Endosomal Trafficking of HIV-1 Gag and Genomic RNAs Regulates Viral Egress

HIV-1 Gag can assemble and generate virions at the plasma membrane, but it is also present in endosomes where its role remains incompletely characterized. Here, we show that HIV-1 RNAs and Gag are transported on endosomal vesicles positive for TiVamp, a v-SNARE involved in fusion events with the plasma membrane. Inhibition of endosomal traffic did not prevent viral release. However, inhibiting lysosomal degradation induced an accumulation of Gag in endosomes and increased viral production 7-fold, indicating that transport of Gag to lysosomes negatively regulates budding. This also suggested that endosomal Gag-RNA complexes could access retrograde pathways to the cell surface and indeed, depleting cells of TiVamp-reduced viral production. Moreover, inhibition of endosomal transport prevented the accumulation of Gag at sites of cellular contact. HIV-1 Gag could thus generate virions using two pathways, either directly from the plasma membrane or through an endosome-dependent route. Endosomal Gag-RNA complexes may be delivered at specific sites to facilitate cell-to-cell viral transmission.The production of infectious retroviral particles is an ordered process that includes many steps (for review see Refs. 1–3). In particular, three major viral components, Gag, the envelope, and genomic RNAs have to traffic inside the cell to reach their assembly site. Viral biogenesis is driven by the polyprotein Gag, which is able to make viral-like particles when expressed alone (4). Upon release, HIV-1⁴ Gag is processed by the viral protease into matrix (MA(p17)), capsid (CA(p24)), nucleocapsid (NC(p7)), p6, and smaller peptides SP1 and SP2. Gag contains several domains that are essential for viral assembly: a membrane binding domain (M) in MA; a Gag-Gag interaction domain in CA; an assembly domain (I) in NC; and a late domain (L) in p6, which recruits the cellular budding machinery. Genomic RNAs are specifically recognized by NC, and they play fundamental roles in viral biogenesis by acting as a scaffold for Gag multimerization (5).It has been demonstrated that retroviruses bud by hijacking the endosomal machinery that sorts proteins into internal vesicles of multivesicular bodies (for review, see Refs. 6, 7). Indeed, these vesicles bud with the same topology as viral particles. Proteins sorted into this pathway are usually destined for degradation in lysosomes, but some can also recycle to the plasma membrane (for review see Refs. 8, 9). They are also frequently ubiquitinated on their cytoplasmic domain (10, 11), allowing their recognition by ESCRT complexes. ESCRT-0 and ESCRT-I recognize ubiquitinated cargo present at the surface of endosomes and recruit other ESCRT complexes (12–14). ESCRT-III is believed to function directly in the formation of multivesicular body intralumenal vesicles (12), even though its mechanism of action is currently not understood. Remarkably, Gag L domains interact directly with components of the multivesicular body-sorting machinery (for review see Ref. 15). HIV-1 Gag uses a PTAP motif to bind Tsg101, a component of ESCRT-I (16–19), and a YPLTSL motif to interact with Alix, a protein linked to ESCRT-I and -III (20–22). Finally, various ubiquitin ligases are also required directly or indirectly during HIV-1 biogenesis (23, 24; for review see Ref. 25).In many cell lines, Gag is found both at the plasma membrane and in endosomes. This has led to the hypothesis that there are several assembly sites for HIV-1 (1, 3). First, Gag can initiate and complete assembly at the plasma membrane. This is thought to occur predominantly in T lymphocytes, and this process is supported by several lines of evidences: (i) disruption of endosomal trafficking with drugs does not prevent viral production (26, 27); (ii) ESCRT complexes can be recruited at the plasma membrane, at sites where Gag accumulates (28–30); (iii) Gag can be seen multimerizing and budding from the plasma membrane in live cells (31). Second, Gag could initiate assembly in endosomes, and then traffic to the cell surface to be released. This is mainly supported by the presence of Gag in endosomes in several cell lines (32–34), including T cells and more strikingly macrophages (32, 35, 36–39). However, we are currently lacking functional experiments addressing the role of this endosomal pool of Gag, and it is still not clear to what extent it contributes to the production of viral particles. Nevertheless, the presence of Gag in endosomes might facilitate recruitment of ESCRT complexes (34, 40), packaging of viral genomic RNAs (32, 41), and incorporation of the envelope (42). It may also be important for polarized budding (43, 44) and to create a viral reservoir in infected cells (45, 46).Despite great progress, the traffic of HIV-1 components is still not fully elucidated. In particular, the transport of the genomic RNAs is poorly understood. In this study, we have used single molecule techniques to investigate the trafficking of HIV-1 RNAs in fixed and live cells, and we show that they are transported on endosomal vesicles. We also obtained functional evidence that Gag and viral RNAs can use at least two trafficking pathways to produce virions, one going directly from the plasma membrane and another one passing through endosomes.     

The Cell Tropism of Human Immunodeficiency Virus Type 1 Determines the Kinetics of Plasma Viremia in SCID Mice Reconstituted with Human Peripheral Blood Leukocytes

Most individuals infected with human immunodeficiency virus type 1 (HIV-1) initially harbor macrophage-tropic, non-syncytium-inducing (M-tropic, NSI) viruses that may evolve into T-cell-tropic, syncytium-inducing viruses (T-tropic, SI) after several years. The reasons for the more efficient transmission of M-tropic, NSI viruses and the slow evolution of T-tropic, SI viruses remain unclear, although they may be linked to expression of appropriate chemokine coreceptors for virus entry. We have examined plasma viral RNA levels and the extent of CD4⁺ T-cell depletion in SCID mice reconstituted with human peripheral blood leukocytes following infection with M-tropic, dual-tropic, or T-tropic HIV-1 isolates. The cell tropism was found to determine the course of viremia, with M-tropic viruses producing sustained high viral RNA levels and sparing some CD4⁺ T cells, dual-tropic viruses producing a transient and lower viral RNA spike and extremely rapid depletion of CD4⁺ T cells, and T-tropic viruses causing similarly lower viral RNA levels and rapid-intermediate rates of CD4⁺ T-cell depletion. A single amino acid change in the V3 region of gp120 was sufficient to cause one isolate to switch from M-tropic to dual-tropic and acquire the ability to rapidly deplete all CD4⁺ T cells.The envelope gene of human immunodeficiency virus type 1 (HIV-1) determines the cell tropism of the virus (11, 32, 47, 62), the use of chemokine receptors as cofactors for viral entry (4, 17), and the ability of the virus to induce syncytia in infected cells (55, 60). Cell tropism is closely linked to but probably not exclusively determined by the ability of different HIV-1 envelopes to bind CD4 and the CC or the CXC chemokine receptors and initiate viral fusion with the target cell. Macrophage-tropic (M-tropic) viruses infect primary cultures of macrophages and CD4⁺ T cells and use CCR5 as the preferred coreceptor (2, 5, 15, 23, 26, 31). T-cell-tropic (T-tropic) viruses can infect primary cultures of CD4⁺ T cells and established T-cell lines, but not primary macrophages. T-tropic viruses use CXCR4 as a coreceptor for viral entry (27). Dual-tropic viruses have both of these properties and can use either CCR5 or CXCR4 (and infrequently other chemokine receptors [25]) for viral entry (24, 37, 57). M-tropic viruses are most frequently transmitted during primary infection of humans and persist throughout the duration of the infection (63). Many, but not all, infected individuals show an evolution of virus cell tropism from M-tropic to dual-tropic and finally to T-tropic with increasing time after infection (21, 38, 57). Increases in replicative capacity of viruses from patients with long-term infection have also been noted (22), and the switch to the syncytium-inducing (SI) phenotype in T-tropic or dual-tropic isolates is associated with more rapid disease progression (10, 20, 60). Primary infection with dual-tropic or T-tropic HIV, although infrequent, often leads to rapid disease progression (16, 51). The viral and host factors that determine the higher transmission rate of M-tropic HIV-1 and the slow evolution of dual- or T-tropic variants remain to be elucidated (4).These observations suggest that infection with T-tropic, SI virus isolates in animal model systems with SCID mice grafted with human lymphoid cells or tissue should lead to a rapid course of disease (1, 8, 44–46). While some studies in SCID mice grafted with fetal thymus and liver are in agreement with this concept (33, 34), our previous studies with the human peripheral blood leukocyte-SCID (hu-PBL-SCID) mouse model have shown that infection with M-tropic isolates (e.g., SF162) causes more rapid CD4⁺ T-cell depletion than infection with T-tropic, SI isolates (e.g., SF33), despite similar proviral copy numbers, and that this property mapped to envelope (28, 41, 43). However, the dual-tropic 89.6 isolate (19) caused extremely rapid CD4⁺ T-cell depletion in infected hu-PBL-SCID mice that was associated with an early and transient increase in HIV-1 plasma viral RNA (29). The relationship between cell tropism of the virus isolate and the pattern of disease in hu-PBL-SCID mice is thus uncertain. We have extended these studies by determining the kinetics of HIV-1 RNA levels in serial plasma samples of hu-PBL-SCID mice infected with primary patient isolates or laboratory stocks that differ in cell tropism and SI properties. The results showed significant differences in the kinetics of HIV-1 replication and CD4⁺ T-cell depletion that are determined by the cell tropism of the virus isolate.     

Evidence that Antibody-Mediated Neutralization of Human Immunodeficiency Virus Type 1 by Sera from Infected Individuals Is Independent of Coreceptor Usage

Human immunodeficiency virus type 1 (HIV-1) uses a variety of chemokine receptors as coreceptors for virus entry, and the ability of the virus to be neutralized by antibody may depend on which coreceptors are used. In particular, laboratory-adapted variants of the virus that use CXCR4 as a coreceptor are highly sensitive to neutralization by sera from HIV-1-infected individuals, whereas primary isolates that use CCR5 instead of, or in addition to, CXCR4 are neutralized poorly. To determine whether this dichotomy in neutralization sensitivity could be explained by differential coreceptor usage, virus neutralization by serum samples from HIV-1-infected individuals was assessed in MT-2 cells, which express CXCR4 but not CCR5, and in mitogen-stimulated human peripheral blood mononuclear cells (PBMC), where multiple coreceptors including CXCR4 and CCR5 are available for use. Our results showed that three of four primary isolates with a syncytium-inducing (SI) phenotype and that use CXCR4 and CCR5 were neutralized poorly in both MT-2 cells and PBMC. The fourth isolate, designated 89.6, was more sensitive to neutralization in MT-2 cells than in PBMC. We showed that the neutralization of 89.6 in PBMC was not improved when CCR5 was blocked by having RANTES, MIP-1α, and MIP-1β in the culture medium, indicating that CCR5 usage was not responsible for the decreased sensitivity to neutralization in PBMC. Consistent with this finding, a laboratory-adapted strain of virus (IIIB) was significantly more sensitive to neutralization in CCR5-deficient PBMC (homozygous Δ32-CCR5 allele) than were two of two SI primary isolates tested. The results indicate that the ability of HIV-1 to be neutralized by sera from infected individuals depends on factors other than coreceptor usage.Human immunodeficiency virus type 1 (HIV-1), the etiologic agent of AIDS, utilizes the HLA class II receptor, CD4, as its primary receptor to gain entry into cells (17, 30). Entry is initiated by a high-affinity interaction between CD4 and the surface gp120 of the virus (32). Subsequent to this interaction, conformational changes that permit fusion of the viral membrane with cellular membranes occur within the viral transmembrane gp41 (9, 58, 59). In addition to CD4, one or more recently described viral coreceptors are needed for fusion to take place. These coreceptors belong to a family of seven-transmembrane G-protein-coupled proteins and include the CXC chemokine receptor CXCR4 (3, 4, 24, 44), the CC chemokine receptors CCR5 (1, 12, 13, 18, 21, 23, 45) and, less commonly, CCR3 and CCR2b (12, 21), and two related orphan receptors termed BONZO/STRL33 and BOB (19, 34). Coreceptor usage by HIV-1 can be blocked by naturally occurring ligands, including SDF-1 for CXCR4 (4, 44), RANTES, MIP-1α, and MIP-1β in the case of CCR5 (13, 45), and eotaxin for CCR3 (12).The selective cellular tropisms of different strains of HIV-1 may be determined in part by coreceptor usage. For example, all culturable HIV-1 variants replicate initially in mitogen-stimulated human peripheral blood mononuclear cells (PBMC), but only a minor fraction are able to infect established CD4⁺ T-cell lines (43). This differential tropism is explained by the expression of CXCR4 together with CCR5 and other CC chemokine coreceptors on PBMC and the lack of expression of CCR5 on most T-cell lines (5, 10, 19, 35, 39, 50, 53). Indeed, low-passage field strains (i.e., primary isolates) of HIV-1 that fail to replicate in T-cell lines use CCR5 as their major coreceptor and are unable to use CXCR4 (1, 12, 18, 21, 23, 28). Because these isolates rarely produce syncytia in PBMC and fail to infect MT-2 cells, they are often classified as having a non-syncytium-inducing (NSI) phenotype. Primary isolates with a syncytium-inducing (SI) phenotype are able to use CXCR4 alone or, more usually, in addition to CCR5 (16, 20, 51). HIV-1 variants that have been passaged multiple times in CD4⁺ T-cell lines, and therefore considered to be laboratory adapted, exhibit a pattern of coreceptor usage that resembles that of SI primary isolates. Most studies have shown that the laboratory-adapted strain IIIB uses CXCR4 alone (3, 13, 20, 24, 51) and that MN and SF-2 use CXCR4 primarily and CCR5 to a lesser degree (11, 13). Sequences within the V3 loop of gp120 have been shown to be important, either directly or indirectly, for the interaction of HIV-1 with both CXCR4 (52) and CCR5 (12, 14, 54, 60). This region of gp120 contains multiple determinants of cellular tropism (43) and is a major target for neutralizing antibodies to laboratory-adapted HIV-1 but not to primary isolates (29, 46, 57).It has been known for some time that the ability of sera from HIV-1-infected individuals to neutralize laboratory-adapted strains of HIV-1 does not predict their ability to neutralize primary isolates in vitro (7). In general, the former viruses are highly sensitive to neutralization whereas the latter viruses are neutralized poorly by antibodies induced in response to HIV-1 infection (7, 43). Importantly, neutralizing antibodies generated by candidate HIV-1 subunit vaccines have been highly specific for laboratory-adapted viruses (26, 37, 38). In principle, the dichotomy in neutralization sensitivity between these two categories of virus could be related to coreceptor usage. To test this, we investigated whether the use of CXCR4 in the absence of CCR5 would render SI primary isolates highly sensitive to neutralization in vitro by sera from HIV-1-infected individuals. Two similar studies using human monoclonal antibodies and soluble CD4 have been reported (31a, 55).     

Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

HIV-1 possesses an exquisite ability to infect cells independently from their cycling status by undergoing an active phase of nuclear import through the nuclear pore. This property has been ascribed to the presence of karyophilic elements present in viral nucleoprotein complexes, such as the matrix protein (MA); Vpr; the integrase (IN); and a cis-acting structure present in the newly synthesized DNA, the DNA flap. However, their role in nuclear import remains controversial at best. In the present study, we carried out a comprehensive analysis of the role of these elements in nuclear import in a comparison between several primary cell types, including stimulated lymphocytes, macrophages, and dendritic cells. We show that despite the fact that none of these elements is absolutely required for nuclear import, disruption of the central polypurine tract-central termination sequence (cPPT-CTS) clearly affects the kinetics of viral DNA entry into the nucleus. This effect is independent of the cell cycle status of the target cells and is observed in cycling as well as in nondividing primary cells, suggesting that nuclear import of viral DNA may occur similarly under both conditions. Nonetheless, this study indicates that other components are utilized along with the cPPT-CTS for an efficient entry of viral DNA into the nucleus.Lentiviruses display an exquisite ability to infect dividing and nondividing cells alike that is unequalled among Retroviridae. This property is thought to be due to the particular behavior or composition of the viral nucleoprotein complexes (NPCs) that are liberated into the cytoplasm of target cells upon virus-to-cell membrane fusion and that allow lentiviruses to traverse an intact nuclear membrane (17, 28, 29, 39, 52, 55, 67, 79). In the case of the human immunodeficiency type I virus (HIV-1), several studies over the years identified viral components of such structures with intrinsic karyophilic properties and thus perfect candidates for mediation of the passage of viral DNA (vDNA) through the nuclear pore: the matrix protein (MA); Vpr; the integrase (IN); and a three-stranded DNA flap, a structure present in neo-synthesized viral DNA, specified by the central polypurine tract-central termination sequence (cPPT-CTS). It is clear that these elements may mediate nuclear import directly or via the recruitment of the host''s proteins, and indeed, several cellular proteins have been found to influence HIV-1 infection during nuclear import, like the karyopherin α2 Rch1 (38); importin 7 (3, 30, 93); the transportin SR-2 (13, 20); or the nucleoporins Nup98 (27), Nup358/RANBP2, and Nup153 (13, 56).More recently, the capsid protein (CA), the main structural component of viral nucleoprotein complexes at least upon their cytoplasmic entry, has also been suggested to be involved in nuclear import or in postnuclear entry steps (14, 25, 74, 90, 92). Whether this is due to a role for CA in the shaping of viral nucleoprotein complexes or to a direct interaction between CA and proteins involved in nuclear import remains at present unknown.Despite a large number of reports, no single viral or cellular element has been described as absolutely necessary or sufficient to mediate lentiviral nuclear import, and important controversies as to the experimental evidences linking these elements to this step exist. For example, MA was among the first viral protein of HIV-1 described to be involved in nuclear import, and 2 transferable nuclear localization signals (NLSs) have been described to occur at its N and C termini (40). However, despite the fact that early studies indicated that the mutation of these NLSs perturbed HIV-1 nuclear import and infection specifically in nondividing cells, such as macrophages (86), these findings failed to be confirmed in more-recent studies (23, 33, 34, 57, 65, 75).Similarly, Vpr has been implicated by several studies of the nuclear import of HIV-1 DNA (1, 10, 21, 43, 45, 47, 64, 69, 72, 73, 85). Vpr does not possess classical NLSs, yet it displays a transferable nucleophilic activity when fused to heterologous proteins (49-51, 53, 77, 81) and has been shown to line onto the nuclear envelope (32, 36, 47, 51, 58), where it can truly facilitate the passage of the viral genome into the nucleus. However, the role of Vpr in this step remains controversial, as in some instances Vpr is not even required for viral replication in nondividing cells (1, 59).Conflicting results concerning the role of IN during HIV-1 nuclear import also exist. Indeed, several transferable NLSs have been described to occur in the catalytic core and the C-terminal DNA binding domains of IN, but for some of these, initial reports of nuclear entry defects (2, 9, 22, 46, 71) were later shown to result from defects at steps other than nuclear import (60, 62, 70, 83). These reports do not exclude a role for the remaining NLSs in IN during nuclear import, and they do not exclude the possibility that IN may mediate this step by associating with components of the cellular nuclear import machinery, such as importin alpha and beta (41), importin 7 (3, 30, 93, 98), and, more recently, transportin-SR2 (20).The central DNA flap, a structure present in lentiviruses and in at least 1 yeast retroelement (44), but not in other orthoretroviruses, has also been involved in the nuclear import of viral DNA (4, 6, 7, 31, 78, 84, 95, 96), and more recently, it has been proposed to provide a signal for viral nucleoprotein complexes uncoating in the proximity of the nuclear pore, with the consequence of providing a signal for import (8). However, various studies showed an absence or weakness of nuclear entry defects in viruses devoid of the DNA flap (24, 26, 44, 61).Overall, the importance of viral factors in HIV-1 nuclear import is still unclear. The discrepancies concerning the role of MA, IN, Vpr, and cPPT-CTS in HIV-1 nuclear import could in part be explained by their possible redundancy. To date, only one comprehensive study analyzed the role of these four viral potentially karyophilic elements together (91). This study showed that an HIV-1 chimera where these elements were either deleted or replaced by their murine leukemia virus (MLV) counterparts was, in spite of an important infectivity defect, still able to infect cycling and cell cycle-arrested cell lines to similar efficiencies. If this result indicated that the examined viral elements of HIV-1 were dispensable for the cell cycle independence of HIV, as infections proceeded equally in cycling and arrested cells, they did not prove that they were not required in nuclear import, because chimeras displayed a severe infectivity defect that precluded their comparison with the wild type (WT).Nuclear import and cell cycle independence may not be as simply linked as previously thought. On the one hand, there has been no formal demonstration that the passage through the nuclear pore, and thus nuclear import, is restricted to nondividing cells, and for what we know, this passage may be an obligatory step in HIV infection in all cells, irrespective of their cycling status. In support of this possibility, certain mutations in viral elements of HIV affect nuclear import in dividing as well as in nondividing cells (4, 6, 7, 31, 84, 95). On the other hand, cell cycle-independent infection may be a complex phenomenon that is made possible not only by the ability of viral DNA to traverse the nuclear membrane but also by its ability to cope with pre- and postnuclear entry events, as suggested by the phenotypes of certain CA mutants (74, 92).Given that the cellular environment plays an important role during the early steps of viral infection, we chose to analyze the role of the four karyophilic viral elements of HIV-1 during infection either alone or combined in a wide comparison between cells highly susceptible to infection and more-restrictive primary cell targets of HIV-1 in vivo, such as primary blood lymphocytes (PBLs), monocyte-derived macrophages (MDM), and dendritic cells (DCs).In this study, we show that an HIV-1-derived virus in which the 2 NLSs of MA are mutated and the IN, Vpr, and cPPT-CTS elements are removed displays no detectable nuclear import defect in HeLa cells independently of their cycling status. However, this mutant virus is partially impaired for nuclear entry in primary cells and more specifically in DCs and PBLs. We found that this partial defect is specified by the cPPT-CTS, while the 3 remaining elements seem to play no role in nuclear import. Thus, our study indicates that the central DNA flap specifies the most important role among the viral elements involved thus far in nuclear import. However, it also clearly indicates that the role played by the central DNA flap is not absolute and that its importance varies depending on the cell type, independently from the dividing status of the cell.     

A Comprehensive Panel of Near-Full-Length Clones and Reference Sequences for Non-Subtype B Isolates of Human Immunodeficiency Virus Type 1

Non-subtype B viruses cause the vast majority of new human immunodeficiency virus type 1 (HIV-1) infections worldwide and are thus the major focus of international vaccine efforts. Although their geographic dissemination is carefully monitored, their immunogenic and biological properties remain largely unknown, in part because well-characterized virological reference reagents are lacking. In particular, full-length clones and sequences are rare, since subtype classification is frequently based on small PCR-derived viral fragments. There are only five proviral clones available for viruses other than subtype B, and these represent only 3 of the 10 proposed (group M) sequence subtypes. This lack of reference sequences also confounds the identification and analysis of mosaic (recombinant) genomes, which appear to be arising with increasing frequency in areas where multiple sequence subtypes cocirculate. To generate a more representative panel of non-subtype B reference reagents, we have cloned (by long PCR or lambda phage techniques) and sequenced 10 near-full-length HIV-1 genomes (lacking less than 80 bp of long terminal repeat sequences) from primary isolates collected at major epicenters of the global AIDS pandemic. Detailed phylogenetic analyses identified six that represented nonrecombinant members of HIV-1 subtypes A (92UG037.1), C (92BR025.8), D (84ZR085.1 and 94UG114.1), F (93BR020.1), and H (90CF056.1), the last two comprising the first full-length examples of these subtypes. Four others were found to be complex mosaics of subtypes A and C (92RW009.6), A and G (92NG083.2 and 92NG003.1), and B and F (93BR029.4), again emphasizing the impact of intersubtype recombination on global HIV-1 diversification. Although a number of clones had frameshift mutations or translational stop codons in major open reading frames, all the genomes contained a complete set of genes and three had intact genomic organizations without inactivating mutations. Reconstruction of one of these (94UG114.1) yielded replication-competent virus that grew to high titers in normal donor peripheral blood mononuclear cell cultures. This panel of non-subtype B reference genomes should prove valuable for structure-function studies of genetically diverse viral gene products, the generation of subtype-specific immunological reagents, and the production of DNA- and protein-based subunit vaccines directed against a broader spectrum of viruses.One critical question facing current AIDS vaccine development efforts is to what extent human immunodeficiency virus type 1 (HIV-1) genetic variation has to be considered in the design of candidate vaccines (11, 21, 41, 72). Phylogenetic analyses of globally circulating viral strains have identified two distinct groups of HIV-1 (M and O) (33, 45, 61, 62), and 10 sequence subtypes (A to J) have been proposed within the major group (M) (29, 30, 45, 72). Sequence variation among viruses belonging to these different lineages is extensive, with envelope amino acid sequence variation ranging from 24% between different subtypes to 47% between the two different groups. Given this extent of diversity, the question has been raised whether immunogens based on a single virus strain can be expected to elicit immune responses effective against a broad spectrum of viruses or whether vaccine preparations should include mixtures of genetically divergent antigens and/or be tailored toward locally circulating strains (11, 21, 41, 72). This is of particular concern in developing countries, where multiple subtypes of HIV-1 are known to cocirculate and where subtype B viruses (which have been the source of most current candidate vaccine preparations [10, 21]) are rare or nonexistent (5, 24, 40, 72).Although the extent of global HIV-1 variation is well defined, little is known about the biological consequences of this genetic diversity and its impact on cellular and humoral immune responses in the infected host. In particular, it remains unknown whether subtype-specific differences in virus biology exist that have to be considered for vaccine design. Thus far, such differences have not been identified. For example, several studies have shown that there is no correlation between HIV-1 genetic subtypes and neutralization serotypes (38, 42, 46, 68). Some viruses are readily neutralized, while most are relatively neutralization resistant (42). Although the reasons for these different susceptibilities remain unknown, it is clear that neutralization is not a function of the viral genotype (38, 42, 46, 68). Similarly, recent studies have identified vigorous cross-clade cytotoxic T-lymphocyte (CTL) reactivities in individuals infected with viruses from several different clades (3, 6), as well as in recipients of a clade B vaccine (15). These results are very encouraging, since they suggest that CTL cross-recognition among HIV-1 clades is much more prevalent than previously anticipated and that immunogens based on a limited number of variants may be able to elicit a broad CTL response (6). Nevertheless, it would be premature to conclude that HIV-1 variation poses no problem for AIDS vaccine design. Only a comprehensive analysis of genetically defined representatives of the various groups and subtypes will allow us to judge whether certain variants differ in fundamental viral properties and whether such differences will have to be incorporated into vaccine strategies. Obviously, such studies require well-characterized reference reagents, in particular full-length and replication-competent molecular clones that can be used for functional and biological studies.Full-length reference sequences representing the various subtypes are also urgently needed for phylogenetic comparisons. Recent analyses of subgenomic (23, 52, 54, 58) as well as full-length (7, 18, 53, 60) HIV-1 sequences identified a surprising number of HIV-1 strains which clustered in different subtypes in different parts of their genome. All of these originated from geographic regions where multiple subtypes cocirculated and are the results of coinfections with highly divergent viruses (52, 60, 62). Detailed phylogenetic characterization revealed that most of them have a complex genome structure with multiple points of crossover (7, 18, 53, 60). Some recombinants, like the “subtype E” viruses, which are in fact A/E recombinants (7, 18), have a widespread geographic dissemination and are responsible for much of the Asian HIV-1 epidemic (69, 70). In other areas, recombinants appear to be generated with increasing frequencies since many randomly chosen isolates exhibit evidence of mosaicism (4, 8, 31, 66, 71). Since recombination provides the opportunity for evolutionary leaps with genetic consequences that are far greater than those of the steady accumulation of individual mutations, the impact of recombination on viral properties must be monitored. We therefore need full-length nonrecombinant reference sequences for all major HIV-1 groups and subtypes before we can map and characterize the extent of intersubtype recombination.The number of molecular reagents for non-subtype B viruses is very limited. There are currently only five full-length, nonrecombinant molecular clones available for viruses other than subtype B (45), and these represent only three of the proposed (group M) subtypes (A, C, and D). Moreover, only three clones (all derived from subtype D viruses) are replication competent and thus useful for studies requiring functional gene products (45, 48, 65). Given the unknown impact of genetic variation on correlates of immune protection, subtype-specific reagents are critically needed for phylogenetic, immunological, and biological studies. In this paper, we report the cloning (by long PCR and lambda techniques) of 10 near-full-length HIV-1 genomes from isolates previously classified as non-subtype B viruses. Detailed phylogenetic analysis showed that six comprise nonmosaic representatives of five major subtypes, including two for which full-length representatives have not been reported. Four others were identified as complex intersubtype recombinants, again emphasizing the prevalence of hybrid genomes among globally circulating HIV-1 strains. We also describe a strategy for the biological evaluation of long-PCR-derived genomes and report the generation of a replication-competent provirus by this approach. The effect of these reagents on vaccine development is discussed.     

Detailed Mechanistic Insights into HIV-1 Sensitivity to Three Generations of Fusion Inhibitors

Peptides based on the second heptad repeat (HR2) of viral class I fusion proteins are effective inhibitors of virus entry. One such fusion inhibitor has been approved for treatment of human immunodeficiency virus-1 (T20, enfuvirtide). Resistance to T20 usually maps to the peptide binding site in HR1. To better understand fusion inhibitor potency and resistance, we combined virological, computational, and biophysical experiments with comprehensive mutational analyses and tested resistance to T20 and second and third generation inhibitors (T1249 and T2635). We found that most amino acid substitutions caused resistance to the first generation peptide T20. Only charged amino acids caused resistance to T1249, and none caused resistance to T2635. Depending on the drug, we can distinguish four mechanisms of drug resistance: reduced contact, steric obstruction, electrostatic repulsion, and electrostatic attraction. Implications for the design of novel antiviral peptide inhibitors are discussed.The HIV-1 envelope glycoprotein complex (Env),³ a class I viral fusion protein, is responsible for viral attachment to CD4⁺ target T cells and subsequent fusion of viral and cellular membranes resulting in release of the viral core in the cell. Other examples of viruses using class I fusion proteins are Coronaviridae (severe acute respiratory syndrome virus), Paramyxoviridae (Newcastle disease virus, human respiratory syncytial virus, Nipah virus, Hendra virus), and Orthomyxoviridae (influenza virus), some of which cause fatal diseases in humans (1–3). The entry process of these viruses is an attractive target for therapeutic intervention.The functional trimeric Env spike on HIV-1 virions consists of three gp120 and three gp41 molecules that are the products of cleavage of the precursor gp160 by cellular proteases such as furin (4, 5). The gp120 surface subunits are responsible for binding to the cellular receptors, whereas the gp41 subunits anchor the complex in the viral membrane and mediate the fusion of viral and cellular membranes. Env undergoes several conformational changes that culminate in membrane fusion. The gp120 subunit binds the CD4 receptor, resulting in creation and/or exposure of the binding site for a coreceptor, usually CCR5 or CXCR4 (6, 7). Two α-helical leucine zipper-like motifs, heptad repeat 1 (HR1) and heptad repeat 2 (HR2), located in the extracellular part of gp41, play a major role in the following conformational changes. Binding of the receptors to gp120 induces formation of the pre-hairpin intermediate of gp41 in which HR1 is exposed and the N-terminal fusion peptide is inserted into the target cell membrane (1, 8–12). Subsequently, three HR1 and three HR2 domains assemble into a highly stable six-helix bundle structure that juxtaposes the viral and cellular membranes for the membrane merger. Other viruses with class I viral fusion proteins use similar HR1-HR2-mediated membrane fusion for target cell entry.Peptides based on the HR domains of class I viral fusion proteins have proven to be efficient inhibitors of virus entry for a broad range of viruses (13–17). The HIV-1 fusion inhibitor T20 (enfuvirtide (Fuzeon)) has been approved for clinical use. T20 mimics HR2 and can bind to HR1, thereby preventing the formation of the six-helix bundle (Fig. 1) (18–21). T1249 is a second-generation fusion inhibitor with improved antiviral potency compared with the first-generation peptide T20 (22–25). Recently, a series of more potent third-generation fusion inhibitors were designed (26, 27). These include T2635, which has an improved helical structure that increases stability and activity against both wild type (WT) HIV-1 and fusion inhibitor resistant variants.Open in a separate windowFIGURE 1.Schematic of the gp41 ectodomain. HR1 and HR2 are represented as cylinders, and position 38 in HR1 is indicated. Residues Gln-142, Asn-145, Glu-146, and Leu-149, which interact with residue 38, are underlined in the HR2 sequence. HR2-based peptide fusion inhibitors are shown underneath. Mutations introduced in T1249^mut and T2635^mut are bold and underlined. Numbering is based on the sequence of HXB2 gp41.Both the in vitro and in vivo selection of resistance has been described for T20 (28–33) and T1249 (23, 34–36). Resistance is often caused by mutations in the HR1 binding site of the fusion inhibitor. In particular, substitutions at positions 36 (G36D/M/S), 38 (V38A/W/M/E), and 43 (N43D/K) of gp41 can cause resistance. Strikingly, substitutions at position 38 can cause resistance to both T20 and T1249, but distinct amino acid substitutions are required. At position 38 only charged amino acids (V38E/R/K) cause resistance to T1249 (35). Surprisingly, none of the known T20 and T1249 resistance mutations at position 38 affect the susceptibility to the third generation inhibitor T2635.We hypothesized that the use of HIV-1 as a model system could provide a more detailed understanding of resistance to fusion inhibitors. We, therefore, analyzed the effect of all 20 amino acids at resistance hotspot 38 on Env function, viral fitness, biochemical properties of gp41, and resistance to the fusion inhibitors. From the results we can propose four resistance mechanisms that differ in the way the drug-target interaction is affected at the molecular level. Furthermore, we can deduce general principles on the mechanisms of resistance against fusion inhibitors and the requirements for effective antiviral drugs.     

Probing HIV-1 Membrane Liquid Order by Laurdan Staining Reveals Producer Cell-dependent Differences

Viruses acquire their envelope by budding from a host cell membrane, but viral lipid composition may differ from that of the budding membrane. We have previously reported that the HIV-1 membrane is highly enriched in cholesterol, sphingolipids, and other raft lipids, suggesting that the virus may bud from pre-existing or virus-induced lipid rafts. Here, we employed the environmentally sensitive fluorescent dye Laurdan to study the membrane lateral structure of HIV-1 derived from different cell lines. Differences in viral membrane order detected by Laurdan staining were shown by mass spectrometry to be due to differences in lipid composition. Isogenic viruses from two different cell lines were both strongly enriched in raft lipids and displayed a liquid-ordered membrane, but these effects were significantly more pronounced for HIV-1 from the T-cell line MT-4 compared with virus from 293T cells. Host-dependent differences in the lipidomes predominantly affected the ratio of sphingomyelins (including dihydrosphingomyelin) to phosphatidylcholine, whereas cholesterol contents were similar. Accordingly, treatment of infectious HIV-1 with the sphingomyelin-binding toxins Equinatoxin-II or lysenin showed differential inhibition of infectivity. Liposomes consisting of lipids that had been extracted from viral particles exhibited slightly less liquid order than the respective viral membranes, which is likely to be due to absence of membrane proteins and to loss of lipid asymmetry. Synthetic liposomes consisting of a quaternary lipid mixture emulating the viral lipids showed a liquid order similar to liposomes derived from virion lipids. Thus, Laurdan staining represents a rapid and quantitative method to probe viral membrane liquid order and may prove useful in the search for lipid active drugs.HIV-1³ is an enveloped retrovirus, which acquires its lipid envelope by budding from the plasma membrane of the infected host cell. Several reports have shown that the viral membrane is enriched in sphingomyelin (SM), including the unusual sphingolipid dihydrosphingomyelin (DHSM) and collectively referred to as sphingomyelins (SMs), glycosphingolipids, cholesterol (CHOL), saturated phosphoglycerolipids and phosphoinositides (1–4). Moreover the CHOL/phospholipid and protein/lipid ratios of the HIV-1 membrane are high, corresponding to a highly ordered membrane, and are presumed to be different from the overall host cell plasma membrane. Accordingly, the HIV-1 envelope has been considered to be a large raft-like membrane microdomain (3). This is in line with previous reports describing enrichment of raft markers in the HIV-1 membrane and its sensitivity to CHOL-depleting agents (5–9). Furthermore, HIV-1 glycoproteins have been suggested to localize within membrane rafts due to palmitoylation of two cysteines (10), and the main structural Gag protein has been shown to rapidly relocalize to detergent-resistant membranes after initial membrane binding (6).Membrane microdomains are dynamic assemblies resulting from the lateral interaction of lipids and proteins. Two phases coexist in the plasma membrane: the liquid-ordered phase (l_o), mainly composed of CHOL and sphingolipids (SPLs), and the liquid disordered phase (l_d), mainly composed of glycerophospholipids (11–13). In the activated state, l_o microdomains can coalesce and serve as platforms for membrane trafficking, signaling, and virus budding (14, 15). The first method to biochemically enrich membrane rafts was the purification of detergent-resistant membranes, based on their resistance to extraction with non-ionic detergent at 4 °C (16). However, this and other methods based on antibody or cholera toxin binding may lead to artificial aggregation of membrane microdomains and thus do not necessarily represent their native state (17, 18). For these reasons and because the association and dissociation of membrane microdomains appears to occur on a rapid time-scale and the raft size is too small to be optically resolved, the raft concept remains controversial. However, the determination of the HIV-1 lipidome, a native membrane purified without any detergent, has provided strong evidence for the existence of these microdomains (3).Fluorescent lipid analogs that partition preferentially into a specialized lipid phase could be an attractive tool to study membrane microdomains. However, partitioning of such dyes mainly depends on the local chemical environment and not on the phase state of the membrane (19–21). In contrast, Laurdan (6-dodecanoyl-2-dimethylaminonapthalene) is a lipophilic dye that binds to membranes independent of their phase state but reports the phase state by a change in its fluorescence emission (20). Laurdan exhibits a blue shift in its emission spectrum with increasing membrane condensation. This is caused by an alteration in the dipole moment of the probe as a consequence of exclusion of water molecules from the lipid bilayer. Thus, excitation of membrane bound Laurdan leads to two emission maxima representing differences in membrane lateral structure. Quantification of membrane order is achieved by computing the Generalized Polarization (GP) value, which is defined as normalized intensity ratio of the two emission channels. GP values range from +1 (most condensed) to −1 (most fluid). They are not biased by probe concentration, membrane ruffles, and surface modifications, such as lipoprotein binding. Furthermore, there is no preferential interaction with a specific lipid, fatty acid, or head group (20, 21). GP value correspondence to different lipid phases was estimated using liposomes with a composition similar to that of cellular membranes (22, 23). Using an equimolar mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine, CHOL, and SM as an l_o membrane, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as an l_d and solid ordered (s_o) phase, GP values below +0.25 were shown to correspond to the l_d phase, GP values between +0.25 and +0.5 to the l_o phase, and GP values above +0.5 to the s_o phase (22, 23).Laurdan has been extensively used to characterize domain formation and lateral lipid segregation in model membranes composed of different phospholipid mixtures or lipids extracted from cellular membranes (19, 22–25). It has also been used to study the membrane structure in living cells. Gaus and coworkers observed l_o domains enriched on membrane protrusions (filopodia), adhesion points, and cell-cell contacts (26). They also used Laurdan to address the physical properties of the plasma membrane around the T-cell receptor in activated T cells, observing larger and more stably ordered membrane domains at sites of T-cell activation (27). Quantitative determination of cellular plasma membrane order by fluorescence spectroscopy is complicated due to the rapid internalization and redistribution of the probes to other cellular membranes, making it difficult to interpret the fluorescence measurements over the whole cell. This problem is not encountered in purified virus particles, because they contain only a single membrane. We therefore developed an assay to study viral membrane lateral structure by fluorescence spectroscopy. For this purpose, isogenic HIV-1 particles were produced in two different cell lines, and their GP profiles were determined. In parallel, the lipid constituents were quantified by mass spectrometry. The viral membrane displayed a l_o structure in both cases, but this was more prominent for the virus derived from the T-cell line MT-4 compared with virus derived from 293T cells. The validity of this result was supported by comparing the lipidome of the two viruses, which revealed a significantly higher SMs/phosphatidylcholine (PC) ratio for the MT-4-derived virus. Accordingly, treatment with SM-binding toxins inactivated MT-4-derived virus more efficiently than 293T-derived virus, whereas both viruses exhibited similar infectivities before treatment. The reported approach allows rapid determination of differences in viral membrane order, permitting screening for compounds that perturb l_o domains, which may act as antivirals of a novel type.