6) orders of magnitude. Two structures have been proposed to support contact-mediated intercellular movement of HIV-1 between T cells: membrane nanotubes (33, 43) and macromolecular adhesive contacts termed virological synapses (VS) (15, 17, 33). VS appear to be the dominant structure involved in T-cell-T-cell spread (33), and both X4 (17) and R5 HIV-1 (6, 15, 42) can spread between T cells via this mechanism.VS assembly and function are dependent on HIV-1 envelope glycoprotein (Env) engaging its primary cellular receptor CD4 (2, 6, 17). This interaction recruits more CD4 and coreceptor to the site of cell-cell contact in an actin-dependent manner (17). Adhesion molecules cluster at the intercellular junction and are thought to stabilize the VS (18). In parallel, viral Env and Gag are recruited to the interface by a microtubule-dependent mechanism (19), where polarized viral budding may release virions into the synaptic space across which the target cell is infected (17). The precise mechanism by which HIV-1 subsequently enters the target T-cell cytoplasm remains unclear: by fusion directly at the plasma membrane, fusion from within an endosomal compartment, or both (4, 6, 15, 25, 34).Viruses from diverse families including herpesviruses (9), poxviruses (22) and hepatitis C virus (44) evade neutralizing antibody attack by direct cell-cell spread, since the tight junctions across which the these viruses move are antibody impermeable. It has been speculated that transfer of HIV-1 across VS may promote evasion from immune or therapeutic intervention with the inference that the junctions formed in retroviral VS may be nonpermissive to antibody entry (39). However, available evidence regarding whether neutralizing antibodies (NAb) and other entry inhibitors can inhibit HIV-1 cell-cell spread is inconsistent (25). An early analysis suggested that HIV-1 T-cell-T-cell spread is relatively resistant to neutralizing monoclonal antibodies (NMAb) (12). A later study agreed with this conclusion by demonstrating a lack of permissivity of HIV-1 T-cell-T-cell spread, measured by transfer of viral Gag, to interference with viral fusion using a gp41-specific NMAb and a peptidic fusion inhibitor (6). In contrast, another analysis reported that anti-gp41-specific NMAb interfered effectively with HIV-1 spread between T cells (26). Inhibitors of the HIV-1 surface glycoprotein (gp120)-CD4 or gp120-CXCR4 interaction reduced X4 HIV-1 VS assembly and viral transfer if applied prior to mixing of infected and receptor-expressing target cells (17, 19), but the effect of these inhibitors has not been tested on preformed VS. Thus, the field is currently unclear on whether direct T-cell-T-cell infectious HIV-1 spread is susceptible or not to antibody and entry inhibitor-mediated disruption of VS assembly, and the related question, whether the VS is permeable to viral entry inhibitors, including NAb. Addressing these questions is of central importance to understanding HIV-1 pathogenesis and informing future drug and vaccine design.Since estimates reported in the literature of the relative efficiency of direct HIV-1 T-cell-T-cell spread compared to cell-free spread vary by approximately 3 orders of magnitude (6, 38, 42), and the evidence for the activity of viral entry inhibitors on cell-cell spread is conflicting, we set out to quantify the efficiency of infection across the T-cell VS and analyze the susceptibility of this structure to NAb and viral entry inhibitors. Assays reporting on events proximal to productive infection show that the R5 HIV-1 T-cell VS is approximately 1 order of magnitude more efficient than cell-free virus infection, and imaging analyses reveal that the VS assembled by HIV-1 is most likely permeable to inhibitors both during, and subsequent to, VS assembly. Thus, we conclude that the T-cell VS does not provide a privileged environment allowing HIV-1 escape from entry inhibition.     

Utilization of Immunoglobulin G Fc Receptors by Human Immunodeficiency Virus Type 1: a Specific Role for Antibodies against the Membrane-Proximal External Region of gp41

Receptors (FcγRs) for the constant region of immunoglobulin G (IgG) are an important link between humoral immunity and cellular immunity. To help define the role of FcγRs in determining the fate of human immunodeficiency virus type 1 (HIV-1) immune complexes, cDNAs for the four major human Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa) were stably expressed by lentiviral transduction in a cell line (TZM-bl) commonly used for standardized assessments of HIV-1 neutralization. Individual cell lines, each expressing a different FcγR, bound human IgG, as evidence that the physical properties of the receptors were preserved. In assays with a HIV-1 multisubtype panel, the neutralizing activities of two monoclonal antibodies (2F5 and 4E10) that target the membrane-proximal external region (MPER) of gp41 were potentiated by FcγRI and, to a lesser extent, by FcγRIIb. Moreover, the neutralizing activity of an HIV-1-positive plasma sample known to contain gp41 MPER-specific antibodies was potentiated by FcγRI. The neutralizing activities of monoclonal antibodies b12 and 2G12 and other HIV-1-positive plasma samples were rarely affected by any of the four FcγRs. Effects with gp41 MPER-specific antibodies were moderately stronger for IgG1 than for IgG3 and were ineffective for Fab. We conclude that FcγRI and FcγRIIb facilitate antibody-mediated neutralization of HIV-1 by a mechanism that is dependent on the Fc region, IgG subclass, and epitope specificity of antibody. The FcγR effects seen here suggests that the MPER of gp41 could have greater value for vaccines than previously recognized.Fc receptors (FcRs) are differentially expressed on a variety of cells of hematopoietic lineage, where they bind the constant region of antibody (Ab) and provide a link between humoral and cellular immunity. Humans possess two classes of FcRs for the constant region of IgG (FcγRs) that, when cross-linked, are distinguished by their ability to either activate or inhibit cell signaling (69, 77, 79). The activating receptors FcγRI (CD64), FcγRIIa (CD32), and FcγRIII (CD16) signal through an immunoreceptor tyrosine-based activation motif (ITAM), whereas FcγRIIb (CD32) contains an inhibitory motif (ITIM) that counters ITAM signals and B-cell receptor signals. It has been suggested that a balance between activating and inhibitory FcγRs coexpressed on the same cells plays an important role in regulating adaptive immunity (23, 68). Moreover, the inhibitory FcγRIIb, being the sole FcγR on B cells, appears to play an important role in regulating self-tolerance (23, 68). The biologic role of FcγRs may be further influenced by differences in their affinity for immunoglobulin G (IgG); thus, FcγRI is a high-affinity receptor that binds monomeric IgG (mIgG) and IgG immune complexes (IC), whereas FcγRIIa, FcγRIIb, and FcγRIIIa are medium- to low-affinity receptors that preferentially bind IgG IC (10, 49, 78). FcγRs also exhibit differences in their relative affinity for the four IgG subclasses (10), which has been suggested to influence the balance between activating and inhibitory FcγRs (67).In addition to their participation in acquired immunity, FcγRs can mediate several innate immune functions, including phagocytosis of opsonized pathogens, Ab-dependent cell cytotoxicity (ADCC), antigen uptake by professional antigen-presenting cells, and the production of inflammatory cytokines and chemokines (26, 35, 41, 48, 69). In some cases, interaction of Ab-coated viruses with FcγRs may be exploited by viruses as a means to facilitate entry into FcγR-expressing cells (2, 33, 47, 84). Several groups have reported FcγR-mediated Ab-dependent enhancement (ADE) of HIV-1 infection in vitro (47, 51, 58, 63, 94, 96), whereas other reports have implicated FcγRs in efficient inhibition of the virus in vitro (19, 21, 29, 44-46, 62, 98) and possibly as having beneficial effects against HIV-1 in vivo (5, 27, 28, 42). These conflicting results are further complicated by the fact that HIV-1-susceptible cells, such as monocytes and macrophages, can coexpress more than one FcγR (66, 77, 79).HIV-1 entry requires sequential interactions between the viral surface glycoprotein, gp120, and its cellular receptor (CD4) and coreceptor (usually CCR5 or CXCR4), followed by membrane fusion that is mediated by the viral transmembrane glycoprotein gp41 (17, 106). Abs neutralize the virus by binding either gp120 or gp41 and blocking entry into cells. Several human monoclonal Abs that neutralize a broad spectrum of HIV-1 variants have attracted considerable interest for vaccine design. Epitopes for these monoclonal Abs include the receptor binding domain of gp120 in the case of b12 (71, 86), a glycan-specific epitope on gp120 in the case of 2G12 (13, 85, 86), and two adjacent epitopes in the membrane-proximal external region (MPER) of g41 in the cases of 2F5 and 4E10 (3, 11, 38, 93). At least three of these monoclonal Abs have been shown to interact with FcRs and to mediate ADCC (42, 43).A highly standardized and validated assay for neutralizing Abs against HIV-1 that quantifies reductions in luciferase (Luc) reporter gene expression after a single round of virus infection in TZM-bl cells has been developed (60, 104). TZM-bl (also called JC53BL-13) is a CXCR4-positive HeLa cell line that was engineered to express CD4 and CCR5 and to contain integrated reporter genes for firefly Luc and Escherichia coli β-galactosidase under the control of the HIV-1 Tat-regulated promoter in the long terminal repeat terminal repeat sequence (74, 103). TZM-bl cells are permissive to infection by a wide variety of HIV-1, simian immunodeficiency virus, and human-simian immunodeficiency virus strains, including molecularly cloned Env-pseudotyped viruses. Here we report the creation and characterization of four new TZM-bl cell lines, each expressing one of the major human FcγRs. These new cell lines were used to gain a better understanding of the individual roles that FcγRs play in determining the fate of HIV-1 IC. Two FcγRs that potentiated the neutralizing activity of gp41 MPER-specific Abs were identified.     

Biological Signature Characteristics of Primary Isolates from Human Immunodeficiency Virus Type 1 Group O in Ex Vivo Human Tonsil Histocultures

Human immunodeficiency virus type 1 (HIV-1) group M viruses have achieved a global distribution, while HIV-1 group O viruses are endemic only in particular regions of Africa. Here, we evaluated biological characteristics of group O and group M viruses in ex vivo models of HIV-1 infection. The replicative capacity and ability to induce CD4 T-cell depletion of eight group O and seven group M primary isolates were monitored in cultures of human peripheral blood mononuclear cells and tonsil explants. Comparative and longitudinal infection studies revealed HIV-1 group-specific activity patterns: CCR5-using (R5) viruses from group M varied considerably in their replicative capacity but showed similar levels of cytopathicity. In contrast, R5 isolates from group O were relatively uniform in their replicative fitness but displayed a high and unprecedented variability in their potential to deplete CD4 T cells. Two R5 group O isolates were identified that cause massive depletion of CD4 T cells, to an extent comparable to CXCR4-using viruses and not documented for any R5 isolate from group M. Intergroup comparisons found a five- to eightfold lower replicative fitness of isolates from group O than for isolates from group M yet a similar overall intrinsic pathogenicity in tonsil cultures. This study establishes biological ex vivo characteristics of HIV-1 group O primary isolates. The current findings challenge the belief that a grossly reduced replicative fitness or inherently impaired cytopathicity of viruses from this group underlies their low global prevalence.Independent cross-species transmission events from simian immunodeficiency virus-infected apes have led to four distinct phylogenetic lineages of human immunodeficiency virus (HIV) in humans (45). The main (M) group of HIV type 1 (HIV-1) is responsible for the HIV pandemic, while HIV-1 group O (outlier) and HIV-2 are endemic only in west and central Africa, and HIV-1 group N (non-M/non-O) infection has been documented only in a small number of Cameroonians (56). These cross-species transmissions are believed to have occurred in western Africa around the same time, but only HIV-1 group M founded the pandemic (33, 37).The global distribution of HIV-1 group O is remarkably restricted. The relative seroprevalence of group O is reported to be highest in the Republic of Cameroon, Equatorial Guinea, and Gabon (7, 42, 57), implicating this area as the possible starting point of this HIV-1 lineage''s epidemic. Rare group O infections have been documented in industrialized countries, the majority comprising patients of Cameroonian descent (8, 25, 30, 40, 46). Notably, the prevalence of group O among HIV-1-positive blood samples in Cameroon showed a marked decline from the period 1986 to 1988 (20.6% of all HIV-1 infections) to the period 1997 to 1998 (1.4%) (7) with evidence of a low, but stabilized, prevalence in the subsequent period up to 2004 (10, 55). Primary isolates from group O and group M display pronounced genetic differences (24, 54), yet the reasons for the decreasing prevalence of HIV-1 group O relative to group M in west Africa and the almost exclusive contribution of group M to the AIDS pandemic are unclear. Many factors could, in principle, have contributed to this variable spread through the human population, including host genetic effects, transmission bottlenecks, behavioral and environmental restrictions, founder effects, and other factors (33, 53).Clinical observations do not suggest major differences in disease progression in patients infected with HIV-1 groups O and M (23, 24, 35, 39). This notion is based on limited data on the immune status and virological parameters for group O-infected individuals. Few experimental in vitro studies have compared the replicative fitness of HIV types or groups (1, 2, 50, 52, 54). In head-to-head replication competition experiments of pairs of primary isolates from group M and group O in peripheral blood mononuclear cell (PBMC) cultures, Arien et al. reported a greater than 100-fold reduced replicative fitness of group O viruses (2). They suggested that grossly reduced “ex vivo pathogenic fitness” and impaired transmission from dendritic cells to cocultured T cells (“ex vivo transmission fitness”) are intrinsic properties of group O viruses that may contribute to their low prevalence and limited geographical spread (2, 3).Here, we evaluated characteristics of a panel of primary isolates from HIV-1 group O compared to a panel from group M in three primary cell models of HIV infection. In addition to replication studies in single-donor PBMCs used in a previous fitness study (2), we employed multidonor pools of PBMCs and an ex vivo human tonsil lymphoid aggregate culture (HLAC) model. HIV readily replicates to high titers in tonsil cultures that maintain the cell composition and cytokine milieu of a lymphoid target organ in vivo (17). Previously, studies in this model have shed light on key pathogenic properties of HIV, including cell tropism and cytopathic effects in relation to coreceptor usage, productive infection of resting CD4 T cells, early host responses to infection, and viral coinfections (5, 6, 14, 18-20, 27, 38, 43, 48-50). A unique characteristic of this ex vivo model is that it allows parallel assessment of an isolate''s replicative fitness and cytopathicity, the latter determined by its ability to deplete CD4 T cells. The current investigation may enhance our understanding of parameters critical for HIV-1 spread in the human population and could thus potentially also provide clues to prevention and therapy.     

Human Immunodeficiency Virus Type 1 Nucleocapsid p1 Confers ESCRT Pathway Dependence

To facilitate the release of infectious progeny virions, human immunodeficiency virus type 1 (HIV-1) exploits the Endosomal Sorting Complex Required for Transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in the C-terminal p6 domain of Gag. However, the L domains in p6 are known to be dispensable for efficient particle production by certain HIV-1 Gag constructs that have the nucleocapsid (NC) domain replaced by a foreign dimerization domain to substitute for the assembly function of NC. We now show that one such L domain-independent HIV-1 Gag construct (termed Z_WT) that has NC-p1-p6 replaced by a leucine zipper domain is resistant to dominant-negative inhibitors of the ESCRT pathway that block HIV-1 particle production. However, Z_WT became dependent on the presence of an L domain when NC-p1-p6 was restored to its C terminus. Furthermore, when the NC domain was replaced by a leucine zipper, the p1-p6 region, but not p6 alone, conferred sensitivity to inhibition of the ESCRT pathway. In an authentic HIV-1 Gag context, the effect of an inhibitor of the ESCRT pathway on particle production could be alleviated by deleting a portion of the NC domain together with p1. Together, these results indicate that the ESCRT pathway dependence of HIV-1 budding is determined, at least in part, by the NC-p1 region of Gag.Human immunodeficiency virus type 1 (HIV-1) and other retroviruses hijack the cellular Endosomal Sorting Complex Required for Transport (ESCRT) pathway to promote the detachment of virions from the cell surface and from each other (3, 21, 42, 44, 47). The ESCRT pathway was initially identified based on its requirement for the sorting of ubiquitinated cargo into multivesicular bodies (MVB) (50, 51). During MVB biogenesis, the ESCRT pathway drives the membrane deformation and fission events required for the inward vesiculation of the limiting membrane of this organelle (26, 29, 50, 51). More recently, it emerged that the ESCRT pathway is also essential for the normal abscission of daughter cells during the final stage of cell division (10, 43). Most of the components of the ESCRT pathway are involved in the formation of four heteromeric protein complexes termed ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. Additional components include ALIX, which interacts both with ESCRT-I and ESCRT-III, and the AAA ATPase Vps4, which mediates the disassembly of ESCRT-III (29, 42).The deformation and scission of endocytic membranes is thought to be mediated by ESCRT-III, which, together with Vps4, constitutes the most conserved element of the pathway (23, 26, 42). Indeed, it was recently shown that purified yeast ESCRT-III induces membrane deformation (52), and in another study three subunits of yeast ESCRT-III were sufficient to promote the formation of intralumenal vesicles in an in vitro assay (61). In mammals, ESCRT-III is formed by the charged MVB proteins (CHMPs), which are structurally related and tightly regulated through autoinhibition (2, 33, 46, 53, 62). The removal of an inhibitory C-terminal domain induces polymerization and association with endosomal membranes and converts CHMPs into potent inhibitors of retroviral budding (34, 46, 53, 60, 62). Alternatively, CHMPs can be converted into strong inhibitors of the ESCRT pathway and of HIV-1 budding through the addition of a bulky tag such as green fluorescent protein (GFP) or red fluorescent protein (RFP) (27, 36, 39, 54). Retroviral budding in general is also strongly inhibited by catalytically inactive Vps4 (22, 41, 55), or upon Vsp4B depletion (31), confirming the crucial role of ESCRT-III.Retroviruses engage the ESCRT pathway through the activity of so-called late assembly (L) domains in Gag. In the case of HIV-1, the primary L domain maps to a conserved PTAP motif in the C-terminal p6 domain of Gag (24, 28) and interacts with the ESCRT-I component Tsg101 (15, 22, 40, 58). HIV-1 p6 also harbors an auxiliary L domain of the LYPx_nL type, which interacts with the V domain of ALIX (20, 35, 39, 54, 59, 63). Interestingly, Tsg101 binding site mutants of HIV-1 can be fully rescued through the overexpression of ALIX, and this rescue depends on the ALIX binding site in p6 (20, 56). In contrast, the overexpression of a specific splice variant of the ubiquitin ligase Nedd4-2 has been shown to rescue the release and infectivity of HIV-1 mutants lacking all known L domains in p6 (12, 57). Nedd4 family ubiquitin ligases had previously been implicated in the function of PPxY-type L domains, which also depend on an intact ESCRT pathway for function (4, 32, 38). However, HIV-1 Gag lacks PPxY motifs, and the WW domains of Nedd4-2, which mediate its interaction with PPxY motifs, are dispensable for the rescue of HIV-1 L domain mutants (57).ALIX also interacts with the nucleocapsid (NC) region of HIV-1 Gag (18, 49), which is located upstream of p6 and the p1 spacer peptide. ALIX binds HIV-1 NC via its Bro1 domain, and the capacity to interact with NC and to stimulate the release of a minimal HIV-1 Gag construct is shared among widely divergent Bro1 domain proteins (48). Based on these findings and the observation that certain mutations in NC cause a phenotype that resembles that of L domain mutants, it has been proposed that NC cooperates with p6 to recruit the machinery required for normal HIV-1 budding (18, 49).NC also plays a role in Gag polyprotein multimerization, and this function of NC depends on its RNA-binding activity (5-8). It has been proposed that the role of the NC-nucleic acid interaction during assembly is to promote the formation of Gag dimers (37), and HIV-1 assembly in the absence of NC can indeed be efficiently rescued by leucine zipper dimerization domains (65). Surprisingly, in this setting the L domains in p6 also became dispensable, since particle production remained efficient even when the entire NC-p1-p6 region of HIV-1 Gag was replaced by a leucine zipper (1, 65). These findings raised the possibility that the reliance of wild-type (WT) HIV-1 Gag on a functional ESCRT pathway is, at least in part, specified by NC-p1-p6. However, it also remained possible that the chimeric Gag constructs engaged the ESCRT pathway in an alternative manner.In the present report, we provide evidence supporting the first of those two possibilities. Particle production became independent of ESCRT when the entire NC-p1-p6 region was replaced by a leucine zipper, and reversion to ESCRT dependence was shown to occur as a result of restoration of p1-p6 but not of p6 alone. Furthermore, although the deletion of p1 alone had little effect in an authentic HIV-1 Gag context, the additional removal of a portion of NC improved particle production in the presence of an inhibitor of the ESCRT pathway. Together, these data imply that the NC-p1 region plays an important role in the ESCRT-dependence of HIV-1 particle production.     

Selective Expression of Human Immunodeficiency Virus Nef in Specific Immune Cell Populations of Transgenic Mice Is Associated with Distinct AIDS-Like Phenotypes

We previously reported that CD4C/human immunodeficiency virus (HIV)^Nef transgenic (Tg) mice, expressing Nef in CD4⁺ T cells and cells of the macrophage/dendritic cell (DC) lineage, develop a severe AIDS-like disease, characterized by depletion of CD4⁺ T cells, as well as lung, heart, and kidney diseases. In order to determine the contribution of distinct populations of hematopoietic cells to the development of this AIDS-like disease, five additional Tg strains expressing Nef through restricted cell-specific regulatory elements were generated. These Tg strains express Nef in CD4⁺ T cells, DCs, and macrophages (CD4E/HIV^Nef); in CD4⁺ T cells and DCs (mCD4/HIV^Nef and CD4F/HIV^Nef); in macrophages and DCs (CD68/HIV^Nef); or mainly in DCs (CD11c/HIV^Nef). None of these Tg strains developed significant lung and kidney diseases, suggesting the existence of as-yet-unidentified Nef-expressing cell subset(s) that are responsible for inducing organ disease in CD4C/HIV^Nef Tg mice. Mice from all five strains developed persistent oral carriage of Candida albicans, suggesting an impaired immune function. Only strains expressing Nef in CD4⁺ T cells showed CD4⁺ T-cell depletion, activation, and apoptosis. These results demonstrate that expression of Nef in CD4⁺ T cells is the primary determinant of their depletion. Therefore, the pattern of Nef expression in specific cell population(s) largely determines the nature of the resulting pathological changes.The major cell targets and reservoirs for human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) infection in vivo are CD4⁺ T lymphocytes and antigen-presenting cells (macrophages and dendritic cells [DC]) (21, 24, 51). The cell specificity of these viruses is largely dependent on the expression of CD4 and of its coreceptors, CCR5 and CXCR-4, at the cell surface (29, 66). Infection of these immune cells leads to the severe disease, AIDS, showing widespread manifestations, including progressive immunodeficiency, immune activation, CD4⁺ T-cell depletion, wasting, dementia, nephropathy, heart and lung diseases, and susceptibility to opportunistic pathogens, such as Candida albicans (1, 27, 31, 37, 41, 82, 93, 109). It is reasonable to assume that the various pathological changes in AIDS result from the expression of one or many HIV-1/SIV proteins in these immune target cells. However, assigning the contribution of each infected cell subset to each phenotype has been remarkably difficult, despite evidence that AIDS T-cell phenotypes can present very differently depending on the strains of infecting HIV-1 or SIV or on the cells targeted by the virus (4, 39, 49, 52, 72). For example, the T-cell-tropic X4 HIV strains have long been associated with late events and severe CD4⁺ T-cell depletion (22, 85, 96). However, there are a number of target cell subsets expressing CD4 and CXCR-4, and identifying which one is responsible for this enhanced virulence has not been achieved in vivo. Similarly, the replication of SIV in specific regions of the thymus (cortical versus medullary areas), has been associated with very different outcomes but, unfortunately, the critical target cells of the viruses were not identified either in these studies (60, 80). The task is even more complex, because HIV-1 or SIV can infect several cell subsets within a single cell population. In the thymus, double (CD4⁻ CD8⁻)-negative (DN) or triple (CD3⁻ CD4⁻ CD8⁻)-negative (TN) T cells, as well as double-positive (CD4⁺ CD8⁺) (DP) T cells, are infectible by HIV-1 in vitro (9, 28, 74, 84, 98, 99, 110) and in SCID-hu mice (2, 5, 91, 94). In peripheral organs, gut memory CCR5⁺ CD4⁺ T cells are primarily infected with R5 SIV, SHIV, or HIV, while circulating CD4⁺ T cells can be infected by X4 viruses (13, 42, 49, 69, 70, 100, 101, 104). Moreover, some detrimental effects on CD4⁺ T cells have been postulated to originate from HIV-1/SIV gene expression in bystander cells, such as macrophages or DC, suggesting that other infected target cells may contribute to the loss of CD4⁺ T cells (6, 7, 32, 36, 64, 90).Similarly, the infected cell population(s) required and sufficient to induce the organ diseases associated with HIV-1/SIV expression (brain, heart, and kidney) have not yet all been identified. For lung or kidney disease, HIV-specific cytotoxic CD8⁺ T cells (1, 75) or infected podocytes (50, 95), respectively, have been implicated. Activated macrophages have been postulated to play an important role in heart disease (108) and in AIDS dementia (35), although other target cells could be infected by macrophage-tropic viruses and may contribute significantly to the decrease of central nervous system functions (11, 86, 97), as previously pointed out (25).Therefore, because of the widespread nature of HIV-1 infection and the difficulty in extrapolating tropism of HIV-1/SIV in vitro to their cell targeting in vivo (8, 10, 71), alternative approaches are needed to establish the contribution of individual infected cell populations to the multiorgan phenotypes observed in AIDS. To this end, we developed a transgenic (Tg) mouse model of AIDS using a nonreplicating HIV-1 genome expressed through the regulatory sequences of the human CD4 gene (CD4C), in the same murine cells as those targeted by HIV-1 in humans, namely, in immature and mature CD4⁺ T cells, as well as in cells of the macrophage/DC lineages (47, 48, 77; unpublished data). These CD4C/HIV Tg mice develop a multitude of pathologies closely mimicking those of AIDS patients. These include a gradual destruction of the immune system, characterized among other things by thymic and lymphoid organ atrophy, depletion of mature and immature CD4⁺ T lymphocytes, activation of CD4⁺ and CD8⁺ T cells, susceptibility to mucosal candidiasis, HIV-associated nephropathy, and pulmonary and cardiac complications (26, 43, 44, 57, 76, 77, 79, 106). We demonstrated that Nef is the major determinant of the HIV-1 pathogenicity in CD4C/HIV Tg mice (44). The similarities of the AIDS-like phenotypes of these Tg mice to those in human AIDS strongly suggest that such a Tg mouse approach can be used to investigate the contribution of distinct HIV-1-expressing cell populations to their development.In the present study, we constructed and characterized five additional mouse Tg strains expressing Nef, through distinct regulatory elements, in cell populations more restricted than in CD4C/HIV Tg mice. The aim of this effort was to assess whether, and to what extent, the targeting of Nef in distinct immune cell populations affects disease development and progression.