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.     

Breadth of Neutralizing Antibodies Elicited by Stable,Homogeneous Clade A and Clade C HIV-1 gp140 Envelope Trimers in Guinea Pigs

The native envelope (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) is trimeric, and thus trimeric Env vaccine immunogens are currently being explored in preclinical immunogenicity studies. Key challenges have included the production and purification of biochemically homogeneous and stable trimers and the evaluation of these immunogens utilizing standardized virus panels for neutralization assays. Here we report the binding and neutralizing antibody (NAb) responses elicited by clade A (92UG037.8) and clade C (CZA97.012) Env gp140 trimer immunogens in guinea pigs. These trimers have been selected and engineered for optimal biochemical stability and have defined antigenic properties. Purified gp140 trimers with Ribi adjuvant elicited potent, cross-clade NAb responses against tier 1 viruses as well as detectable but low-titer NAb responses against select tier 2 viruses from clades A, B, and C. In particular, the clade C trimer elicited NAbs that neutralized 27%, 20%, and 47% of tier 2 viruses from clades A, B, and C, respectively. Heterologous DNA prime, protein boost as well as DNA prime, recombinant adenovirus boost regimens expressing these antigens, however, did not result in an increased magnitude or breadth of NAb responses in this system. These data demonstrate the immunogenicity of stable, homogeneous clade A and clade C gp140 trimers and exemplify the utility of standardized tier 1 and tier 2 virus panels for assessing the NAb responses of candidate HIV-1 Env immunogens.The development and evaluation of novel HIV-1 Env immunogens are critical priorities of the HIV-1 vaccine field (2, 10, 25). The major antigenic target for neutralizing antibodies (NAbs) is the trimeric Env glycoprotein on the virion surface (4, 18, 30). Monomeric gp120 immunogens have not elicited broadly reactive NAbs in animal models (5, 13, 28, 29) or humans (16, 31), and thus several groups have focused on generating trimer immunogens that better mimic the native Env spike found on virions (3, 7, 14, 15, 20, 22, 27). It has, however, proven difficult to produce stable and conformationally homogeneous Env trimers. Strategies to modify Env immunogens have therefore been explored, including the removal of the cleavage site between gp120 and gp41 (3, 7, 23, 39, 40), the incorporation of an intramolecular disulfide bond to stabilize cleaved gp120 and gp41 moieties (6), and the addition of trimerization motifs such as the T4 bacteriophage fibritin “fold-on” (Fd) domain (8, 17, 39).Preclinical evaluation of candidate Env immunogens is critical for concept testing and for the prioritization of vaccine candidates. Luciferase-based virus neutralization assays with TZM.bl cells (21, 24) have been developed as high-throughput assays that can be standardized (26). However, the optimal use of this assay requires the generation of standardized virus panels derived from multiple clades that reflect both easy-to-neutralize (tier 1) and primary isolate (tier 2) viruses (21, 24). A tiered approach for the evaluation of novel Env immunogens has been proposed, in which tier 1 viruses represent homologous vaccine strains and a small number of heterologous neutralization-sensitive viruses while tier 2 viruses provide a greater measure of neutralization breadth for the purpose of comparing immunogens (24).We screened a large panel of primary HIV-1 isolates for Env stability and identified two viruses, CZA97.012 (clade C) (32) and 92UG037.8 (clade A) (17), that yielded biochemically homogeneous and stable Env trimers with well defined and uniform antigenic properties (17). The addition of the T4 bacteriophage fibritin “fold-on” (Fd) trimerization domain further increased their yield and purity (17). In the present study, we assessed the immunogenicity of these stable clade A and clade C gp140 trimers in guinea pigs. Both trimers elicited high-titer binding antibody responses and cross-clade neutralization of select tier 1 viruses as well as low-titer but detectable NAb responses against select tier 2 viruses from clades A, B, and C. These data demonstrate the immunogenicity of these stable gp140 trimers and highlight the utility of standardized virus panels in the evaluation of novel HIV-1 Env immunogens.     

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.     

Investigation of Clade B New World Arenavirus Tropism by Using Chimeric GP1 Proteins

Clade B of the New World arenaviruses contains both pathogenic and nonpathogenic members, whose surface glycoproteins (GPs) are characterized by different abilities to use the human transferrin receptor type 1 (hTfR1) protein as a receptor. Using closely related pairs of pathogenic and nonpathogenic viruses, we investigated the determinants of the GP1 subunit that confer these different characteristics. We identified a central region (residues 85 to 221) in the Guanarito virus GP1 that was sufficient to interact with hTfR1, with residues 159 to 221 being essential. The recently solved structure of part of the Machupo virus GP1 suggests an explanation for these requirements.Arenaviruses are bisegmented, single-stranded RNA viruses that use an ambisense coding strategy to express four proteins: NP (nucleoprotein), Z (matrix protein), L (polymerase), and GP (glycoprotein). The viral GP is sufficient to direct entry into host cells, and retroviral vectors pseudotyped with GP recapitulate the entry pathway of these viruses (5, 13, 24, 31). GP is a class I fusion protein comprising two subunits, GP1 and GP2, cleaved from the precursor protein GPC (4, 14, 16, 18, 21). GP1 contains the receptor binding domain (19, 28), while GP2 contains structural elements characteristic of viral membrane fusion proteins (8, 18, 20, 38). The N-terminal stable signal peptide (SSP) remains associated with the mature glycoprotein after cleavage (2, 39) and plays a role in transport, maturation, and pH-dependent fusion (17, 35, 36, 37).The New World arenaviruses are divided into clades A, B, and C based on phylogenetic relatedness (7, 9, 11). Clade B contains the human pathogenic viruses Junin (JUNV), Machupo (MACV), Guanarito (GTOV), Sabia, and Chapare, which cause severe hemorrhagic fevers in South America (1, 10, 15, 26, 34). Clade B also contains the nonpathogenic viruses Amapari (AMAV), Cupixi, and Tacaribe (TCRV), although mild disease has been reported for a laboratory worker infected with TCRV (29).Studies with both viruses and GP-pseudotyped retroviral vectors have shown that the pathogenic clade B arenaviruses use the human transferrin receptor type 1 (hTfR1) to gain entry into human cells (19, 30). In contrast, GPs from nonpathogenic viruses, although capable of using TfR1 orthologs from other species (1), cannot use hTfR1 (1, 19) and instead enter human cells through as-yet-uncharacterized hTfR1-independent pathways (19). In addition, human T-cell lines serve as useful tools to distinguish these GPs, since JUNV, GTOV, and MACV pseudotyped vectors readily transduce CEM cells, while TCRV and AMAV GP vectors do not (27; also unpublished data). These properties of the GPs do not necessarily reflect a tropism of the pathogenic viruses for human T cells, since viral tropism is influenced by many factors and T cells are not a target for JUNV replication in vivo (3, 22, 25).     

The ESCRT-Associated Protein Alix Recruits the Ubiquitin Ligase Nedd4-1 To Facilitate HIV-1 Release through the LYPXnL L Domain Motif

The p6 region of HIV-1 Gag contains two late (L) domains, PTAP and LYPX_nL, that bind Tsg101 and Alix, respectively. Interactions with these two cellular proteins recruit members of the host''s fission machinery (ESCRT) to facilitate HIV-1 release. Other retroviruses gain access to the host ESCRT components by utilizing a PPXY-type L domain that interacts with cellular Nedd4-like ubiquitin ligases. Despite the absence of a PPXY motif in HIV-1 Gag, interaction with the ubiquitin ligase Nedd4-2 was recently shown to stimulate HIV-1 release. We show here that another Nedd4-like ubiquitin ligase, Nedd4-1, corrected release defects resulting from the disruption of PTAP (PTAP⁻), suggesting that HIV-1 Gag also recruits Nedd4-1 to facilitate virus release. Notably, Nedd4-1 remediation of HIV-1 PTAP⁻ budding defects is independent of cellular Tsg101, implying that Nedd4-1''s function in HIV-1 release does not involve ESCRT-I components and is therefore distinct from that of Nedd4-2. Consistent with this finding, deletion of the p6 region decreased Nedd4-1-Gag interaction, and disruption of the LYPX_nL motif eliminated Nedd4-1-mediated restoration of HIV-1 PTAP⁻. This result indicated that both Nedd4-1 interaction with Gag and function in virus release occur through the Alix-binding LYPX_nL motif. Mutations of basic residues located in the NC domain of Gag that are critical for Alix''s facilitation of HIV-1 release, also disrupted release mediated by Nedd4-1, further confirming a Nedd4-1-Alix functional interdependence. In fact we found that Nedd4-1 binds Alix in both immunoprecipitation and yeast-two-hybrid assays. In addition, Nedd4-1 requires its catalytic activity to promote virus release. Remarkably, RNAi knockdown of cellular Nedd4-1 eliminated Alix ubiquitination in the cell and impeded its ability to function in HIV-1 release. Together our data support a model in which Alix recruits Nedd4-1 to facilitate HIV-1 release mediated through the LYPX_nL/Alix budding pathway via a mechanism that involves Alix ubiquitination.Retroviral Gag polyproteins bear short conserved sequences that control virus budding and release. As such, these motifs have been dubbed late or L domains (49). Three types of L domains have thus far been characterized: PT/SAP, LYPX_nL, and PPPY motifs (5, 9, 32). They recruit host proteins known to function in the vacuolar protein sorting (vps) of cargo proteins and the generation of multivesicular bodies (MVB) compartments (2). It is currently accepted that budding of vesicles into MVB involves the sequential recruitment of endosomal sorting complexes required for transport (ESCRT-I, -II, and -III) and the activity of the VPS4 AAA-ATPase (22). These sorting events are believed to be triggered by recognition of ubiquitin molecules conjugated to cargo proteins (20, 24, 41). For retrovirus budding, L domain motifs are the primary signals in Gag that elicit the recruitment of ESCRT components to facilitate viral budding. Consequently, mutations in L domain motifs or dominant-negative interference with the function of ESCRT-III members or the VPS4 ATPase adversely affect virus release. This indicates that Gag interactions with the ESCRT machinery are necessary for virus budding and separation from the cell (7, 10, 15, 16, 21, 28, 44).Two late domains have been identified within the p6 region of human immunodeficiency virus type 1 (HIV-1) Gag protein: the PTAP and LYPX_nL motifs. The PTAP motif binds the cellular protein Tsg101 (15, 39, 40, 47), whereas the LYPX_nL motif is the docking site for Alix (44). Tsg101 functions in HIV-1 budding (15) as a member of ESCRT-I (30, 48), a soluble complex required for the generation of MVB. This process is topologically similar to HIV-1 budding and requires the recruitment of ESCRT-III members called the charged-multivesicular body proteins (3, 29, 48) and the activity of the VPS4 AAA-ATPase (4, 48). In addition to binding the LYPX_nL motif, Alix also interacts with the nucleocapsid (NC) domain of HIV-1 Gag (13, 38), thus linking Gag to components of ESCRT-III that are critical for virus release (13).Other retroviruses, including the human T-cell leukemia virus (HTLV) and the Moloney murine leukemia virus (MoMLV), utilize the PPPY-type L domain to efficiently release virus (7, 26, 51). The PPPY motif binds members of the Nedd4-like ubiquitin ligase family (6, 7, 16, 19, 25, 43), whose normal cellular function is to ubiquitinate cargo proteins and target them into the MVB sorting pathway (11, 12, 20). Members of the Nedd4-like ubiquitin ligase family include Nedd4-1, Nedd4-2 (also known as Nedd4L), WWP-1/2, and Itch. They contain three distinct domains: an N-terminal membrane binding C2 domain (12), a central PPPY-interacting WW domain (43), and a C-terminal HECT domain that contains the ubiquitin ligase active site (42). The functional requirement for the binding of Nedd4-like ubiquitin ligases to the PPPY motif in virus budding has been demonstrated (7, 16, 18, 19, 25, 26, 28, 50, 51). Overexpression of dominant-negative mutants of Nedd4-like ligases, ESCRT-III components, or VPS4 cause a potent inhibition of PPPY-dependent virus release (7, 19, 29, 31, 52) and induce assembly and budding defects similar to those observed after perturbation of the PPPY motif (26, 51). These observations demonstrated that Nedd4-like ligases connect Gag encoding PPPY motif to ESCRT-III and VPS4 proteins to facilitate virus release.Whereas the role of Nedd4-like ubiquitin ligases in virus budding has been established, the protein interactions that link them to the cell''s ESCRT-III pathway are still unknown. Evidence for associations of Nedd4-like ligases with ESCRT proteins have been previously reported and include: the binding of Nedd4-like ubiquitin ligases LD1 and Nedd4-1 to ESCRT-I member Tsg101 (6, 31), the colocalization of multiple Nedd4-like ubiquitin ligases with endosomal compartments (1, 28), the requirement of the cell''s ESCRT pathway for Itch mediated L domain independent stimulation of MoMLV release (23), and the ubiquitination of ESCRT-I components with a shorter isoform, Nedd4-2s (8). Therefore, Nedd4-like ubiquitin ligase interactions with members of the cell''s ESCRT pathway may provide retroviral Gag with access to the host budding machinery required for virus release.Although HIV-1 Gag does not carry the PPPY canonical sequence known to interact with Nedd4-like ubiquitin ligases, both Nedd4-1 and Nedd4-2 were shown to restore the release of the HIV-1 PTAP⁻ mutant, albeit Nedd4-1 with less efficiency than Nedd4-2 (8, 46). These findings suggested that HIV-1 might utilize cellular Nedd4-like ubiquitin ligases to increase virus release. We present here evidence demonstrating that Nedd4-1 interacts with Gag and enhances HIV-1 PTAP⁻ virus release. Furthermore, we show that Nedd4-1''s function in HIV-1 release is distinct from that of Nedd4-2 in both its viral and cellular requirements. Notably, we found that Nedd4-1 enhancement of HIV-1 release requires the Alix-binding LYPX_nL L domain motif in the p6 region and basic residues in the NC domain. In addition, Alix''s facilitation of HIV-1 release requires cellular Nedd4-1, since mutations in NC that prevented Alix-mediated HIV-1 release also eliminated release by overexpression of Nedd4-1. This suggested a Nedd4-1-Alix physical and functional interdependence. In agreement with this, we found Nedd4-1 to bind and ubiquitinate Alix in the cell. Taken together, these results support a model in which Alix recruits Nedd4-1 to facilitate late steps of HIV-1 release through the LYPX_nL L domain motif via a mechanism that involves Alix ubiquitination.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

DivIC Stabilizes FtsL against RasP Cleavage

The essential cell division protein FtsL is a substrate of the intramembrane protease RasP. Using heterologous coexpression experiments, we show here that the division protein DivIC stabilizes FtsL against RasP cleavage. Degradation seems to be initiated upon accessibility of a cytosolic substrate recognition motif.Cell division in bacteria is a highly regulated process (1). The division site selection as well as assembly and disassembly of the divisome have to be strictly controlled (1, 4). Although the spatial control of the divisome is relatively well understood (2, 4, 14, 17), mechanisms governing the temporal control of division are still mainly elusive. Regulatory proteolysis was thought to be a potential modulatory mechanism (8, 9). The highly unstable division protein FtsL was shown to be rate limiting for division and would make an ideal candidate for a regulatory factor in the timing of bacterial cell division (7, 9). In Bacillus subtilis, FtsL is an essential protein of the membrane part of the divisome (5, 7, 8). It is necessary for the assembly of the membrane-spanning division proteins, and a knockout is lethal (8, 9, 12). We have previously reported that FtsL is a substrate of the intramembrane protease RasP (5).These findings raised the question of whether RasP can regulate cell division by cleaving FtsL from the division complex. In order to mimic the situation in which FtsL is bound to at least one of its interaction partners, we used a heterologous coexpression system in which we synthesized FtsL and DivIC. It has been reported before that DivIC and FtsL are intimate binding partners in various organisms (6, 9, 15, 21, 22, 26) and that FtsL and DivIC (together with DivIB) can form complexes even in the absence of the other divisome components (6, 21). We therefore asked whether RasP is able to cleave FtsL in the presence of its major interaction partner DivIC, which would argue for the possibility that RasP could cleave FtsL within a mature divisome. In contrast, if interaction with DivIC could stabilize FtsL against RasP cleavage, this result would bring such a model into question. An alternative option for the role of RasP might be the removal of FtsL from the membrane. It has been shown that divisome disassembly and prevention of reassembly are crucial to prevent minicell formation close to the new cell poles (3, 16).     

Pseudotyping Incompatibility between HIV-1 and Gibbon Ape Leukemia Virus Env Is Modulated by Vpu

The Env protein from gibbon ape leukemia virus (GaLV) has been shown to be incompatible with human immunodeficiency virus type 1 (HIV-1) in the production of infectious pseudotyped particles. This incompatibility has been mapped to the C-terminal cytoplasmic tail of GaLV Env. Surprisingly, we found that the HIV-1 accessory protein Vpu modulates this incompatibility. The infectivity of HIV-1 pseudotyped with murine leukemia virus (MLV) Env was not affected by Vpu. However, the infectivity of HIV-1 pseudotyped with an MLV Env with the cytoplasmic tail from GaLV Env (MLV/GaLV Env) was restricted 50- to 100-fold by Vpu. A Vpu mutant containing a scrambled membrane-spanning domain, Vpu_RD, was still able to restrict MLV/GaLV Env, but mutation of the serine residues at positions 52 and 56 completely alleviated the restriction. Loss of infectivity appeared to be caused by reduced MLV/GaLV Env incorporation into viral particles. The mechanism of this downmodulation appears to be distinct from Vpu-mediated CD4 downmodulation because Vpu-expressing cells that failed to produce infectious HIV-1 particles nonetheless continued to display robust surface MLV/GaLV Env expression. In addition, if MLV and HIV-1 were simultaneously introduced into the same cells, only the HIV-1 particle infectivity was restricted by Vpu. Collectively, these data suggest that Vpu modulates the cellular distribution of MLV/GaLV Env, preventing its recruitment to HIV-1 budding sites.The gammaretrovirus gibbon ape leukemia virus (GaLV) has been widely used for gene therapy because of its wide host cell tropism and nonpathogenicity (1, 6, 10, 12, 13, 20). The host cell receptor for GaLV Env has been cloned and identified as a sodium-dependent phosphate transporter protein (25, 26). Like other retroviruses, GaLV encodes a single transmembrane surface glycoprotein (GaLV Env), which is cleaved into surface (SU) and transmembrane (TM) subunits (Fig. ​(Fig.1).1). The TM domain of GaLV Env contains a short 30-amino-acid C-terminal cytoplasmic tail. Although GaLV Env functions well when coupled (pseudotyped) with murine leukemia virus (MLV)-based retroviral vectors, it has been shown to be completely incompatible with HIV-1 (4, 35). When GaLV Env is expressed with HIV-1, essentially no infectious HIV-1 particles are produced (4, 35). The mechanism for this infectivity downmodulation is unknown, but the component of GaLV Env responsible for the restriction has been mapped to the cytoplasmic tail. Replacing the cytoplasmic tail of GaLV Env with the equivalent sequence from MLV Env ameliorates the restriction. Likewise, replacing the cytoplasmic tail of MLV Env with that from GaLV Env confers the restriction (4).Open in a separate windowFIG. 1.Schematic of MLV Env protein. Sequences are the C-terminal cytoplasmic tails of MLV Env, GaLV Env, and human CD4. GaLV sequences in boldface are residues that have been shown to modulate the HIV-1 incompatibility (4). Underlined sequences in CD4 are amino acids required for Vpu-mediated downmodulation (2, 15). Arrows denote the location of MLV/GaLV tail substitution. SU, surface domain; TM, transmembrane domain.Vpu is an 81-amino-acid HIV-1 accessory protein produced from the same mRNA as the HIV-1 Env gene. The N terminus of Vpu contains a membrane-spanning domain, followed by a 50-amino-acid cytoplasmic domain. Vpu is unique to HIV-1 and a few closely related SIV strains. The best-characterized roles for Vpu in the HIV-1 life cycle are modulation of host proteins CD4 and tetherin (also known as BST-2, CD317, and HM1.24) (24, 38, 39). Vpu promotes the degradation of CD4 in the endoplasmic reticulum through a proteasome-dependent mechanism (29). The cytoplasmic tail of Vpu physically interacts with the cytoplasmic tail of CD4 and recruits the human β-transducing repeat-containing protein (β-TrCP) and E3 ubiquitin ligase components to polyubiquitinate and ultimately trigger the degradation of CD4 (18). Two serine residues at positions 52 and 56 of Vpu are phosphorylated by casein kinase-2 and are required for CD4 degradation (31, 32). The membrane-spanning domain of Vpu is not specifically required for CD4 degradation. A mutant protein containing a scrambled membrane-spanning sequence, Vpu_RD, is still able to trigger the degradation of CD4 (32). The region of CD4 that is targeted by Vpu is approximately 17 to 13 amino acids from the C terminus in the cytoplasmic tail (Fig. ​(Fig.1)1) (2, 15).In addition to degrading CD4, Vpu has also long been known to result in enhanced viral release (EVR) in certain cell lines (14, 36). Recently, the type I interferon-induced host protein tetherin was identified as being responsible for this Vpu-modulated restriction (24, 38). In the absence of Vpu, tetherin causes particles to remain tethered (hence the name) to the host cell postfission. Although Vpu counteracts the function of tetherin, the exact mechanism has not been fully elucidated. However, the mechanism for tetherin antagonism appears to be distinct from that for modulating CD4. Mutation of the serines 52 and 56 of Vpu abolish CD4 degradation, but only reduce EVR activity (5, 17, 21, 32). Some EVR activity remains even when much of the Vpu cytoplasmic tail is deleted (30). In addition, many mutations in the membrane-spanning domain, such as Vpu_RD, do not affect CD4 degradation and yet completely abolish EVR activity (27, 30, 37). The critical residues in tetherin for recognition by Vpu appear to be in the membrane-spanning domain and not the cytoplasmic tail (9, 19, 28). Although β-TrCP is required for complete EVR activity, there is no consensus whether the degradation of tetherin is proteasome or lysosome mediated (5, 7, 21) or whether degradation is required at all. In some cases there can be some EVR activity in the absence of tetherin degradation (17, 22).We demonstrate here that Vpu is responsible for the incompatibility between HIV-1 and GaLV Env. Glycoproteins containing the cytoplasmic tail from GaLV Env are prevented from being incorporated into HIV-1 particles by Vpu, effectively reducing infectious particle production by 50- to 100-fold. The serines at positions 52 and 56 are required for this restriction, but the membrane-spanning domain is not. Although the mechanism for this restriction appears similar to CD4 degradation, there are apparent differences. Vpu does not prevent surface expression, and it does not prevent its incorporation into MLV particles. Therefore, the mechanism of restriction appears to involve a system that does not rely directly on global protein degradation.     

Human Immunodeficiency Virus Type 1 Elite Neutralizers: Individuals with Broad and Potent Neutralizing Activity Identified by Using a High-Throughput Neutralization Assay together with an Analytical Selection Algorithm

The development of a rapid and efficient system to identify human immunodeficiency virus type 1 (HIV-1)-infected individuals with broad and potent HIV-1-specific neutralizing antibody responses is an important step toward the discovery of critical neutralization targets for rational AIDS vaccine design. In this study, samples from HIV-1-infected volunteers from diverse epidemiological regions were screened for neutralization responses using pseudovirus panels composed of clades A, B, C, and D and circulating recombinant forms (CRFs). Initially, 463 serum and plasma samples from Australia, Rwanda, Uganda, the United Kingdom, and Zambia were screened to explore neutralization patterns and selection ranking algorithms. Samples were identified that neutralized representative isolates from at least four clade/CRF groups with titers above prespecified thresholds and ranked based on a weighted average of their log-transformed neutralization titers. Linear regression methods selected a five-pseudovirus subset, representing clades A, B, and C and one CRF01_AE, that could identify top-ranking samples with 50% inhibitory concentration (IC₅₀) neutralization titers of ≥100 to multiple isolates within at least four clade groups. This reduced panel was then used to screen 1,234 new samples from the Ivory Coast, Kenya, South Africa, Thailand, and the United States, and 1% were identified as elite neutralizers. Elite activity is defined as the ability to neutralize, on average, more than one pseudovirus at an IC₅₀ titer of 300 within a clade group and across at least four clade groups. These elite neutralizers provide promising starting material for the isolation of broadly neutralizing monoclonal antibodies to assist in HIV-1 vaccine design.Since the identification of human immunodeficiency virus type 1 (HIV-1) as the cause of AIDS, one of the greatest challenges has been the development of a vaccine that will prevent infection and/or ameliorate disease progression (38, 43). Although over 100 phase I, II, and III vaccine clinical trials of different candidates have been conducted all over the world, only a few candidates have advanced to efficacy testing and none has yet to show any benefit in prevention or control of HIV-1 (HIV Vaccine Database; www.iavi.org). In other viral diseases (such as polio, influenza, and measles), neutralizing antibodies are generated as part of either the natural immune response to infection or the response to immunization, and their role in protective immunity is well established (10, 12, 15, 22, 37, 42, 45, 47, 49, 52). For HIV-1, studies in animal models indicate that both broadly neutralizing antibodies and cell-mediated responses may be required to provide vaccine protection (7, 14, 16, 20, 29, 31, 33, 34, 39, 53). Unlike many other viruses, HIV-1 is highly variable, with multiple subtypes and recombinant forms circulating in different regions of the world. This high level of HIV-1 genetic variability, particularly in the envelope glycoproteins (gp120 and gp41), has been one of the greatest obstacles in development of a safe and effective HIV-1 vaccine and in particular in the elicitation of broadly neutralizing antibodies. In addition, HIV-1 has other mechanisms of immune escape preventing elicitation of broadly neutralizing antibodies, including the heavy glycosylation of the envelope glycoproteins, instability of such glycoproteins, and conformational masking of receptor-binding sites (6, 25, 32).Despite the enormous diversity of HIV-1, a relatively small number of broadly neutralizing monoclonal antibodies (bnMAbs) have been isolated, providing evidence that broad neutralization by single antibody specificities can be achieved (3-5, 8, 9, 17, 21, 23, 24, 29, 35, 36, 40, 41, 44, 50, 51, 55). Structures for such bnMAbs have been determined in complex with HIV-1 Env (26, 54) and provide starting points for the design of immunogens capable of eliciting broadly neutralizing antibodies. However, since there are only a few such bnMAbs, we established a global program as part of International AIDS Vaccine Initiative''s (IAVI''s) Neutralizing Antibody Consortium (6), aimed at screening HIV-1⁺ subjects with the goal of identifying individuals with broad and potent neutralizing activities as a potential source of novel bnMAbs, with an emphasis placed on individuals infected with non-clade B viruses. This paper describes the screening algorithm implemented to successfully identify HIV-1⁺ subjects with broadly neutralizing antibodies, including a subset of individuals termed “elite neutralizers.” These volunteers will be studied further to characterize the specificities of serum antibodies and will provide source materials for isolation of bnMAbs.     

Role of Env in resistance of feline immunodeficiency virus (FIV)-infected cats to superinfection by a second FIV strain as determined by using a chimeric virus

A more or less pronounced resistance to superinfection by a second strain of the infecting virus has been observed in many lentivirus-infected hosts. We used a chimeric feline immunodeficiency virus (FIV), designated FIV_χ, containing a large part of the env gene of a clade B virus (strain M2) and all the rest of the genome of a clade A virus (a p34TF10 molecular clone of the Petaluma strain modified to grow in lymphoid cells), to gain insights into such resistance. FIV_χ was infectious and moderately pathogenic for cats and in vitro exhibited the neutralization specificity of the env donor. The experiments performed were bidirectional, in that cats preinfected with either parental virus were challenged with FIV_χ and vice versa. The preinfected animals were partially or completely protected relative to what was observed in naïve control animals, most likely due, at least in part, to the circumstance that in all the preinfecting/challenge virus combinations examined, the first and the second virus shared significant viral components. Based on the proportions of complete protection observed, the role of a strongly matched viral envelope appeared to be modest and possibly dependent on the time interval between the first and the second infection. Furthermore, complete protection and the presence of measurable neutralizing antibodies capable of blocking the second virus in vitro were not associated.Recent reportsman immunodeficiency virus type 1 (HIV-1)-infected individuals can become superinfected with a second strain of the virus more frequently than previously estimated (1, 9, 26, 28, 46, 50). On the other hand, there is also convincing evidence that an established HIV-1 infection may confer on the host the ability to ward off acquisition of another HIV-1, especially if it belongs to the same clade as the initial virus and if exposure occurs after a sufficient time interval from the first infection (reviewed in references 8 and 50). The latter evidence is corroborated by studies with several animal lentiviruses, including simian immunodeficiency virus (SIV) in macaques, equine infectious anemia virus in ponies, and feline immunodeficiency virus (FIV) in cats, showing that prior infection with live attenuated and even wild-type viruses can prevent subsequent infection by fully virulent strains of the same viruses or at least afford substantial protection against their pathological effects (10, 11, 13, 18, 38 reviewed in reference 17). The mechanisms mediating such protection are, however, unresolved in both the primate and nonprimate systems (4, 27, 29, 32, 33, 55). The issue is important, since a clear understanding of the nature of these mechanisms might help in identifying immune correlates of protection against lentiviruses. That such correlates have so far remained elusive is considered a major obstacle to development and testing of candidate AIDS vaccines (2, 7, 21, 22, 37).FIV is both a significant pathogen of domestic cats (42) and a widely used model to investigate HIV pathogenesis and approaches to AIDS vaccination (14, 16, 51, 54). In the present study, we developed a chimeric FIV, designated FIV_χ, having most of the env gene of a clade B virus and the rest of the genome of a clade A virus and used it in an attempt to gain insight into the variables that may affect the resistance to superinfection by a second strain of virus in this system, including the neutralization specificity of the viral envelope (Env).     

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.     

Heavily Isotype-Dependent Protective Activities of Human Antibodies against Vaccinia Virus Extracellular Virion Antigen B5

Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro—and protection in vivo in a mouse model—by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.The smallpox vaccine, live vaccinia virus (VACV), is frequently considered the gold standard of human vaccines and has been enormously effective in preventing smallpox disease. The smallpox vaccine led to the worldwide eradication of the disease via massive vaccination campaigns in the 1960s and 1970s, one of the greatest successes of modern medicine (30). However, despite the efficacy of the smallpox vaccine, the mechanisms of protection remain unclear. Understanding those mechanisms is key for developing immunologically sound vaccinology principles that can be applied to the design of future vaccines for other infectious diseases (3, 101).Clinical studies of fatal human cases of smallpox disease (variola virus infection) have shown that neutralizing antibody titers were either low or absent in patient serum (24, 68). In contrast, neutralizing antibody titers for the VACV intracellular mature virion (MV or IMV) were correlated with protection of vaccinees against smallpox (68). VACV immune globulin (VIG) (human polyclonal antibodies) is a promising treatment against smallpox (47), since it was able to reduce the number of smallpox cases ∼80% among variola-exposed individuals in four case-controlled clinical studies (43, 47, 52, 53, 69). In animal studies, neutralizing antibodies are crucial for protecting primates and mice against pathogenic poxviruses (3, 7, 17, 21, 27, 35, 61, 66, 85).The specificities and the functions of protective antipoxvirus antibodies have been areas of intensive research, and the mechanics of poxvirus neutralization have been debated for years. There are several interesting features and problems associated with the antibody response to variola virus and related poxviruses, including the large size of the viral particles and the various abundances of many distinct surface proteins (18, 75, 91, 93). Furthermore, poxviruses have two distinct virion forms, intracellular MV and extracellular enveloped virions (EV or EEV), each with a unique biology. Most importantly, MV and EV virions share no surface proteins (18, 93), and therefore, there is no single neutralizing antibody that can neutralize both virion forms. As such, an understanding of virion structure is required to develop knowledge regarding the targets of protective antibodies.Neutralizing antibodies confer protection mainly through the recognition of antigens on the surface of a virus. A number of groups have discovered neutralizing antibody targets of poxviruses in animals and humans (3). The relative roles of antibodies against MV and EV in protective immunity still remain somewhat unclear. There are compelling data that antibodies against MV (21, 35, 39, 66, 85, 90, 91) or EV (7, 16, 17, 36, 66, 91) are sufficient for protection, and a combination of antibodies against both targets is most protective (66). It remains controversial whether antibodies to one virion form are more important than those to the other (3, 61, 66). The most abundant viral particles are MV, which accumulate in infected cells and are released as cells die (75). Neutralization of MV is relatively well characterized (3, 8, 21, 35). EV, while less abundant, are critical for viral spread and virulence in vivo (93, 108). Neutralization of EV has remained more enigmatic (3).B5R (also known as B5 or WR187), one of five known EV-specific proteins, is highly conserved among different strains of VACV and in other orthopoxviruses (28, 49). B5 was identified as a protective antigen by Galmiche et al., and the available evidence indicated that the protection was mediated by anti-B5 antibodies (36). Since then, a series of studies have examined B5 as a potential recombinant vaccine antigen or as a target of therapeutic monoclonal antibodies (MAbs) (1, 2, 7, 17, 40, 46, 66, 91, 110). It is known that humans immunized with the smallpox vaccine make antibodies against B5 (5, 22, 62, 82). It is also known that animals receiving the smallpox vaccine generate antibodies against B5 (7, 20, 27, 70). Furthermore, previous neutralization assays have indicated that antibodies generated against B5 are primarily responsible for neutralization of VACV EV (5, 83). Recently Chen at al. generated chimpanzee-human fusion MAbs against B5 and showed that the MAbs can protect mice from lethal challenge with virulent VACV (17). We recently reported, in connection with a study using murine monoclonal antibodies, that neutralization of EV is highly complement dependent and the ability of anti-B5 MAbs to protect in vivo correlated with their ability to neutralize EV in a complement-dependent manner (7).The focus of the study described here was to elucidate the mechanisms of EV neutralization, focusing on the human antibody response to B5. Our overall goal is to understand underlying immunobiological and virological parameters that determine the emergence of protective antiviral immune responses in humans.     

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.