Matrix and Envelope Coevolution Revealed in a Patient Monitored since Primary Infection with Human Immunodeficiency Virus Type 1

Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), typically encode envelope glycoproteins (Env) with long cytoplasmic tails (CTs). The strong conservation of CT length in primary isolates of HIV-1 suggests that this factor plays a key role in viral replication and persistence in infected patients. However, we report here the emergence and dominance of a primary HIV-1 variant carrying a natural 20-amino-acid truncation of the CT in vivo. We demonstrated that this truncation was deleterious for viral replication in cell culture. We then identified a compensatory amino acid substitution in the matrix protein that reversed the negative effects of CT truncation. The loss or rescue of infectivity depended on the level of Env incorporation into virus particles. Interestingly, we found that a virus mutant with defective Env incorporation was able to spread by cell-to-cell transfer. The effects on viral infectivity of compensation between the CT and the matrix protein have been suggested by in vitro studies based on T-cell laboratory-adapted virus mutants, but we provide here the first demonstration of the natural occurrence of similar mechanisms in an infected patient. Our findings provide insight into the potential of HIV-1 to evolve in vivo and its ability to overcome major structural alterations.The envelope glycoprotein complex of the human immunodeficiency virus type 1 (HIV-1) is involved principally in virion attachment to target cell surfaces and in the entry process (15, 18, 27, 29, 52). Envelope glycoproteins (Env) are initially translated as a gp160 precursor glycoprotein, which is then processed during its trafficking through the secretory pathway, to yield a surface subunit gp120 noncovalently attached to a transmembrane subunit gp41. During HIV-1 assembly, Env proteins are incorporated at the surface of the viral particle as a trimeric structure consisting of three gp120/gp41 dimers (59, 62).The gp41 consists of an ectodomain, a hydrophobic transmembrane anchor, and a cytoplasmic tail (CT). Lentiviruses, including HIV-1 and simian immunodeficiency virus (SIV), are unusual in having a transmembrane subunit with much longer CTs (∼150 amino acids) than most other retroviruses (20 to 50 amino acids) (27). Early studies with T-cell laboratory-adapted HIV-1 mutants showed that the gp41 CT region played an important role in regulating Env functions, the incorporation of Env into virus particles and, consequently, viral replication (16, 21, 35, 63). The integrity of the gp41 CT thus appears to be crucial for replication in primary T cells, macrophages, and in many transformed T-cell lines (1, 44). Viral variants with truncated gp41 are rarely isolated from infected patients. One study reported the isolation of a CD4-independent variant harboring a sharply truncated CT (64). However, this atypical isolate existed as a minority variant in the original quasispecies of the patient (54). SIV variants with truncated CTs obtained in cell culture in vitro have also been shown to revert rapidly (to full-length CT) when introduced into macaques (39). These observations indicate that the long CTs of lentiviruses, such as HIV-1 and SIV, have functions specific to viral replication and persistence in vivo.Two groups of conserved sequence motifs have been identified in the gp41 CT that are likely to be involved in its functions. The first group, involved in regulating the intracellular trafficking of Env, includes a membrane-proximal tyrosine-based endocytic motif, Y₇₁₂SPL, (9, 47); a diaromatic motif, Y₈₀₂W₈₀₃, implicated in the retrograde transport of Env to the trans-Golgi network (8), and a C-terminal dileucine motif recently identified as a second endocytic motif (7, 10, 60). We have also provided evidence for the existence of additional as-yet-unidentified signals in studies of primary HIV-1 (34). The second group of motifs consists of three structurally conserved amphipathic α-helical domains: lentivirus lytic peptides 1, 2, and 3 (LLP-1, LLP-2, and LLP-3) (11, 17, 33). LLP domains have been implicated in various functions, including Env fusogenicity and the incorporation of Env into HIV-1 particles (28, 32, 43, 45, 50, 61).Several lines of evidence suggest that Env incorporation requires direct or indirect interactions between the matrix domain of the structural protein precursor Pr55^Gag (matrix) and the gp41 CT during HIV-1 assembly. This possibility was first suggested by the observation that HIV-1 Env drives the basolateral budding of Gag in polarized cells (37, 48). A direct interaction between the matrix and a glutathione S-transferase fusion protein containing Env CT was subsequently observed in vitro (13). Synthetic peptides corresponding to various domains of the gp41 CT have also been shown to interact directly with Pr55^Gag molecules (26). Furthermore, effects on viral infectivity of compensation between the CT and the matrix protein have been suggested by studies based on T-cell laboratory-adapted virus mutants (19, 40, 43). Finally, the cellular protein TIP47 was recently implicated in Env incorporation, based on its ability to bind both the matrix protein and the gp41 CT (38).In a previous study describing the evolutionary dynamics of the glycan shield of HIV-1 Env, we identified a patient (patient 153) for whom the 15 env clones obtained during primary infection (early stage) encoded full-length Env, whereas the 15 env sequences from the HIV-1 present 6 years later (late stage) encoded truncated gp41 CTs (14). These late-stage sequences contained a deletion introducing an in-frame stop codon, resulting in a 20-amino-acid truncation of the Env. Note that, unlike a point mutation, this deletion cannot easily revert to the full-length form. Such a deletion affecting various known motifs of the gp41 CT would be expected to impair viral replication. However, the plasma viral load measured in patient 153 demonstrated that the virus had retained its ability to replicate.In the present study, we explored the molecular mechanisms by which a primary HIV-1 maintained its capacity to replicate efficiently in this patient and demonstrated for the first time the occurrence of matrix and Env coevolution in vivo, providing insight into the ability of HIV-1 to overcome major structural alterations.     

Env-Expressing Autologous T Lymphocytes Induce Neutralizing Antibody and Afford Marked Protection against Feline Immunodeficiency Virus

The envelope (Env) glycoproteins of HIV and other lentiviruses possess neutralization and other protective epitopes, yet all attempts to induce protective immunity using Env as the only immunogen have either failed or afforded minimal levels of protection. In a novel prime-boost approach, specific-pathogen-free cats were primed with a plasmid expressing Env of feline immunodeficiency virus (FIV) and feline granulocyte-macrophage colony-stimulating factor and then boosted with their own T lymphocytes transduced ex vivo to produce the same Env and interleukin 15 (3 × 10⁶ to 10 × 10⁶ viable cells/cat). After the boost, the vaccinees developed elevated immune responses, including virus-neutralizing antibodies (NA). Challenge with an ex vivo preparation of FIV readily infected all eight control cats (four mock vaccinated and four naïve) and produced a marked decline in the proportion of peripheral CD4 T cells. In contrast, five of seven vaccinees showed little or no traces of infection, and the remaining two had reduced viral loads and underwent no changes in proportions of CD4 T cells. Interestingly, the viral loads of the vaccinees were inversely correlated to the titers of NA. The findings support the concept that Env is a valuable immunogen but needs to be administered in a way that permits the expression of its full protective potential.Despite years of intense research, a truly protective AIDS vaccine is far away. Suboptimal immunogenicity, inadequate antigen presentation, and inappropriate immune system activation are believed to have contributed to these disappointing results. However, several lines of evidence suggest that the control or prevention of infection is possible. For example, despite repeated exposures, some individuals escape infection or delay disease progression after being infected (1, 14, 15). Furthermore, passively infused neutralizing antibodies (NA) (28, 42, 51) or endogenously expressed NA derivatives (29) have been shown to provide protection against intravenous simian immunodeficiency virus challenge. On the other hand, data from several vaccine experiments suggest that cellular immunity is an important factor for protection (6, 32). Therefore, while immune protection against human immunodeficiency virus (HIV) and other lentiviruses appears feasible, the strategies for eliciting it remain elusive.Because of its crucial role in viral replication and infectivity, the HIV envelope (Env) is an attractive immunogen and has been included in nearly all vaccine formulations tested so far (28, 30, 31). Env surface (SU) and transmembrane glycoproteins (gp) are actively targeted by the immune system (9, 10, 47), and Env-specific antibodies and cytotoxic T lymphocytes (CTLs) are produced early in infection. The appearance of these effectors also coincides with the decline of viremia during the acute phase of infection (30, 32). Individuals who control HIV infection in the absence of antiretroviral therapy have Env-specific NA and CTL responses that are effective against a wide spectrum of viral strains (14, 23, 35, 52, 60). At least some of the potentially protective epitopes in Env appear to interact with the cellular receptors during viral entry and are therefore highly conserved among isolates (31, 33, 39, 63). However, these epitopes have complex secondary and tertiary structures and are only transiently exposed by the structural changes that occur during the interaction between Env and its receptors (10, 11, 28). As a consequence, these epitopes are usually concealed from the immune system, and this may explain, at least in part, why Env-based vaccines have failed to show protective efficacy. Indeed, data from previous studies suggested that protection may be most effectively triggered by nascent viral proteins (22, 28, 30, 48, 62).We have conducted a proof-of-concept study to evaluate whether presenting Env to the immune system in a manner as close as possible to what occurs in the context of a natural infection may confer some protective advantage. The study was carried out with feline immunodeficiency virus (FIV), a lentivirus similar to HIV that establishes persistent infections and causes an AIDS-like disease in domestic cats. As far as it is understood, FIV evades immune surveillance through mechanisms similar to those exploited by HIV, and attempts to develop an effective FIV vaccine have met with difficulties similar to those encountered with AIDS vaccines (25, 37, 66). In particular, attempts to use FIV Env as a protective immunogen have repeatedly failed (13, 38, 58). Here we report the result of one experiment in which specific-pathogen-free (SPF) cats primed with a DNA immunogen encoding FIV Env and feline granulocyte-macrophage colony-stimulating factor (GM-CSF) and boosted with viable, autologous T lymphocytes ex vivo that were transduced to express Env and feline interleukin 15 (IL-15) showed a remarkable level of protection against challenge with ex vivo FIV. Consistent with recent findings indicating the importance of NA in controlling lentiviral infections (1, 59, 63), among the immunological parameters investigated, only the titers of NA correlated inversely with protection. Collectively, the findings support the notion that Env is a valuable vaccine immunogen but needs to be administered in a way that permits the expression of its full protective potential.     

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.     

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.     

Opposing Effects of a Tyrosine-Based Sorting Motif and a PDZ-Binding Motif Regulate Human T-Lymphotropic Virus Type 1 Envelope Trafficking

Human T-lymphotropic virus type 1 (HTLV-1) envelope (Env) glycoprotein mediates binding of the virus to its receptor on the surface of target cells and subsequent fusion of virus and cell membranes. To better understand the mechanisms that control HTLV-1 Env trafficking and activity, we have examined two protein-protein interaction motifs in the cytoplasmic domain of Env. One is the sequence YSLI, which matches the consensus YXXΦ motifs that are known to interact with various adaptor protein complexes; the other is the sequence ESSL at the C terminus of Env, which matches the consensus PDZ-binding motif. We show here that mutations that destroy the YXXΦ motif increased Env expression on the cell surface and increased cell-cell fusion activity. In contrast, mutation of the PDZ-binding motif greatly diminished Env expression in cells, which could be restored to wild-type levels either by mutating the YXXΦ motif or by silencing AP2 and AP3, suggesting that interactions with PDZ proteins oppose an Env degradation pathway mediated by AP2 and AP3. Silencing of the PDZ protein hDlg1 did not affect Env expression, suggesting that hDlg1 is not a binding partner for Env. Substitution of the YSLI sequence in HTLV-1 Env with YXXΦ elements from other cell or virus membrane-spanning proteins resulted in alterations in Env accumulation in cells, incorporation into virions, and virion infectivity. Env variants containing YXXΦ motifs that are predicted to have high-affinity interaction with AP2 accumulated to lower steady-state levels. Interestingly, mutations that destroy the YXXΦ motif resulted in viruses that were not infectious by cell-free or cell-associated routes of infection. Unlike YXXΦ, the function of the PDZ-binding motif manifests itself only in the producer cells; AP2 silencing restored the incorporation of PDZ-deficient Env into virus-like particles (VLPs) and the infectivity of these VLPs to wild-type levels.Human T-lymphotropic virus type 1 (HTLV-1) envelope (Env), like most retroviral envelopes, is synthesized as a precursor protein in the endoplasmic reticulum, forms trimers, and is cleaved by a cellular furin-like protease as it transits through the trans-Golgi network on its way to the plasma membrane (7, 21, 31). Cleavage of the HTLV-1 Env precursor generates a 46-kDa surface subunit (SU, gp46) and a 21-kDa transmembrane protein (TM, gp21) (8, 43). SU contains the receptor-binding domain and is linked by a disulfide bond to TM, which anchors Env to the membrane and mediates fusion of virus and cell membranes after receptor engagement (11, 28, 40, 51). TM consists of extracellular, membrane-spanning, and cytoplasmic domains (31); the last contains motifs that direct Env trafficking, membrane targeting, and virion incorporation. HTLV-1 is poorly transmitted as cell-free virus, and there is good evidence supporting a model in which virions are transmitted in a polarized fashion between lymphocytes that are in close contact (22, 30). Unlike murine leukemia virus (MLV) and Mason-Pfizer monkey virus (MPMV) Envs, in which the cytoplasmic domain (CD) is cleaved by the virus-encoded protease to activate fusogenic activity (3, 6, 19, 42), the HTLV-1 Env cytoplasmic domain is not cleaved and HTLV-1 Env exists on the cell surface in a highly fusogenic state. In many respects, HTLV-1 Env resembles versions of MLV or MPMV Envs that lack C-terminal amino acids, e.g., with elevated cell-cell fusion activity and low virion infectivity. It is not exactly clear how HTLV-1 Env is controlled such that virus infection can proceed without cell-cell fusion, but it is probable that Env trafficking plays an important role. The cytoplasmic domain of HTLV-1 Env is relatively short and contains two important trafficking motifs: a YXXΦ motif (YSLI), which is involved in membrane protein trafficking and basolateral sorting in polarized epithelial cells (10), and a PDZ-binding motif (ESSL), which can interact with numerous PDZ proteins but is not found in other retroviral Envs (2).The tyrosine-based sorting motif (YXXΦ, where Y is tyrosine, X is any amino acid, and Φ is a bulky hydrophobic amino acid) determines the trafficking and turnover of many membrane-spanning proteins in the cell (5, 39) and is present in most retroviral Env proteins (7). The YXXΦ motif interacts with the μ subunit of the heterotetrameric adaptor protein complexes AP1, AP2, AP3, and AP4. Each adaptor complex is involved in a specific trafficking pathway: AP1 and AP4 deliver cargo from the trans-Golgi network to the plasma membrane (13, 33, 48), AP2 directs the endocytosis of proteins from the cell surface, and AP3 is involved in lysosomal sorting (5, 12, 24, 35). Each type of μ subunit interacts with a distinct but overlapping type of tyrosine-based motif; the tyrosine and the Φ residues are most critical, but affinity is determined in large part by the variable amino acids at positions +1 and +2 relative to tyrosine and also by surrounding amino acids (5, 37). Furthermore, interactions between AP2 and the YXXΦ motif may be regulated by phosphorylation of μ2 (38, 47), by localized changes in phosphoinositide concentration, or by interactions between AP2 and docking factors (47). Although most retroviral Env proteins contain YXXΦ-sorting motifs, the sequences of the motifs and their roles in Env trafficking and function appear to vary widely among different retroviruses. For example, mutation of the YXXΦ motif in MLV Env interferes with basolateral targeting of Env and diminishes viral pathogenesis in vivo but has little effect on Env accumulation at the plasma membrane (9, 16, 23, 25, 29). Mutations in the YXXΦ motif in MPMV Env are similar to those in MLV Evn and also were reported to affect Env incorporation into virions (45). Mutation of the YXXΦ motif in HTLV-1 Env was previously shown to decrease Env endocytosis, increase cell-cell fusion, increase Env incorporation into virions, abolish basolateral targeting, and decrease virus infectivity (1, 10).The most abundant protein-protein interaction domains in mammalian cells are the PDZ domains; more than 400 PDZ proteins are encoded in the human genome. PDZ domains are modular, recognize short C-terminal peptide motifs, and are often found in multiple copies or in combination with other protein interaction domains (36, 46, 50). PDZ proteins have the ability to form supramolecular scaffolds that coordinate signaling, synapse formation, cell polarity, and trafficking of interacting proteins (26, 44, 53). With respect to the last, it is important to note that PDZ proteins can delay the internalization of G protein-coupled receptors, ion channels, and membrane transporters (17, 41, 49, 52). Among retroviral Env proteins, only HTLV and simian T-lymphotropic virus (STLV) Envs contain putative PDZ-binding motifs. A yeast two-hybrid screen using the HTLV-1 Env cytoplasmic domain (CD) as bait identified the PDZ protein hDlg (human homolog of disc large protein) as a potential binding partner (2). In vitro pulldown experiments showed that a glutathione S-transferase (GST)-EnvCD fusion protein interacted with several PDZ proteins from cell lysates, one of which was hDlg. In one study, mutation of the PDZ-binding motif in HTLV-1 Env inhibited cell-cell fusion (2); in another study, hDlg small interfering RNA (siRNA) silencing caused a modest reduction in syncytium formation (54). Neither study examined how the PDZ-binding motif controls Env expression, membrane targeting, trafficking, or virus infectivity. Thus, it is still unclear which PDZ proteins interact with HTLV-1 Env in vivo and how those interactions affect Env trafficking and activity.In this paper, functional interactions between the YXXΦ motif and the PDZ-binding motif in the cytoplasmic domain of HTLV-1 Env were investigated by mutagenesis of Env and by siRNA silencing of potential cellular interacting proteins. The YXXΦ motif in HTLV-1 Env appears to interact primarily with AP2 and AP3, which regulate Env endocytosis and lysosomal degradation, respectively. Mutations that ablated the YXXΦ motif increased Env accumulation on the cell surface. The PDZ-binding motif at the C terminus of Env appears to delay Env turnover. Mutation of the PDZ-binding element diminished Env accumulation in cells to very low levels, indicating that loss of the PDZ-binding motif accelerates Env degradation. Expression of Env with a mutated PDZ-binding motif could be restored to normal levels by also mutating the YXXΦ motif or by silencing AP2 or AP3. The ability of the PDZ-binding motif to alter the activity of the YXXΦ motif depends on the particular sequence of the latter. The attenuating effect of the PDZ-binding motif on Env endocytosis could be overcome by substitution of the YSLI motif in HTLV-1 Env with YXXΦ elements from other cell or virus proteins that are predicted to have higher affinities for AP2 than the YSLI motif of HTLV-1 Env.     

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.     

Identification of a Feline Leukemia Virus Variant That Can Use THTR1, FLVCR1, and FLVCR2 for Infection

The pathogenic subgroup C feline leukemia virus (FeLV-C) arises in infected cats as a result of mutations in the envelope (Env) of the subgroup A FeLV (FeLV-A). To better understand emergence of FeLV-C and potential FeLV intermediates that may arise, we characterized FeLV Env sequences from the primary FY981 FeLV isolate previously derived from an anemic cat. Here, we report the characterization of the novel FY981 FeLV Env that is highly related to FeLV-A Env but whose variable region A (VRA) receptor recognition sequence partially resembles the VRA sequence from the prototypical FeLV-C/Sarma Env. Pseudotype viruses bearing FY981 Env were capable of infecting feline, human, and guinea pig cells, suggestive of a subgroup C phenotype, but also infected porcine ST-IOWA cells that are normally resistant to FeLV-C and to FeLV-A. Analysis of the host receptor used by FY981 suggests that FY981 can use both the FeLV-C receptor FLVCR1 and the feline FeLV-A receptor THTR1 for infection. However, our results suggest that FY981 infection of ST-IOWA cells is not mediated by the porcine homologue of FLVCR1 and THTR1 but by an alternative receptor, which we have now identified as the FLVCR1-related protein FLVCR2. Together, our results suggest that FY981 FeLV uses FLVCR1, FLVCR2, and THTR1 as receptors. Our findings suggest the possibility that pathogenic FeLV-C arises in FeLV-infected cats through intermediates that are multitropic in their receptor use.Feline leukemia viruses (FeLVs) are pathogenic retroviruses of domestic cats that induce proliferative, degenerative, and immunosuppressive disorders (17, 18, 27). Infected cats often contain a mixture of FeLVs that have been categorized into subgroups A, B, and C based on their interference and in vitro host range properties (39). These subgroups have been shown to be tightly associated with distinct feline diseases (17, 27). A fourth subgroup (T) that is highly related to FeLV-A has also been reported (26) and has been shown to be associated with immune deficiency (30). FeLV-A is the primary strain that is transmitted between cats and is the progenitor virus from which other subgroups arise. FeLV-B arises by recombination between endogenous retroviral sequences present in the cat genome and the gene encoding the FeLV-A envelope (Env) protein (32, 40, 42) that is responsible for host cell surface receptor recognition. FeLV-C and FeLV-T are formed by mutations in the FeLV-A Env gene (10, 27, 38). Env recombination and mutations are a major determinant of the host receptors used by the different FeLV subgroups (3, 23, 34, 46, 47).The emergence of pathogenic FeLV-C in cats infected with FeLV-A provides a classic example of Env mutations that switch the host receptor used for infection, leading to a fatal pathogenic disease in the host. The emergence of FeLV-C is tightly associated with red blood cell aplasia, a fatal feline anemia characterized by a specific disruption in erythroid progenitor cell development (2, 12, 19, 28). Moreover, FeLV-C emergence coincides with a switch in the host receptor used for infection from the thiamine transporter THTR1 (FeLV-A receptor) (23) to the heme exporter FLVCR1 (FeLV-C receptor) (34, 35, 46). Previous studies have suggested that the feline anemia is caused by the FeLV-C Env protein binding to, and disrupting, the cellular function of FLVCR1 (1, 34, 46). Indeed, expression of FeLV-C Env in hematopoietic stem cells (36) or disruption of FLVCR1 (21, 35) specifically disrupts early erythropoiesis, which mimics the anemia observed in cats with FeLV-C. Interestingly, the emergence of FeLV-C from FeLV-A is analogous to the emergence of cytopathic X4 strains of human immunodeficiency virus type 1 (HIV-1) in individuals infected with the R5 strain of HIV-1. Analogous to the emergence of FeLV-C, X4 HIV-1 arises by Env mutations in the R5 HIV-1 strain, which leads to a switch in the coreceptor used for infection allowing an expansion in HIV-1 cell tropism and subsequently to accelerated AIDS (4, 8). However, whereas HIV-1 pathogenesis also involves emergence of variants or X4/R5 intermediates that can use multiple related coreceptors for infection (5, 8, 11), the mechanism of how FeLV-C emerges, in terms of the presence of FeLV-A/FeLV-C intermediates, has yet to be elucidated.To better understand the emergence of pathogenic FeLV-C in infected cats and potential FeLV variants/intermediates that may arise, we isolated and characterized FeLV Env sequences from a primary FeLV isolate derived from a cat with pure red cell aplasia. In this study, we report the isolation and characterization of the novel FY981 Env, a hybrid FeLV-A/FeLV-C Env that, when pseudotyped, can use FLVCR1, THTR1, and the FLVCR1-related FLVCR2 for infection. Our findings suggest that the FY981 Env could represent a potential FeLV intermediate that arises during the emergence of pathogenic FeLV-C.     

Effect of Mutations in the Human Immunodeficiency Virus Type 1 Protease on Cleavage of the gp41 Cytoplasmic Tail

We previously reported that human immunodeficiency virus type 1 (HIV-1) develops resistance to the cholesterol-binding compound amphotericin B methyl ester (AME) by acquiring mutations (P203L and S205L) in the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 that create cleavage sites for the viral protease (PR). In the present study, we observed that a PR inhibitor-resistant (PIR) HIV-1 mutant is unable to efficiently cleave the gp41 cytoplasmic tail in P203L and S205L virions, resulting in loss of AME resistance. To define the pathway to AME resistance in the context of the PIR PR, we selected for resistance with an HIV-1 isolate expressing the mutant enzyme. We identified a new gp41 mutation, R236L, that results in cleavage of the gp41 tail by the PIR PR. These results highlight the central role of gp41 cleavage as the primary mechanism of AME resistance.Cholesterol-enriched membrane microdomains, often referred to as lipid rafts (4, 18, 24), play an important role in the replication of many enveloped viruses, including human immunodeficiency virus type 1 (HIV-1) (22, 30). Lipid rafts are involved in both HIV-1 entry and egress (reviewed in references 6, 22, and 30), and the lipid bilayer of HIV-1 virions is significantly enriched in cholesterol and highly saturated lipids characteristic of lipid rafts (3, 5, 8). We recently demonstrated that the cholesterol-binding polyene fungal antibiotic amphotericin B methyl ester (AME) potently inhibits HIV-1 replication. The antiviral activity of AME is due to a profound inhibition of viral entry (27, 28) and impairment of virus particle production (29).In our previous studies, we showed that the propagation of HIV-1 in the presence of AME leads to viral escape from this compound. The mutations that confer resistance map to the cytoplasmic tail (CT) of the gp41 transmembrane envelope (Env) glycoprotein (27, 28). AME-resistant mutants (P203L and S205L) overcome the defect in viral entry imposed by AME by a novel mechanism of resistance whereby the gp41 CT is cleaved by the viral protease (PR) after incorporation of Env into virions (28). The introduction of stop codons into the gp41-coding region that prematurely truncate the CT also renders virions AME resistant. In the present study, we evaluated the interplay between protease inhibitor resistance (PIR) mutations and AME resistance.     

Balancing Reversion of Cytotoxic T-Lymphocyte and Neutralizing Antibody Escape Mutations within Human Immunodeficiency Virus Type 1 Env upon Transmission

Human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is subject to both neutralizing antibody (NAb) and CD8 T-cell (cytotoxic T-lymphocyte [CTL]) immune pressure. We studied the reversion of the Env CTL escape mutant virus to the wild type and the relationship between the reversion of CTL mutations with N-linked glycosylation site (NLGS)-driven NAb escape in pigtailed macaques. Env CTL mutations either did not revert to the wild type or only transiently reverted 5 to 7 weeks after infection. The CTL escape mutant reversion was coincident, for the same viral clones, with the loss of NLGS mutations. At one site studied, both CTL and NLGS mutations were needed to confer NAb escape. We conclude that CTL and NAb escape within Env can be tightly linked, suggesting opportunities to induce effective multicomponent anti-Env immunity.CD8 T-cell responses against human immunodeficiency virus (HIV) have long been observed to select for viral variants that avoid cytotoxic T-lymphocyte (CTL) recognition (2, 5, 15, 18, 27). These immune escape mutations may, however, result in reduced replication competence (“fitness cost”) (11, 20, 26). CTL escape variants have been shown to revert to the wild type (WT) upon passage to major histocompatibility complex-mismatched hosts, both in macaques with simian immunodeficiency virus (SIV) or chimeric SIV/HIV (SHIV) infection (11, 12) and in humans with HIV type 1 (HIV-1) infection (1, 19).Most analyses of CTL escape and reversion have studied Gag CTL epitopes known to facilitate control of viremia (7, 14, 21, 30). Fewer analyses have studied Env-specific CTL epitopes. Recent sequencing studies suggest the potential for mutations within predicted HIV-1 Env-specific CTL epitopes to undergo reversion to the WT (16, 23). Env-specific CTL responses may, however, have less impact on viral control of both HIV-1 and SIV/SHIV than do Gag CTL responses (17, 24, 25), presumably reflecting either less-potent inhibition of viral replication or minimal fitness cost of escape (9).Serial viral escape from antibody pressure also occurs in both macaques and humans (3, 13, 28). Env is extensively glycosylated, and this “evolving glycan shield” can sterically block antibody binding without mutation at the antibody-binding site (8, 16, 31). Mutations at glycosylation sites, as well as other mutations, are associated with escape from neutralizing antibody (NAb) responses (4, 13, 29). Mutations in the amino acid sequences of N-linked glycosylation sites (NLGS) can alter the packing of the glycan cloud that surrounds the virion, by a loss, gain, or shift of an NLGS (32), thus facilitating NAb escape.Env is the only viral protein targeted by both CTL and NAb responses. The serial viral escape from both Env-specific CTL and NAb responses could have implications for viral fitness and the reversion of multiple mutations upon transmission to naïve hosts.We previously identified three common HIV-1 Env-specific CD8 T cell epitopes, RY8_788-795, SP9_110-118, and NL9_671-679, and their immune escape patterns in pigtail macaques (Macaca nemestrina) infected with SHIV_mn229 (25). SHIV_mn229 is a chimeric virus constructed from an SIV_mac239 backbone and an HIV-1_HXB2 env fragment that was passaged through macaques to become pathogenic (11). This earlier work provided an opportunity for detailed studies of how viruses with Env-specific CTL escape mutations, as well as mutations in adjacent NLGS, evolve when transmitted to naïve pigtail macaques.     

Ebola Virus Glycoprotein Counteracts BST-2/Tetherin Restriction in a Sequence-Independent Manner That Does Not Require Tetherin Surface Removal

BST-2/tetherin is an interferon-inducible protein that restricts the release of enveloped viruses from the surface of infected cells by physically linking viral and cellular membranes. It is present at both the cell surface and in a perinuclear region, and viral anti-tetherin factors including HIV-1 Vpu and HIV-2 Env have been shown to decrease the cell surface population. To map the domains of human tetherin necessary for both virus restriction and sensitivity to viral anti-tetherin factors, we constructed a series of tetherin derivatives and assayed their activity. We found that the cytoplasmic tail (CT) and transmembrane (TM) domains of tetherin alone produced its characteristic cellular distribution, while the ectodomain of the protein, which includes a glycosylphosphatidylinositol (GPI) anchor, was sufficient to restrict virus release when presented by the CT/TM regions of a different type II membrane protein. To counteract tetherin restriction and remove it from the cell surface, HIV-1 Vpu required the specific sequence present in the TM domain of human tetherin. In contrast, the HIV-2 Env required only the ectodomain of the protein and was sensitive to a point mutation in this region. Strikingly, the anti-tetherin factor, Ebola virus GP, was able to overcome restriction conferred by both tetherin and a series of functional tetherin derivatives, including a wholly artificial tetherin molecule. Moreover, GP overcame restriction without significantly removing tetherin from the cell surface. These findings suggest that Ebola virus GP uses a novel mechanism to circumvent tetherin restriction.Pathogenic viruses often have evolved mechanisms to neutralize host defenses that act at the cellular level to interfere with the virus life cycle. Such cellular restriction factors have been most extensively characterized for HIV-1 (38) and include the interferon-inducible membrane protein BST-2/HM1.24/CD317/tetherin (28, 40). If unchecked, tetherin blocks the release of newly formed HIV-1 particles from cells by physically tethering them at the cell surface (7, 28, 32, 40). In addition, tetherin has been shown to act against a broad range of enveloped viral particles, including retroviruses, filoviruses, arenaviruses, and herpesviruses (17, 18, 23, 35). In turn, certain viruses that are targeted by tetherin appear to have evolved counteracting activities, and anti-tetherin factors so far identified include HIV-1 Vpu; HIV-2 Env; simian immunodeficiency virus (SIV) Nef, Vpu, and Env proteins; Ebola virus GP; and Kaposi''s sarcoma-associated herpesvirus (KSHV) K5 (11, 16, 18, 20, 23, 28, 36, 40, 44, 45).Tetherin is a homodimeric type II integral membrane protein containing an N-terminal cytoplasmic tail (CT), a single-pass transmembrane domain (TM), an ectodomain-containing predicted coiled-coil regions, two glycoslyation sites, three conserved cysteines, and a C-terminal glycosylphosphatidylinositol (GPI) anchor (2, 19, 31). This unusual topology, with two independent membrane anchors, has led to the suggestion that the retention of virions at the cell surface arises from tetherin''s ability to be inserted simultaneously in both host and viral membranes (28, 32, 41) or, alternatively, that dimers or higher-order complexes of tetherin conferred by the ectodomain mediate this effect (39). Interestingly, an artificial tetherin containing the same structural features as the native protein but constructed from unrelated sequences was able to restrict both HIV-1 and Ebola virus particles (32). This suggests that the viral lipid envelope is the target of tetherin and provides an explanation for tetherin''s broad activity against diverse enveloped viruses.A fraction of tetherin is present at the plasma membrane of cells (9, 14), and it has been proposed that viral anti-tetherin factors function by removing this cell surface fraction (40). This now has been shown to occur in the presence of HIV-1 Vpu (5, 7, 15, 26, 34, 40, 44), HIV-2 Env (5, 20), SIV Env (11), SIV Nef (15), and KSHV K5 (3, 23). In addition, certain anti-tetherin factors also may promote the degradation of tetherin, as has been observed for both HIV-1 Vpu (3, 5, 7, 10, 22, 26, 27) and KSHV K5 (3, 23), although Vpu also appears able to block tetherin restriction in the absence of degradation (8), and no effects on tetherin steady-state levels have been observed in the presence of either the HIV-2 or SIVtan Env (11, 20). Simply keeping tetherin away from the cell surface, or targeting it for degradation, may not be the only mechanism used by anti-tetherin factors, since it also has been reported that Vpu does not affect the levels of surface tetherin or its total cellular levels in certain T-cell lines (27).The interactions between tetherin and viral anti-tetherin factors show evidence of species specificity, suggesting ongoing evolution between viruses and their hosts. HIV-1 Vpu is active against human and chimpanzee tetherin but not other primate tetherins (10, 25, 34, 36, 44, 45), while SIV Nef proteins are active against primate but not human tetherins (16, 36, 44, 45). This suggests that, unlike tetherin restriction, the action of the anti-tetherin factors may involve specific sequence interactions. Indeed, the TM domain has been recognized as a target for HIV-1 Vpu (10, 15, 16, 25, 34), while a single point mutation introduced into the extracellular domain of human tetherin can block its antagonism by the SIVtan Env (11).In the present study, we investigated the roles of the different domains of tetherin in both promoting virus restriction and conferring susceptibility to the anti-tetherin factors encoded by HIV-1, HIV-2, and Ebola virus. We confirmed that tetherin restriction can be conferred by proteins that retain the two distinct membrane anchors, while signals for the cellular localization of the protein reside in the CT/TM domains of the protein. We found that the Vpu protein targets the TM domain of tetherin, while the HIV-2 Env targets the ectodomain of the protein. In contrast, the Ebola virus GP appears to use a non-sequence-specific mechanism to counteract tetherin restriction, since even an artificial tetherin could be successfully overcome by GP expression. Interestingly, Ebola virus GP counteracted tetherin restriction without removing the protein from the cell surface, suggesting that it is possible to overcome this restriction by mechanisms other than blocking tetherin''s cell surface expression.     

Fundamental Difference in the Content of High-Mannose Carbohydrate in the HIV-1 and HIV-2 Lineages

The virus-encoded envelope proteins of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) typically contain 26 to 30 sites for N-linked carbohydrate attachment. N-linked carbohydrate can be of three major types: high mannose, complex, or hybrid. The lectin proteins from Galanthus nivalis (GNA) and Hippeastrum hybrid (HHA), which specifically bind high-mannose carbohydrate, were found to potently inhibit the replication of a pathogenic cloned SIV from rhesus macaques, SIVmac239. Passage of SIVmac239 in the presence of escalating concentrations of GNA and HHA yielded a lectin-resistant virus population that uniformly eliminated three sites (of 26 total) for N-linked carbohydrate attachment (Asn-X-Ser or Asn-X-Thr) in the envelope protein. Two of these sites were in the gp120 surface subunit of the envelope protein (Asn244 and Asn460), and one site was in the envelope gp41 transmembrane protein (Asn625). Maximal resistance to GNA and HHA in a spreading infection was conferred to cloned variants that lacked all three sites in combination. Variant SIV gp120s exhibited dramatically decreased capacity for binding GNA compared to SIVmac239 gp120 in an enzyme-linked immunosorbent assay (ELISA). Purified gp120s from six independent HIV type 1 (HIV-1) isolates and two SIV isolates from chimpanzees (SIVcpz) consistently bound GNA in ELISA at 3- to 10-fold-higher levels than gp120s from five SIV isolates from rhesus macaques or sooty mangabeys (SIVmac/sm) and four HIV-2 isolates. Thus, our data indicate that characteristic high-mannose carbohydrate contents have been retained in the cross-species transmission lineages for SIVcpz-HIV-1 (high), SIVsm-SIVmac (low), and SIVsm-HIV-2 (low).The envelope proteins of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) are heavily glycosylated. N-linked carbohydrate is attached to the nascent protein at the asparagine of the consensus sequence N-X-S or N-X-T, where X is any amino acid except a proline (31, 52, 53). The number of potential N-linked carbohydrate attachment sites in the surface subunit of Env (gp120) ranges from 18 to 33, with a median of 25 (34, 65). There are typically 3 or 4 potential N-linked sites in the ectodomain of the Env transmembrane protein (gp41) (34).N-linked glycosylation of a protein consists of the en bloc transfer of the carbohydrate core oligosaccharide (two N-acetylglucosamines, nine mannoses, and three glucoses) from dolichol to the asparagine of the N-linked attachment site (8, 60). Initially the attached carbohydrate is processed into the high-mannose type (8). In the Golgi complex, high-mannose carbohydrate may be further processed into complex or hybrid oligosaccharides (58). Incomplete processing of N-linked carbohydrate results in the production of high-mannose carbohydrate chains, which terminate in mannose (58). Fully processed complex carbohydrate chains terminate in galactose, N-acetylglucosamine, sialic acid, or glucose (33, 57). Hybrid carbohydrate chains have two branches from the core, one that terminates in mannose and one that terminates in a sugar of the complex type (63).Glycoproteins exist as a heterogeneous population, exhibiting heterogeneity with respect to the proportion of potential glycosylation sites that are occupied and to the oligosaccharide structure observed at each site. Factors that influence the type of carbohydrate chain that is attached at any one N-linked site are the accessibility of the carbohydrate chain to processing enzymes (49), protein sequences surrounding the site (5, 40), and the type of cell from which the protein is produced (19).The N-linked carbohydrate chains of HIV and SIV Env are critical for the proper folding and cleavage of the fusion-competent envelope spike (20, 59, 61). After Env is assembled, enzymatic removal of N-linked carbohydrate does not dramatically affect the functional conformation (2, 6, 7, 13, 24, 38). It is generally accepted that the carbohydrate attached to Env limits the ability of the underlying protein to be recognized by B cells (11, 48, 62). This carbohydrate also shields protein epitopes that would otherwise be the direct targets of antibodies that neutralize viral infection (41, 48, 62, 64). Furthermore, the high-mannose carbohydrates of HIV and SIV Env bind dynamically to an array of lectin proteins that are part of the host lymphoreticular system. The interaction of viral high-mannose carbohydrate with host lectin proteins has been associated with the enhancement (9, 16, 17, 43-45) or suppression (42, 56) of viral infection of CD4-positive T cells. The high-mannose carbohydrate of Env is also known to activate the release of immune-modulatory proteins from a subset of host antigen-presenting cells (12, 54).The plant lectin proteins from Galanthus nivalis (GNA) and Hippeastrum hybrid (HHA) specifically bind terminal α-1,3- and/or α-1,6-mannose of high-mannose oligosaccharides but not hybrid oligosaccharides (28, 55). GNA and HHA inhibit the replication of HIV-1 and SIVmac251, and uncloned, resistant populations of virus have been selected (3, 14). In this report, we define two N-linked sites in the external surface glycoprotein gp120 and one in the transmembrane glycoprotein gp41 whose mutation imparts high-level resistance to the inhibitory effects of GNA and HHA to cloned SIVmac239. Furthermore, using a GNA-binding enzyme-linked immunosorbent assay (ELISA), we show that assorted HIV-1 and SIVcpz gp120s consistently are considerably higher in mannose content than assorted gp120s from SIVmac, SIVsm, and HIV-2. These results shed new light on the impact of virus-host evolutionary dynamics on viral carbohydrate composition, and they may have important implications for the mechanisms by which long-standing natural hosts such as sooty mangabeys can resist generalized lymphoid activation and disease despite high levels of SIV replication.     

Immunization with Wild-Type or CD4-Binding-Defective HIV-1 Env Trimers Reduces Viremia Equivalently following Heterologous Challenge with Simian-Human Immunodeficiency Virus

We recently reported that rhesus macaques inoculated with CD4-binding-competent and CD4-binding-defective soluble YU2-derived HIV-1 envelope glycoprotein (Env) trimers in adjuvant generate comparable levels of Env-specific binding antibodies (Abs) and T cell responses. We also showed that Abs directed against the Env coreceptor binding site (CoRbs) were elicited only in animals immunized with CD4-binding-competent trimers and not in animals immunized with CD4-binding-defective trimers, indicating that a direct interaction between Env and CD4 occurs in vivo. To investigate both the overall consequences of in vivo Env-CD4 interactions and the elicitation of CoRbs-directed Abs for protection against heterologous simian-human immunodeficiency virus (SHIV) challenge, we exposed rhesus macaques immunized with CD4-binding-competent and CD4-binding-defective trimers to the CCR5-tropic SHIV-SF162P4 challenge virus. Compared to unvaccinated controls, all vaccinated animals displayed improved control of plasma viremia, independent of the presence or absence of CoRbs-directed Abs prior to challenge. Immunization resulted in plasma responses that neutralized the heterologous SHIV challenge stock in vitro, with similar neutralizing Ab titers elicited by the CD4-binding-competent and CD4-binding-defective trimers. The neutralizing responses against both the SHIV-SF162P4 stock and a recombinant virus pseudotyped with a cloned SHIV-SF162P4-derived Env were significantly boosted by the SHIV challenge. Collectively, these results suggest that the capacity of soluble Env trimers to interact with primate CD4 in vivo and to stimulate the production of moderate titers of CoRbs-directed Abs did not influence the magnitude of the neutralizing Ab recall response after viral challenge or the subsequent control of viremia in this heterologous SHIV challenge model.The external glycoprotein gp120 and the membrane-anchored glycoprotein gp41 of human immunodeficiency virus type 1 (HIV-1), collectively referred to as the envelope glycoproteins (Env), mediate viral entry and are the sole virally encoded targets for neutralizing antibodies (NAbs). Prior to binding the primary host cell receptor, CD4, the trimeric Env spike may sample multiple conformations on the surface of the virus. Which of these potential conformations display neutralizing Ab epitopes and are recognized by broadly reactive NAbs is currently unclear. A substantial conformational change occurs when the functional Env spike interacts with CD4, leading to the exposure and the formation of the bridging sheet, a highly conserved and immunogenic structure spanning the inner and outer domains of gp120 that contributes to coreceptor interaction (6, 14, 25, 30). CD4 binding is also thought to lead to the displacement of variable region 3 (V3) from a less exposed conformation in the packed functional spike to a more protruding conformation. Exposure of V3 is necessary for viral entry, as it also contributes to Env interaction with coreceptor (21). Additional or concurrent rearrangements of the functional spike structure may occur upon CD4 binding, as suggested by cryotomography (38), However, these rearrangements are less well understood due to the absence of a high-resolution structure of the static or CD4-liganded trimeric spike.In attempts to elicit broadly reactive NAbs against HIV-1 through vaccination, a range of recombinant Env variants were designed and tested (reviewed in references 15, 26, 49, and 50). The capacity of such immunogens to elicit broadly reactive NAbs is often determined using standardized in vitro neutralization assays (34). However, the ability of HIV-1 Env vaccine-elicited B cell responses to mediate actual protective and functional responses against in vivo virus challenge is evaluated less frequently, since this requires the use of nonhuman primates (NHPs) and infection with chimeric simian-human immunodeficiency viruses (SHIVs). A series of SHIVs was developed, including those based on the HIV-1 Env glycoproteins from SF162 (40), 89.6 (54), ADA (45), BaL (48), DH12 (59), and 1157i (27). So far, few of these models, if any, fully mimic HIV-1 infection in humans. Currently, serially passaged CCR5-using SHIV-SF162 (SHIV-SF162P), which establishes transient or more prolonged viremia in macaques, represent a frequently used model to evaluate the protective effect of Env-based immunogens (2-5, 19, 20, 23, 24, 29, 53, 67). Depending on the number and nature of passages that this virus has been exposed to, the SHIV-SF162P stocks are more or less neutralization resistant (19, 62), allowing one to test the efficacy of a given vaccine candidate against a more or less rigorous form of viral challenge. Protection against mucosal SHIV-SF162P4 challenge after homologous SF162ΔV2 Env protein immunization of rhesus macaques was recently reported (2, 3). However, the nature and specificities of the vaccine-induced immune responses that mediate this effect remain incompletely defined.We recently showed that Abs against the HIV-1 gp120 coreceptor binding site (CoRbs) are elicited as a consequence of in vivo interactions between Env and primate CD4 during immunization with soluble CD4 (sCD4)-binding-competent Env trimers (14). We subsequently showed that rhesus macaques inoculated with CD4-binding competent and CD4-binding defective soluble YU2-derived gp140-F trimers in adjuvant generate comparable levels of Env-specific binding Abs and T cell responses but that CoRbs-directed Abs are elicited only in animals immunized with wild-type (wt) CD4-binding competent Env trimers (13). So far, the impact of Env-CD4 in vivo interactions during Env immunization and the role of CoRbs-directed Abs in protection against SHIV infection remain incompletely understood. A majority of the well-characterized CoRbs-directed monoclonal Abs (MAbs) lack the capacity to neutralize primary viruses in vitro (7, 31). However, it has been suggested that Abs directed against this region may contribute to the neutralizing Ab response seen in some HIV-1-infected individuals (18, 35, 58) and to the protection observed in some SHIV challenge experiments (12).The distinct difference in the capacity of the CD4-binding competent and CD4-binding defective Env trimers to elicit CoRbs-directed Abs described in our previous study presented an opportunity to evaluate the protective effect of CoRbs-directed Abs in the SHIV model. The availability of animals immunized with these Env immunogens also allowed us to ask the more general question about whether in vivo interactions between soluble Env trimers and CD4-expressing host cells would influence the outcome of heterologous SHIV-SF162P4 infection. We show here that Env trimer-immunized animals displayed improved control of SHIV-SF162P4 viremia compared to unimmunized control animals, independent of whether they were inoculated with CD4-binding-competent or CD4-binding-defective trimers. These results suggest that the capacity of soluble Env trimers to interact with CD4 in vivo and to stimulate the production of CoRbs-directed Abs did not measurably influence the protective effect of the vaccine-elicited immune responses in this SHIV challenge model.     

Caveolin-1 Modulates HIV-1 Envelope-Induced Bystander Apoptosis through gp41

Human immunodeficiency virus (HIV) envelope (Env)-mediated bystander apoptosis is known to cause the progressive, severe, and irreversible loss of CD4⁺ T cells in HIV-1-infected patients. Env-induced bystander apoptosis has been shown to be gp41 dependent and related to the membrane hemifusion between envelope-expressing cells and target cells. Caveolin-1 (Cav-1), the scaffold protein of specific membrane lipid rafts called caveolae, has been reported to interact with gp41. However, the underlying pathological or physiological meaning of this robust interaction remains unclear. In this report, we examine the interaction of cellular Cav-1 and HIV gp41 within the lipid rafts and show that Cav-1 modulates Env-induced bystander apoptosis through interactions with gp41 in SupT1 cells and CD4⁺ T lymphocytes isolated from human peripheral blood. Cav-1 significantly suppressed Env-induced membrane hemifusion and caspase-3 activation and augmented Hsp70 upregulation. Moreover, a peptide containing the Cav-1 scaffold domain sequence markedly inhibited bystander apoptosis and apoptotic signal pathways. Our studies shed new light on the potential role of Cav-1 in limiting HIV pathogenesis and the development of a novel therapeutic strategy in treating HIV-1-infected patients.HIV infection causes a progressive, severe, and irreversible depletion of CD4⁺ T cells, which is responsible for the development of AIDS (9). The mechanism through which HIV infection induces cell death involves a variety of processes (58). Among these processes, apoptosis is most likely responsible for T-cell destruction in HIV-infected patients (33), because active antiretroviral therapy has been associated with low levels of CD4⁺ T-cell apoptosis (7), and AIDS progression was shown previously to correlate with the extent of immune cell apoptosis (34). Importantly, bystander apoptosis of uninfected cells was demonstrated to be one of the major processes involved in the destruction of immune cells (58), with the majority of apoptotic CD4⁺ T cells in the peripheral blood and lymph nodes being uninfected in HIV patients (22).Binding to uninfected cells or the entry of viral proteins released by infected cells is responsible for the virus-mediated killing of innocent-bystander CD4⁺ T cells (2-4, 9, 65). The HIV envelope glycoprotein complex, consisting of gp120 and gp41 subunits expressed on an HIV-infected cell membrane (73), is believed to induce bystander CD4⁺ T-cell apoptosis (58). Although there is a soluble form of gp120 in the blood, there is no conclusive agreement as to whether the concentration is sufficient to trigger apoptosis (57, 58). The initial step in HIV infection is mediated by the Env glycoprotein gp120 binding with high affinity to CD4, the primary receptor on the target cell surface, which is followed by interactions with the chemokine receptor CCR5 or CXCR4 (61). This interaction triggers a conformational change in gp41 and the insertion of its N-terminal fusion peptide into the target membrane (30). Next, a prehairpin structure containing leucine zipper-like motifs is formed by the two conserved coiled-coil domains, called the N-terminal and C-terminal heptad repeats (28, 66, 70). This structure quickly collapses into a highly stable six-helix bundle structure with an N-terminal heptad repeat inside and a hydrophobic C-terminal heptad repeat outside (28, 66, 70). The formation of the six-helix bundle leads to a juxtaposition and fusion with the target cell membrane (28, 66, 70). The fusogenic potential of HIV Env is proven to correlate with the pathogenesis of both CXCR4- and CCR5-tropic viruses by not only delivering the viral genome to uninfected cells but also mediating Env-induced bystander apoptosis (71). Initial infection is dominated by the CCR5-tropic strains, with the CXCR4-tropic viruses emerging in the later stages of disease (20). Studies have shown that CXCR4-tropic HIV-1 triggers more depletion of CD4⁺ T cells than CCR5-tropic strains (36).Glycolipid- and cholesterol-enriched membrane microdomains, termed lipid rafts, are spatially organized plasma membranes and are known to have many diverse functions (26, 53). These functions include membrane trafficking, endocytosis, the regulation of cholesterol and calcium homeostasis, and signal transduction in cellular growth and apoptosis. Lipid rafts have also been implicated in HIV cell entry and budding processes (19, 46, 48, 51). One such organelle is the caveola, which is a small, flask-shaped (50 to 100 nm in diameter) invagination in the plasma membrane (5, 62). The caveola structure, which is composed of proteins known as caveolins, plays a role in various functions by serving as a mobile platform for many receptors and signal proteins (5, 62). Caveolin-1 (Cav-1) is a 22- to 24-kDa major coat protein responsible for caveola assembly (25, 47). This scaffolding protein forms a hairpin-like structure and exists as an oligomeric complex of 14 to 16 monomers (21). Cav-1 has been shown to be expressed by a variety of cell types, mostly endothelial cells, type I pneumocytes, fibroblasts, and adipocytes (5, 62). In addition, Cav-1 expression is evident in immune cells such as macrophages and dendritic cells (38, 39). However, Cav-1 is not expressed in isolated thymocytes (49). Furthermore, Cav-1 and caveolar structures are absent in human or murine T-cell lines (27, 41, 68). Contrary to this, there has been one report showing evidence of Cav-1 expression in bovine primary cell subpopulations of CD4⁺, CD8⁺, CD21⁺, and IgM⁺ cells with Cav-1 localized predominantly in the perinuclear region (38). That report also demonstrated a membrane region staining with Cav-1-specific antibody of human CD21⁺ and CD26⁺ peripheral blood lymphocytes (PBLs). Recently, the expression of Cav-1 in activated murine B cells, with a potential role in the development of a thymus-independent immune response, was also reported (56). It remains to be determined whether Cav-1 expression is dependent on the activation state of lymphocytes. For macrophages, however, which are one of the main cell targets for HIV infection, Cav-1 expression has been clearly documented (38).The scaffolding domain of Cav-1, located in the juxtamembranous region of the N terminus, is responsible for its oligomerization and binding to various proteins (5, 62, 64). It recognizes a consensus binding motif, ΦXΦXXXXΦ, ΦXXXXΦXXΦ, or ΦXΦXXXXΦXXΦ, where Φ indicates an aromatic residue (F, W, or Y) and X indicates any residue (5, 62, 64). A Cav-1 binding motif (WNNMTWMQW) has been identified in the HIV-1 envelope protein gp41 (42, 43). Cav-1 has been shown to associate with gp41 by many different groups under various circumstances, including the immunoprecipitation of gp41 and Cav-1 in HIV-infected cells (42, 43, 52). However, the underlying pathological or physiological functions of this robust interaction between Cav-1 and gp41 remain unclear.Here, we report that the interaction between Cav-1 and gp41 leads to a modification of gp41 function, which subsequently regulates Env-induced T-cell bystander apoptosis. Moreover, we show that a peptide containing the Cav-1 scaffold domain sequence is capable of modulating Env-induced bystander apoptosis, which suggests a novel therapeutic application for HIV-1-infected patients.     

Receptor Binding and Low pH Coactivate Oncogenic Retrovirus Envelope-Mediated Fusion

Fusion of enveloped viruses with host cells is triggered by either receptor binding or low pH but rarely requires both except for avian sarcoma leukosis virus (ASLV). We recently reported that membrane fusion mediated by an oncogenic Jaagsiekte sheep retrovirus (JSRV) envelope (Env) requires an acidic pH, yet receptor overexpression is required for this process to occur. Here we show that a soluble form of the JSRV receptor, sHyal2, promoted JSRV Env-mediated fusion at a low pH in normally fusion-negative cells and that this effect was blocked by a synthetic peptide analogous to the C-terminal heptad repeat of JSRV Env. In contrast to the receptor of ASLV, sHyal2 induced pronounced shedding of the JSRV surface subunit, as well as unstable conformational rearrangement of its transmembrane (TM) subunit, yet full activation of JSRV Env fusogenicity, associated with strong TM oligomerization, required both sHyal2 and low pH. Consistently, sHyal2 enabled transduction of nonpermissive cells by JSRV Env pseudovirions, with low efficiency, but substantially blocked viral entry into permissive cells at both binding and postbinding steps, indicating that sHyal2 prematurely activates JSRV Env-mediated fusion. Altogether, our study supports a model that receptor priming promotes fusion activation of JSRV Env at a low pH, and that the underlying mechanism is likely to be different from that of ASLV. Thus, JSRV may provide a useful alternate model for the better understanding of virus fusion and cell entry.Fusion is a fundamental event in the life cycle of enveloped viruses and is essential for viral replication. While viral fusion proteins are highly divergent in primary sequence, their structures and modes of activation share striking similarities, permitting their classification into two major groups (41). Class I fusion proteins, as exemplified by the retrovirus envelope (Env) and influenza virus hemagglutinin (HA), are composed mainly of alpha-helices, and they are present as metastable trimers on the viral surface (11). Class II fusion proteins, represented by alphavirus E1 and flavivirus E, contain predominantly beta-sheets and exist as dimers in the prefusion state (16). Of note, the vesicular stomatitis virus G (VSV-G) and herpesvirus gB proteins were recently assigned to a newly established class III, for fusion proteins combining properties of both class I and class II (13, 30). Despite these differences, one common and intriguing characteristic of all viral fusion proteins is their ability to undergo dramatic conformational rearrangements upon activation, i.e., the formation of trimers of hairpins, which drive fusion between viral and cellular membranes (11, 17).Retrovirus Env is a typical type I transmembrane protein composed of surface (SU) and transmembrane (TM) subunits and belongs to the class I fusion proteins. SU is responsible for binding to cognate cellular receptors or cofactors, while TM directly mediates membrane fusion (6). Most retroviruses use a pH-independent pathway for entry, during which receptor binding relieves the ability of SU to restrain TM, resulting in conformational changes in TM and subsequent fusion with the cell membrane (11). Interestingly, increasing numbers of retroviruses have recently been shown to require a low pH (3, 15, 24, 28, 31) or pH-dependent protease activities to trigger fusion (18); the latter property has also been demonstrated for some other enveloped viruses (2, 14, 18, 26, 27, 33, 34). Among these, avian sarcoma leukosis virus (ASLV) is unique in that it uses a two-step mechanism for fusion, in which receptor binding primes the second trigger of low pH (24).Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovirus etiologically responsible for contagious lung tumors in sheep (12). The native Env protein of JSRV functions as a potent oncogene that induces cell transformation in vitro and in animals (4, 9, 21, 29, 42). The cell entry receptor for JSRV has been identified as hyaluronidase 2 (Hyal2), a glycosylphosphatidylinositol (GPI)-anchored protein belonging to the hyaluronidase family (29); Hyal2 itself has low hyaluronidase activity, and this activity is not associated with JSRV entry and infection (38). Intrigued by the oncogenic nature of JSRV Env, we recently examined the mechanism of JSRV entry and found that JSRV Env-mediated fusion and cell entry require a low pH (1, 8). These observations led us to hypothesize that the pH-dependent fusion activation of JSRV Env may be advantageous for its oncogenesis, given that extreme cell-cell fusion of the plasma membrane at a neutral pH would result in syncytium formation and often cell death. Curiously, we noticed that overexpression of Hyal2 is necessary for JSRV Env to induce membrane fusion at a low pH in vitro, suggesting that Hyal2 may play an active role in the pH-dependent fusion process. Here we provide direct evidence that Hyal2 functions in cooperation with a low pH to trigger the JSRV Env-mediated fusion activation yet exhibits some striking differences from the mechanism of ASLV fusion. The multistep pathway for JSRV Env-mediated fusion activation might be important for its replication fitness and oncogenesis.     

Borna Disease Virus Requires Cholesterol in both Cellular Membrane and Viral Envelope for Efficient Cell Entry

Borna disease virus (BDV), the prototypic member of the family Bornaviridae within the order Mononegavirales, provides an important model for the investigation of viral persistence within the central nervous system (CNS) and of associated brain disorders. BDV is highly neurotropic and enters its target cell via receptor-mediated endocytosis, a process mediated by the virus surface glycoprotein (G), but the cellular factors and pathways determining BDV cell tropism within the CNS remain mostly unknown. Cholesterol has been shown to influence viral infections via its effects on different viral processes, including replication, budding, and cell entry. In this work, we show that cell entry, but not replication and gene expression, of BDV was drastically inhibited by depletion of cellular cholesterol levels. BDV G-mediated attachment to BDV-susceptible cells was cholesterol independent, but G localized to lipid rafts (LR) at the plasma membrane. LR structure and function critically depend on cholesterol, and hence, compromised structural integrity and function of LR caused by cholesterol depletion likely inhibited the initial stages of BDV cell internalization. Furthermore, we also show that viral-envelope cholesterol is required for BDV infectivity.Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization (3′-N-p10/P-M-G-L-5′) is characteristic of mononegaviruses (6, 28, 46, 48). However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales (8, 28, 46, 49).BDV can infect a variety of cell types in cell culture but in vivo exhibits exquisite neurotropism and causes central nervous system (CNS) disease in different vertebrate species, which is frequently manifested in behavioral abnormalities (19, 33, 44, 53). Both host and viral factors contribute to a variable period of incubation and heterogeneity in the symptoms and pathology associated with BDV infection (14, 16, 29, 42, 44). BDV provides an important model for the investigation of both immune-mediated pathological events associated with virus-induced neurological disease and mechanisms whereby noncytolytic viruses induce neurodevelopmental and behavioral disturbances in the absence of inflammation (15, 18, 41). Moreover, serological data and molecular epidemiological studies suggest that BDV, or a BDV-like virus, can infect humans and that it might be associated with certain neuropsychiatric disorders (17, 24), which further underscores the interest in understanding the mechanisms underlying BDV persistence in the CNS and its effect on brain cell functions. The achievement of these goals will require the elucidation of the determinants of BDV cell tropism within the CNS.BDV enters its target cell via receptor-mediated endocytosis, a process in which the BDV G protein plays a central role (1, 5, 13, 14, 39). Cleavage of BDV G by the cellular protease furin generates two functional subunits: GP1 (GP_N), involved in virus interaction with a yet-unidentified cell surface receptor (1, 39), and GP2 (GP_C), which mediates a pH-dependent fusion event between viral and cellular membranes (13). However, a detailed characterization of cellular factors and pathways involved in BDV cell entry remains to be done.Besides cell surface molecules that serve as viral receptors, many other cell factors, including nonproteinaceous molecules, can influence cell entry by virus (52). In this regard, cholesterol, which plays a critical role in cellular homeostasis (55), has also been identified as a key factor required for productive infection by different viruses. Accordingly, cholesterol participates in a variety of processes in virus-infected cells, including fusion events between viral and cellular membranes (3), viral replication (23), and budding (35, 37), as well as maintenance of lipid rafts (LR) (12) as scaffold structures where the viral receptor and coreceptor associate (11, 26, 32, 36). LR are specialized microdomains within cellular membranes constituted principally of proteins, sphingolipids, and cholesterol. LR facilitate the close proximity and interaction of specific sets of proteins and contribute to different processes associated with virus multiplication (38). Cholesterol can also influence virus infection by contributing to the maintenance of the properties of the viral envelope required for virus particle infectivity (21, 54). Here, we show for the first time that cholesterol plays a critical role in BDV infection. Depletion of cellular cholesterol prior to, but not after, BDV cell entry prevented productive BDV infection, likely due to disruption of plasma membrane LR that appear to be the cell entry point for BDV. In addition, we document that cholesterol also plays an essential role in the properties of the BDV envelope required for virus particle infectivity.     

Crystallographic Definition of the Epitope Promiscuity of the Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2F5: Vaccine Design Implications

The quest to create a human immunodeficiency virus type 1 (HIV-1) vaccine capable of eliciting broadly neutralizing antibodies against Env has been challenging. Among other problems, one difficulty in creating a potent immunogen resides in the substantial overall sequence variability of the HIV envelope protein. The membrane-proximal region (MPER) of gp41 is a particularly conserved tryptophan-rich region spanning residues 659 to 683, which is recognized by three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, Z13, and 4E10. In this study, we first describe the variability of residues in the gp41 MPER and report on the invariant nature of 15 out of 25 amino acids comprising this region. Subsequently, we evaluate the ability of the bnMAb 2F5 to recognize 31 varying sequences of the gp41 MPER at a molecular level. In 19 cases, resulting crystal structures show the various MPER peptides bound to the 2F5 Fab′. A variety of amino acid substitutions outside the ⁶⁶⁴DKW⁶⁶⁶ core epitope are tolerated. However, changes at the ⁶⁶⁴DKW⁶⁶⁶ motif itself are restricted to those residues that preserve the aspartate''s negative charge, the hydrophobic alkyl-π stacking arrangement between the β-turn lysine and tryptophan, and the positive charge of the former. We also characterize a possible molecular mechanism of 2F5 escape by sequence variability at position 667, which is often observed in HIV-1 clade C isolates. Based on our results, we propose a somewhat more flexible molecular model of epitope recognition by bnMAb 2F5, which could guide future attempts at designing small-molecule MPER-like vaccines capable of eliciting 2F5-like antibodies.Eliciting broadly neutralizing antibodies (bnAbs) against primary isolates of human immunodeficiency virus type I (HIV-1) has been identified as a major milestone to attain in the quest for a vaccine in the fight against AIDS (12, 28). These antibodies would need to interact with HIV-1 envelope glycoproteins gp41 and/or gp120 (Env), target conserved regions and functional conformations of gp41/gp120 trimeric complexes, and prevent new HIV-1 fusion events with target cells (21, 57, 70, 71). Although a humoral response generating neutralizing antibodies against HIV-1 can be detected in HIV-1-positive individuals, the titers are often very low, and virus control is seldom achieved by these neutralizing antibodies (22, 51, 52, 66, 67). The difficulty in eliciting a broad and potent neutralizing antibody response against HIV-1 is thought to reside in the high degree of genetic diversity of the virus, in the heterogeneity of Env on the surface of HIV-1, and in the masking of functional regions by conformational covering, by an extensive glycan shield, or by the ability of some conserved domains to partition to the viral membrane (24, 25, 29, 30, 38, 39, 56, 68, 69). So far, vaccine trials using as immunogens mimics of Env in different conformations have primarily elicited antibodies with only limited neutralization potency across different HIV-1 clades although recent work has demonstrated more encouraging results (4, 12, 61).The use of conserved regions on gp41 and gp120 Env as targets for vaccine design has been mostly characterized by the very few anti-HIV-1 broadly neutralizing monoclonal antibodies (bnMAbs) that recognize them: the CD4 binding-site on gp120 (bnMAb b12), a CD4-induced gp120 coreceptor binding site (bnMAbs 17b and X5), a mannose cluster on the outer face of gp120 (bnMAb 2G12), and the membrane proximal external region (MPER) of gp41 (bnMAbs 2F5, Z13 and 4E10) (13, 29, 44, 58, 73). The gp41 MPER region is a particularly conserved part of Env that spans residues 659 to 683 (HXB2 numbering) (37, 75). Substitution and deletion studies have linked this unusually tryptophan-rich region to the fusion process of HIV-1, possibly involving a series of conformational changes (5, 37, 41, 49, 54, 74). Additionally, the gp41 MPER has been implicated in gp41 oligomerization, membrane leakage ability facilitating pore formation, and binding to the galactosyl ceramide receptor on epithelial cells for initial mucosal infection mediated by transcytosis (2, 3, 40, 53, 63, 64, 72). This wide array of roles for the gp41 MPER will put considerable pressure on sequence conservation, and any change will certainly lead to a high cost in viral fitness.Monoclonal antibody 2F5 is a broadly neutralizing monoclonal anti-HIV-1 antibody isolated from a panel of sera from naturally infected asymptomatic individuals. It reacts with a core gp41 MPER epitope spanning residues 662 to 668 with the linear sequence ELDKWAS (6, 11, 42, 62, 75). 2F5 immunoglobulin G binding studies and screening of phage display libraries demonstrated that the DKW core is essential for 2F5 recognition and binding (15, 36, 50). Crystal structures of 2F5 with peptides representing its core gp41 epitope reveal a β-turn conformation involving the central DKW residues, flanked by an extended conformation and a canonical α-helical turn for residues located at the N terminus and C terminus of the core, respectively (9, 27, 45, 47). In addition to binding to its primary epitope, evidence is accumulating that 2F5 also undergoes secondary interactions: multiple reports have demonstrated affinity of 2F5 for membrane components, possibly through its partly hydrophobic flexible elongated complementarity-determining region (CDR) H3 loop, and it has also been suggested that 2F5 might interact in a secondary manner with other regions of gp41 (1, 10, 23, 32, 33, 55). Altogether, even though the characteristics of 2F5 interaction with its linear MPER consensus epitope have been described extensively, a number of questions persist about the exact mechanism of 2F5 neutralization at a molecular level.One such ambiguous area of the neutralization mechanism of 2F5 is investigated in this study. Indeed, compared to bnMAb 4E10, 2F5 is the more potent neutralizing antibody although its breadth across different HIV-1 isolates is more limited (6, 35). In an attempt to shed light on the exact molecular requirements for 2F5 recognition of its primary gp41 MPER epitope, we performed structural studies of 2F5 Fab′ with a variety of peptides. The remarkable breadth of possible 2F5 interactions reveals a somewhat surprising promiscuity of the 2F5 binding site. Furthermore, we link our structural observations with the natural variation observed within the gp41 MPER and discuss possible routes of 2F5 escape from a molecular standpoint. Finally, our discovery of 2F5''s ability to tolerate a rather broad spectrum of amino acids in its binding, a spectrum that even includes nonnatural amino acids, opens the door to new ways to design small-molecule immunogens potentially capable of eliciting 2F5-like neutralizing antibodies.     

Characterization of Lassa Virus Glycoprotein Oligomerization and Influence of Cholesterol on Virus Replication

Mature glycoprotein spikes are inserted in the Lassa virus envelope and consist of the distal subunit GP-1, the transmembrane-spanning subunit GP-2, and the signal peptide, which originate from the precursor glycoprotein pre-GP-C by proteolytic processing. In this study, we analyzed the oligomeric structure of the viral surface glycoprotein. Chemical cross-linking studies of mature glycoprotein spikes from purified virus revealed the formation of trimers. Interestingly, sucrose density gradient analysis of cellularly expressed glycoprotein showed that in contrast to trimeric mature glycoprotein complexes, the noncleaved glycoprotein forms monomers and oligomers spanning a wide size range, indicating that maturation cleavage of GP by the cellular subtilase SKI-1/S1P is critical for formation of the correct oligomeric state. To shed light on a potential relation between cholesterol and GP trimer stability, we performed cholesterol depletion experiments. Although depletion of cholesterol had no effect on trimerization of the glycoprotein spike complex, our studies revealed that the cholesterol content of the viral envelope is important for the infectivity of Lassa virus. Analyses of the distribution of viral proteins in cholesterol-rich detergent-resistant membrane areas showed that Lassa virus buds from membrane areas other than those responsible for impaired infectivity due to cholesterol depletion of lipid rafts. Thus, derivation of the viral envelope from cholesterol-rich membrane areas is not a prerequisite for the impact of cholesterol on virus infectivity.Lassa virus (LASV) is a member of the family Arenaviridae, of which Lymphocytic choriomeningitis virus (LCMV) is the prototype. Arenaviruses comprise more than 20 species, divided into the Old World and New World virus complexes (19). The Old World arenaviruses include the human pathogenic LASV strains, Lujo virus, which was first identified in late 2008 and is associated with an unprecedented high case fatality rate in humans, the nonhuman pathogenic Ippy, Mobala, and Mopeia viruses, and the recently described Kodoko virus (10, 30, 49). The New World virus complex contains, among others, the South American hemorrhagic fever-causing viruses Junín virus, Machupo virus, Guanarito virus, Sabiá virus, and the recently discovered Chapare virus (22).Arenaviruses contain a bisegmented single-stranded RNA genome encoding the polymerase L, matrix protein Z, nucleoprotein NP, and glycoprotein GP. The bipartite ribonucleoprotein of LASV is surrounded by a lipid envelope derived from the plasma membrane of the host cell. The matrix protein Z has been identified as a major budding factor, which lines the interior of the viral lipid membrane, in which GP spikes are inserted (61, 75). The glycoprotein is synthesized as precursor protein pre-GP-C and is cotranslationally cleaved by signal peptidase into GP-C and the signal peptide, which exhibits unusual length, stability, and topology (3, 27, 28, 33, 70, 87). Moreover, the arenaviral signal peptide functions as trans-acting maturation factor (2, 26, 33). After processing by signal peptidase, GP-C of both New World and Old World arenaviruses is cleaved by the cellular subtilase subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P) into the distal subunit GP-1 and the membrane-anchored subunit GP-2 within the secretory pathway (5, 52, 63). For LCMV, it has been shown that GP-1 subunits are linked to each other by disulfide bonds and are noncovalently connected to GP-2 subunits (14, 24, 31). GP-1 is responsible for binding to the host cell receptor, while GP-2 mediates fusion between the virus envelope and the endosomal membrane at low pH due to a bipartite fusion peptide near the amino terminus (24, 36, 44). Sequence analysis of the LCMV GP-2 ectodomain revealed two heptad repeats that most likely form amphipathic helices important for this process (34, 86).In general, viral class I fusion proteins have triplets of α-helical structures in common, which contain heptad repeats (47, 73). In contrast, class II fusion proteins are characterized by β-sheets that form dimers in the prefusion status and trimers in the postfusion status (43). The class III fusion proteins are trimers that, unlike class I fusion proteins, were not proteolytically processed N-terminally of the fusion peptide, resulting in a fusion-active membrane-anchored subunit (39, 62). Previous studies with LCMV described a tetrameric organization of the glycoprotein spikes (14), while more recent data using a bacterially expressed truncated ectodomain of the LCMV GP-2 subunit pointed toward a trimeric spike structure (31). Due to these conflicting data regarding the oligomerization status of LCMV GP, it remains unclear to which class of fusion proteins the arenaviral glycoproteins belong.The state of oligomerization and the correct conformation of viral glycoproteins are crucial for membrane fusion during virus entry. The early steps of infection have been shown for several viruses to be dependent on the cholesterol content of the participating membranes (i.e., either the virus envelope or the host cell membrane) (4, 9, 15, 20, 21, 23, 40, 42, 53, 56, 76, 78, 79). In fact, it has been shown previously that entry of both LASV and LCMV is susceptible to cholesterol depletion of the target host cell membrane using methyl-β-cyclodextrin (MβCD) treatment (64, 71). Moreover, cholesterol not only plays an important role in the early steps during entry in the viral life cycle but also is critical in the virus assembly and release process. Several viruses of various families, including influenza virus, human immunodeficiency virus type 1 (HIV-1), measles virus, and Ebola virus, use the ordered environment of lipid raft microdomains. Due to their high levels of glycosphingolipids and cholesterol, these domains are characterized by insolubility in nonionic detergents under cold conditions (60, 72). Recent observations have suggested that budding of the New World arenavirus Junin virus occurs from detergent-soluble membrane areas (1). Assembly and release from distinct membrane microdomains that are detergent soluble have also been described for vesicular stomatitis virus (VSV) (12, 38, 68). At present, however, it is not known whether LASV requires cholesterol in its viral envelope for successful virus entry or whether specific membrane microdomains are important for LASV assembly and release.In this study, we first investigated the oligomeric state of the premature and mature LASV glycoprotein complexes. Since it has been shown for several membrane proteins that the oligomerization and conformation are dependent on cholesterol (58, 59, 76, 78), we further analyzed the dependence of the cholesterol content of the virus envelope on glycoprotein oligomerization and virus infectivity. Finally, we characterized the lipid membrane areas from which LASV is released.     

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

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