Cell Type-Dependent Escape of Capsid Inhibitors by Simian Immunodeficiency Virus SIVcpz.

Pandemic human immunodeficiency virus type 1 (HIV-1) is the result of the zoonotic transmission of simian immunodeficiency virus (SIV) from the chimpanzee subspecies Pan troglodytestroglodytes (SIVcpzPtt). The related subspecies Pan troglodytesschweinfurthii is the host of a similar virus, SIVcpzPts, which did not spread to humans. We tested these viruses with small-molecule capsid inhibitors (PF57, PF74, and GS-CA1) that interact with a binding groove in the capsid that is also used by CPSF6. While HIV-1 was sensitive to capsid inhibitors in cell lines, human macrophages, and peripheral blood mononuclear cells (PBMCs), SIVcpzPtt was resistant in rhesus FRhL-2 cells and human PBMCs but was sensitive to PF74 in human HOS and HeLa cells. SIVcpzPts was insensitive to PF74 in FRhL-2 cells, HeLa cells, PBMCs, and macrophages but was inhibited by PF74 in HOS cells. A truncated version of CPSF6 (CPSF6-358) inhibited SIVcpzPtt and HIV-1, while in contrast, SIVcpzPts was resistant to CPSF6-358. Homology modeling of HIV-1, SIVcpzPtt, and SIVcpzPts capsids and binding energy estimates suggest that these three viruses bind similarly to the host proteins cyclophilin A (CYPA) and CPSF6 as well as the capsid inhibitor PF74. Cyclosporine treatment, mutation of the CYPA-binding loop in the capsid, or CYPA knockout eliminated the resistance of SIVcpzPts to PF74 in HeLa cells. These experiments revealed that the antiviral capacity of PF74 is controlled by CYPA in a virus- and cell type-specific manner. Our data indicate that SIVcpz viruses can use infection pathways that escape the antiviral activity of PF74. We further suggest that the antiviral activity of PF74 capsid inhibitors depends on cellular cofactors.IMPORTANCE HIV-1 originated from SIVcpzPtt but not from the related virus SIVcpzPts, and thus, it is important to describe molecular infection by SIVcpzPts in human cells to understand the zoonosis of SIVs. Pharmacological HIV-1 capsid inhibitors (e.g., PF74) bind a capsid groove that is also a binding site for the cellular protein CPSF6. SIVcpzPts was resistant to PF74 in HeLa cells but sensitive in HOS cells, thus indicating cell line-specific resistance. Both SIVcpz viruses showed resistance to PF74 in human PBMCs. Modulating the presence of cyclophilin A or its binding to capsid in HeLa cells overcame SIVcpzPts resistance to PF74. These results indicate that early cytoplasmic infection events of SIVcpzPts may differ between cell types and affect, in an unknown manner, the antiviral activity of capsid inhibitors. Thus, capsid inhibitors depend on the activity or interaction of currently uncharacterized cellular factors.

Segmentation by classification: A novel and reliable approach for semi-automatic selection of HIV/SIV envelope spikes.

We describe a semiautomated approach to segment Env spikes from the membrane envelope of Simian Immunodeficiency Virus visualized by cryoelectron tomography of frozen-hydrated specimens. Multivariate data analysis is applied to a large set of overlapping subvolumes extracted semiautomatically from the viral envelope and does not utilize a template of the target structure. The major manual step used in the method involves determination of six points that define an ellipsoid approximating the virion shape. The approach is robust to departures of the actual virion from this starting ellipsoid. A point cage of sufficient density is generated to ensure that any spike-like protein is identified multiple times. Subsequently translational alignment of class averages to a cylindrical reference on a curved surface separates subvolumes with spikes from those without. Spike containing subvolumes identified multiple times are removed by proximity analysis. Slightly different procedures segment spikes in the equatorial and the polar regions. Once all spikes are segmented, further alignment of class averages using separately the polar and spin angles produces recognizable spike images. Our approach localized 96% of the equatorial spikes and 85% of all spikes identified manually; it identifies a significant number of additional spikes missed by manual selection. Two types of spike shapes were segmented, one with near 3-fold symmetry resembling the conventional spike, the other had a T-shape resembling the spike structure obtained when antibodies such as PG9 bind to HIV Env. The approach should be applicable to segmentation of any protein spikes extending from a cellular or virion envelope.

Immunological and virological mechanisms of vaccine-mediated protection against SIV and HIV.

A major challenge for the development of a highly effective AIDS vaccine is the identification of mechanisms of protective immunity. To address this question, we used a nonhuman primate challenge model with simian immunodeficiency virus (SIV). We show that antibodies to the SIV envelope are necessary and sufficient to prevent infection. Moreover, sequencing of viruses from breakthrough infections revealed selective pressure against neutralization-sensitive viruses; we identified a two-amino-acid signature that alters antigenicity and confers neutralization resistance. A similar signature confers resistance of human immunodeficiency virus (HIV)-1 to neutralization by monoclonal antibodies against variable regions 1 and 2 (V1V2), suggesting that SIV and HIV share a fundamental mechanism of immune escape from vaccine-elicited or naturally elicited antibodies. These analyses provide insight into the limited efficacy seen in HIV vaccine trials.

Structural basis of lentiviral subversion of a cellular protein degradation pathway.

Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4(+) T cells by reducing the cellular deoxynucleoside 5'-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell's ubiquitination machinery to destroy the cell's protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell's normal protein degradation pathway to inactivate the cellular viral defence system.

A Helical Short-Peptide Fusion Inhibitor with Highly Potent Activity against Human Immunodeficiency Virus Type 1 (HIV-1), HIV-2, and Simian Immunodeficiency Virus.

Human immunodeficiency virus type 2 (HIV-2) has already spread to different regions worldwide, and currently about 1 to 2 million people have been infected, calling for new antiviral agents that are effective on both HIV-1 and HIV-2 isolates. T20 (enfuvirtide), a 36-mer peptide derived from the C-terminal heptad repeat region (CHR) of gp41, is the only clinically approved HIV-1 fusion inhibitor, but it easily induces drug resistance and is not active on HIV-2. In this study, we first demonstrated that the M-T hook structure was also vital to enhancing the binding stability and inhibitory activity of diverse CHR-based peptide inhibitors. We then designed a novel short peptide (23-mer), termed 2P23, by introducing the M-T hook structure, HIV-2 sequences, and salt bridge-forming residues. Promisingly, 2P23 was a highly stable helical peptide with high binding to the surrogate targets derived from HIV-1, HIV-2, and simian immunodeficiency virus (SIV). Consistent with this, 2P23 exhibited potent activity in inhibiting diverse subtypes of HIV-1 isolates, T20-resistant HIV-1 mutants, and a panel of primary HIV-2 isolates, HIV-2 mutants, and SIV isolates. Therefore, we conclude that 2P23 has high potential to be further developed for clinical use, and it is also an ideal tool for exploring the mechanisms of HIV-1/2- and SIV-mediated membrane fusion. IMPORTANCE: The peptide drug T20 is the only approved HIV-1 fusion inhibitor, but it is not active on HIV-2 isolates, which have currently infected 1 to 2 million people and continue to spread worldwide. Recent studies have demonstrated that the M-T hook structure can greatly enhance the binding and antiviral activities of gp41 CHR-derived inhibitors, especially for short peptides that are otherwise inactive. By combining the hook structure, HIV-2 sequence, and salt bridge-based strategies, the short peptide 2P23 has been successfully designed. 2P23 exhibits prominent advantages over many other peptide fusion inhibitors, including its potent and broad activity on HIV-1, HIV-2, and even SIV isolates, its stability as a helical, oligomeric peptide, and its high binding to diverse targets. The small size of 2P23 would benefit its synthesis and significantly reduce production cost. Therefore, 2P23 is an ideal candidate for further development, and it also provides a novel tool for studying HIV-1/2- and SIV-mediated cell fusion.

DNA Prime-Boost Vaccine Regimen To Increase Breadth, Magnitude, and Cytotoxicity of the Cellular Immune Responses to Subdominant Gag Epitopes of Simian Immunodeficiency Virus and HIV.

HIV sequence diversity and the propensity of eliciting immunodominant responses targeting variable regions of the HIV proteome are hurdles in the development of an effective AIDS vaccine. An HIV-derived conserved element (CE) p24gag plasmid DNA (pDNA) vaccine is able to redirect immunodominant responses to otherwise subdominant and often more vulnerable viral targets. By homology to the HIV immunogen, seven CE were identified in SIV p27Gag Analysis of 31 rhesus macaques vaccinated with full-length SIV gag pDNA showed inefficient induction (58% response rate) of cellular responses targeting these CE. In contrast, all 14 macaques immunized with SIV p27CE pDNA developed robust T cell responses recognizing CE. Vaccination with p27CE pDNA was also critical for the efficient induction and increased the frequency of Ag-specific T cells with cytotoxic potential (granzyme B+ CD107a+) targeting subdominant CE epitopes, compared with the responses elicited by the p57gag pDNA vaccine. Following p27CE pDNA priming, two booster regimens, gag pDNA or codelivery of p27CE+gag pDNA, significantly increased the levels of CE-specific T cells. However, the CE+gag pDNA booster vaccination elicited significantly broader CE epitope recognition, and thus, a more profound alteration of the immunodominance hierarchy. Vaccination with HIV molecules showed that CE+gag pDNA booster regimen further expanded the breadth of HIV CE responses. Hence, SIV/HIV vaccine regimens comprising CE pDNA prime and CE+gag pDNA booster vaccination significantly increased cytotoxic T cell responses to subdominant highly conserved Gag epitopes and maximized response breadth.

Rationally Designed Vaccines Targeting the V2 Region of HIV-1 gp120 Induce a Focused, Cross-Clade-Reactive, Biologically Functional Antibody Response.

Strong antibody (Ab) responses against V1V2 epitopes of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (Env) correlated with reduced infection rates in studies of HIV, simian-human immunodeficiency virus (SHIV), and simian immunodeficiency virus (SIV). In order to focus the Ab response on V1V2, we used six V1V2 sequences and nine scaffold proteins to construct immunogens which were tested using various immunization regimens for their ability to induce cross-reactive and biologically active V2 Abs in rabbits. A prime/boost immunization strategy was employed using gp120 DNA and various V1V2-scaffold proteins. The rabbit polyclonal Ab responses (i) were successfully focused on the V1V2 region, with weak or only transient responses to other Env epitopes, (ii) displayed broad cross-reactive binding activity with gp120s and the V1V2 regions of diverse strains from clades B, C, and E, (iii) included V2 Abs with specificities similar to those found in HIV-infected individuals, and (iv) remained detectable ≥1 year after the last boosting dose. Importantly, sera from rabbits receiving V1V2-scaffold immunogens displayed Ab-dependent cellular phagocytosis whereas sera from rabbits receiving only gp120 did not. The results represent the first fully successful example of reverse vaccinology in the HIV vaccine field with rationally designed epitope scaffold immunogens inducing Abs that recapitulate the epitope specificity and biologic activity of the human monoclonal Abs from which the immunogens were designed. Moreover, this is the first immunogenicity study using epitope-targeting, rationally designed vaccine constructs that induced an Fc-mediated activity associated with protection from infection with HIV, SIV, and SHIV. IMPORTANCE: Novel immunogens were designed to focus the antibody response of rabbits on the V1V2 epitopes of HIV-1 gp120 since such antibodies were associated with reduced infection rates of HIV, SIV, and SHIV. The vaccine-induced antibodies were broadly cross-reactive with the V1V2 regions of HIV subtypes B, C and E and, importantly, facilitated Fc-mediated phagocytosis, an activity not induced upon immunization of rabbits with gp120. This is the first immunogenicity study of vaccine constructs that focuses the antibody response on V1V2 and induces V2-specific antibodies with the ability to mediate phagocytosis, an activity that has been associated with protection from infection with HIV, SIV, and SHIV.

Mechanistic differences between HIV-1 and SIV nucleocapsid proteins and cross-species HIV-1 genomic RNA recognition.

BACKGROUND: The nucleocapsid (NC) domain of HIV-1 Gag is responsible for specific recognition and packaging of genomic RNA (gRNA) into new viral particles. This occurs through specific interactions between the Gag NC domain and the Psi packaging signal in gRNA. In addition to this critical function, NC proteins are also nucleic acid (NA) chaperone proteins that facilitate NA rearrangements during reverse transcription. Although the interaction with Psi and chaperone activity of HIV-1 NC have been well characterized in vitro, little is known about simian immunodeficiency virus (SIV) NC. Non-human primates are frequently used as a platform to study retroviral infection in vivo; thus, it is important to understand underlying mechanistic differences between HIV-1 and SIV NC. RESULTS: Here, we characterize SIV NC chaperone activity for the first time. Only modest differences are observed in the ability of SIV NC to facilitate reactions that mimic the minus-strand annealing and transfer steps of reverse transcription relative to HIV-1 NC, with the latter displaying slightly higher strand transfer and annealing rates. Quantitative single molecule DNA stretching studies and dynamic light scattering experiments reveal that these differences are due to significantly increased DNA compaction energy and higher aggregation capability of HIV-1 NC relative to the SIV protein. Using salt-titration binding assays, we find that both proteins are strikingly similar in their ability to specifically interact with HIV-1 Psi RNA. In contrast, they do not demonstrate specific binding to an RNA derived from the putative SIV packaging signal. CONCLUSIONS: Based on these studies, we conclude that (1) HIV-1 NC is a slightly more efficient NA chaperone protein than SIV NC, (2) mechanistic differences between the NA interactions of highly similar retroviral NC proteins are revealed by quantitative single molecule DNA stretching, and (3) SIV NC demonstrates cross-species recognition of the HIV-1 Psi RNA packaging signal.

Accumulation of Cytotoxic CD16+ NK Cells in Simian Immunodeficiency Virus-Infected Lymph Nodes Associated with In Situ Differentiation and Functional Anergy.

UNLABELLED: Recent evidence suggests that even in treated infections, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication may continue in lymph nodes (LN), serving as a potential virus reservoir. Here we investigated the effects of lentivirus infection on natural killer (NK) cell frequencies, phenotypes, and functions in naive and acutely or chronically SIVmac239-infected rhesus macaques. Compared to that in naive animals, we observed a 3-fold-greater frequency of cytotoxic CD16(+) CD56(-) NK cells in LN of chronically infected macaques. However, NK cells did not appear to be trafficking to LN, as homing markers CD62L and CCR7 did not increase on circulating NK cells during infection. LN NK cells demonstrated enhanced cytotoxicity in acute infection, with 2-fold increases in perforin expression and 3-fold increases in CD107a expression following mitogen stimulation. Lysis of K562 cells by LN NK cells from acutely infected animals was greater than lysis by preinfection samples from the same animals. LN NK cells from chronically infected animals lysed K562 cells more efficiently than LN NK cells from uninfected animals, but importantly, surrogate markers of cytotoxicity in infected macaques were disproportionately greater than ex vivo killing. Furthermore, Tim-3, an indicator of activation and/or exhaustion, was upregulated 3-fold on LN NK cells in chronically infected animals. Collectively, these data suggest that LN NK cells are skewed toward a cytotoxic phenotype during SIV infection but may become dysfunctional and exhausted in chronic disease. IMPORTANCE: The accumulation of CD16(+) CD56(-) NK cells in the SIV-infected lymph node without changes in NK homing to the LN could suggest that these cells are differentiating in situ. Surprisingly, this increase in frequency of the cytotoxic subset of NK cells is not accompanied by an increase of similar magnitude in the cytolytic function of LN lymphocytes. This functional modulation, together with the higher Tim-3 expression observed on LN NK cells isolated from chronically infected animals than on those from naive macaques, is indicative of an exhausted phenotype. This exhaustion could contribute to the robust replication of HIV and SIV in the LN during acute and chronic stages of infection, allowing the survival of infected cells and maintenance of a viral reservoir.

Contribution of glutamine residues in the helix 4-5 loop to capsid-capsid interactions in simian immunodeficiency virus of macaques.

UNLABELLED: Following retrovirus entry, the viral capsid (CA) disassembles into its component capsid proteins. The rate of this uncoating process, which is regulated by CA-CA interactions and by the association of the capsid with host cell factors like cyclophilin A (CypA), can influence the efficiency of reverse transcription. Inspection of the CA sequences of lentiviruses reveals that several species of simian immunodeficiency viruses (SIVs) have lost the glycine-proline motif in the helix 4-5 loop important for CypA binding; instead, the helix 4-5 loop in these SIVs exhibits an increase in the number of glutamine residues. In this study, we investigated the role of these glutamine residues in SIVmac239 replication. Changes in these residues, particularly glutamine 89 and glutamine 92, resulted in a decreased efficiency of core condensation, decreased stability of the capsids in infected cells, and blocks to reverse transcription. In some cases, coexpression of two different CA mutants produced chimeric virions that exhibited higher infectivity than either parental mutant virus. For this complementation of infectivity, glutamine 89 was apparently required on one of the complementing pair of mutants and glutamine 92 on the other. Modeling suggests that glutamines 89 and 92 are located on the distal face of hexameric capsid spokes and thus are well positioned to contribute to interhexamer interactions. Requirements to evade host restriction factors like TRIMCyp may drive some SIV lineages to evolve means other than CypA binding to stabilize the capsid. One solution used by several SIV strains consists of glutamine-based bonding. IMPORTANCE: The retroviral capsid is an assembly of individual capsid proteins that surrounds the viral RNA. After a retrovirus enters a cell, the capsid must disassemble, or uncoat, at a proper rate. The interactions among capsid proteins contribute to this rate of uncoating. We found that some simian immunodeficiency viruses use arrays of glutamine residues, which can form hydrogen bonds efficiently, to keep their capsids stable. This strategy may allow these viruses to forego the use of capsid-stabilizing factors from the host cell, some of which have antiviral activity.

Evolutionarily conserved requirement for core binding factor beta in the assembly of the human immunodeficiency virus/simian immunodeficiency virus Vif-cullin 5-RING E3 ubiquitin ligase.

UNLABELLED: The human immunodeficiency virus type 1 (HIV-1)-encoded virion infectivity factor (Vif) is required to inactivate the host restriction factor APOBEC3 by engaging Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-ß) is a novel regulator of Vif-CRL5 function; as yet, its mechanism of regulation remains unclear. In the present study, we demonstrate that CBF-ß promotion of Vif-CRL5 assembly is independent of its influence on Vif stability and is also a conserved feature of primate lentiviral Vif proteins. Furthermore, CBF-ß is critical for the formation of the Vif-ElonginB/ElonginC-Cul5 core E3 ubiquitin ligase complex in vitro. CBF-ß from diverse vertebrate species supported HIV-1 Vif function, indicating the conserved nature of Vif-CBF-ß interfaces. Considering the importance of the interaction between Vif and CBF-ß in viral CRL5 function, disrupting this interaction represents an attractive pharmacological intervention against HIV-1. IMPORTANCE: HIV-1 encodes virion infectivity factor (Vif) to inactivate its host's antiviral APOBEC3 proteins. Vif triggers APOBEC3 degradation by forming Vif-Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-ß) is a novel regulator of Vif-CRL5 function whose mechanism of regulation remains poorly defined. In the present study, we demonstrate that the promotion of Vif-CRL5 assembly by CBF-ß can be separated from its influence on Vif stability. The promotion of Vif-CRL5 assembly, but not the influence on Vif stability, is conserved among primate lentiviral Vif proteins: we found that CBF-ß from diverse vertebrate species supported HIV-1 Vif function. Considering the importance of the interaction between Vif and CBF-ß in viral CRL5 function and HIV-1 replication, disrupting this interaction is an attractive strategy against HIV-1.

A single amino acid mutation in the envelope cytoplasmic tail restores the ability of an attenuated simian immunodeficiency virus mutant to deplete mucosal CD4+ T cells.

Disruption of the conserved motif GYxxØ in the simian immunodeficiency virus (SIV) SIVmac239 envelope (Env) cytoplasmic tail resulted in a virus (ΔGY) that exhibited a high plasma peak but uniquely failed to acutely deplete mucosal CD4(+) T cells. Here, we show that ΔGY containing a flanking S727P mutation that was acquired in ΔGY-infected macaques reacquired the ability to rapidly deplete CD4(+) T cells in lamina propria. This suggests that the GYxxØ motif and S727P each contribute to SIV's targeting to mucosal tissues.

Evidence against extracellular exposure of a highly immunogenic region in the C-terminal domain of the simian immunodeficiency virus gp41 transmembrane protein.

The generally accepted model for human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein topology includes a single membrane-spanning domain. An alternate model has been proposed which features multiple membrane-spanning domains. Consistent with the alternate model, a high percentage of HIV-1-infected individuals produce unusually robust antibody responses to a region of envelope, the so-called "Kennedy epitope," that in the conventional model should be in the cytoplasm. Here we show analogous, robust antibody responses in simian immunodeficiency virus SIVmac239-infected rhesus macaques to a region of SIVmac239 envelope located in the C-terminal domain, which in the conventional model should be inside the cell. Sera from SIV-infected rhesus macaques consistently reacted with overlapping oligopeptides corresponding to a region located within the cytoplasmic domain of gp41 by the generally accepted model, at intensities comparable to those observed for immunodominant areas of the surface component gp120. Rabbit serum raised against this highly immunogenic region (HIR) reacted with SIV envelope in cell surface-staining experiments, as did monoclonal anti-HIR antibodies isolated from an SIVmac239-infected rhesus macaque. However, control experiments demonstrated that this surface staining could be explained in whole or in part by the release of envelope protein from expressing cells into the supernatant and the subsequent attachment to the surfaces of cells in the culture. Serum and monoclonal antibodies directed against the HIR failed to neutralize even the highly neutralization-sensitive strain SIVmac316. Furthermore, a potential N-linked glycosylation site located close to the HIR and postulated to be outside the cell in the alternate model was not glycosylated. An artificially introduced glycosylation site within the HIR was also not utilized for glycosylation. Together, these data support the conventional model of SIV envelope as a type Ia transmembrane protein with a single membrane-spanning domain and without any extracellular loops.

Vif proteins of human and simian immunodeficiency viruses require cellular CBFß to degrade APOBEC3 restriction factors.

HIV-1 requires the cellular transcription factor CBFß to stabilize its accessory protein Vif and promote APOBEC3G degradation. Here, we demonstrate that both isoforms of CBFß allow for increased steady-state levels of Vif, enhanced APOBEC3G degradation, and increased viral infectivity. This conserved functional interaction enhances the steady-state levels of Vif proteins from multiple HIV-1 subtypes and is required for the degradation of all human and rhesus Vif-sensitive APOBEC3 proteins by their respective lentiviral Vif proteins.

Lineage-specific differences between human and simian immunodeficiency virus regulation of gp120 trimer association and CD4 binding.

Metastable conformations of the gp120 and gp41 envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) must be maintained in the unliganded state of the envelope glycoprotein trimer. Binding of gp120 to the primary receptor, CD4, triggers the transition to an open conformation of the trimer, promoting interaction with the CCR5 chemokine receptor and ultimately leading to gp41-mediated virus-cell membrane fusion and entry. Topological layers in the gp120 inner domain contribute to gp120-trimer association in the unliganded state and to CD4 binding. Here we describe similarities and differences between HIV-1 and SIVmac gp120. In both viruses, the gp120 N/C termini and the inner domain ß-sandwich and layer 2 support the noncovalent association of gp120 with the envelope glycoprotein trimer. Layer 1 of the SIVmac gp120 inner domain contributes more to trimer association than the corresponding region of HIV-1 gp120. On the other hand, layer 1 plays an important role in stabilizing the CD4-bound conformation of HIV-1 but not SIVmac gp120 and thus contributes to HIV-1 binding to CD4. In SIVmac, CD4 binding is instead enhanced by tryptophan 375, which fills the Phe 43 cavity of gp120. Activation of SIVmac by soluble CD4 is dependent on tryptophan 375 and on layer 1 residues that determine a tight association of gp120 with the trimer. Distinct biological requirements for CD4 usage have resulted in lineage-specific differences in the HIV-1 and SIV gp120 structures that modulate trimer association and CD4 binding.

Two sorting motifs, a ubiquitination motif and a tyrosine motif, are involved in HIV-1 and simian immunodeficiency virus Nef-mediated receptor endocytosis.

HIV-1 and SIV Nef proteins downregulate cell surface CD4 and MHC class I (MHC-I) molecules of infected cells, which are necessary for efficient viral replication and pathogenicity. We previously reported that K144 in HIV-1 Nef is di-ubiquitinated, and K144R substitution impairs Nef-mediated CD4 downregulation. In this report, we extend the role of ubiquitination at this lysine residue from Nef-mediated CD4 downregulation to Nef-mediated MHC-I downregulation and from HIV Nef to SIV Nef. All HIV-1 Nef mutants that contain K144R substitution are inactive in MHC-I downregulation. Tested MHC-I alleles include HLA-ABC endogenously expressed and HLA-A2 exogenously expressed in Jurkat T cells. CD4 downregulation by SIV Nef involves K176 that aligns with K144 in HIV-1 Nef, as well as an N-terminal tyrosine motif Y28Y39 not present in HIV-1 Nef. Dual mutation at K176 and Y28Y39 completely impaired SIV Nef-mediated CD4 and MHC-I downregulation, whereas a single mutation at K176 or Y28Y39 did not. The involvement of tyrosine motif in SIV Nef-mediated CD4 and MHC-I downregulation prompted us to investigate a putative tyrosine motif (Y202Y/F203) in HIV-1 Nef that is conserved among HIV-1 species. Single mutation at the tyrosine motif Y202F203 in HIV-1 Nef (NA7) greatly impaired Nef-mediated CD4 downregulation, which is similar to what we observed previously with the single mutation at lysine K144. Thus, our study demonstrated that Nef-mediated receptor endocytosis involves the ubiquitination motif and tyrosine motif.

Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens.

The envelope spike of HIV is one of the most highly N-glycosylated structures found in nature. However, despite extensive research revealing essential functional roles in infection and immune evasion, the chemical structures of the glycans on the native viral envelope glycoprotein gp120--as opposed to recombinantly generated gp120--have not been described. Here, we report on the identity of the N-linked glycans from primary isolates of HIV-1 (clades A, B, and C) and from the simian immunodeficiency virus. MS analysis reveals a remarkably simple and highly conserved virus-specific glycan profile almost entirely devoid of medial Golgi-mediated processing. In stark contrast to recombinant gp120, which shows extensive exposure to cellular glycosylation enzymes (>70% complex type glycans), the native envelope shows barely detectable processing beyond the biosynthetic intermediate Man5GlcNAc2 (<2% complex type glycans). This oligomannose (Man5-9GlcNAc2) profile is conserved across primary isolates and geographically divergent clades but is not reflected in the current generation of gp120 antigens used for vaccine trials. In the context of vaccine design, we also note that Manalpha1-->2Man-terminating glycans (Man6-9GlcNAc2) of the type recognized by the broadly neutralizing anti-HIV antibody 2G12 are 3-fold more abundant on the native envelope than on the recombinant monomer and are also found on isolates not neutralized by 2G12. The Manalpha1-->2Man residues of gp120 therefore provide a vaccine target that is physically larger and antigenically more conserved than the 2G12 epitope itself. This study revises and extends our understanding of the glycan shield of HIV with implications for AIDS vaccine design.

Simian immunodeficiency virus from the sooty mangabey and rhesus macaque is modified with O-linked carbohydrate.

Although stretches of serine and threonine are sometimes sites for O-linked carbohydrate attachment, specific sequence and structural determinants for O-linked attachment remain ill defined. The gp120 envelope protein of SIVmac239 contains a serine-threonine-rich stretch of amino acids at positions 128 to 139. Here we show that lectin protein from jackfruit seed (jacalin), which binds to non- and monosialylated core 1 O-linked carbohydrate, potently inhibited the replication of SIVmac239. Selection of a jacalin-resistant SIVmac239 variant population resulted in virus with specific substitutions within amino acids 128 to 139. Cloned simian immunodeficiency virus (SIV) variants with substitutions in the 128-to-139 region had infectivities equivalent to, or within 1 log unit of, that of SIVmac239 and were resistant to the inhibitory effects of jacalin. Characterization of the SIVmac239 gp120 O-linked glycome showed the presence of core 1 and core 2 O-linked carbohydrate; a 128-to-139-substituted variant gp120 from jacalin-resistant SIV lacked O-linked carbohydrate. Unlike that of SIVmac239, the replication of HIV-1 strain NL4-3 was resistant to inhibition by jacalin. Purified gp120s from four SIVmac and SIVsm strains bound jacalin strongly in an enzyme-linked immunosorbent assay, while nine different HIV-1 gp120s, two SIVcpz gp120s, and 128-to-139-substituted SIVmac239 gp120 did not bind jacalin. The ability or inability to bind jacalin thus correlated with the presence of the serine-threonine-rich stretch in the SIVmac and SIVsm gp120s and the absence of such stretches in the SIVcpz and HIV-1 gp120s. Consistent with sequence predictions, two HIV-2 gp120s bound jacalin, while one did not. These data demonstrate the presence of non- and monosialylated core 1 O-linked carbohydrate on the gp120s of SIVmac and SIVsm and the lack of these modifications on HIV-1 and SIVcpz gp120s.

Molecular architectures of trimeric SIV and HIV-1 envelope glycoproteins on intact viruses: strain-dependent variation in quaternary structure.

The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ~20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively "open" conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses.

Distribution and three-dimensional structure of AIDS virus envelope spikes.

Envelope glycoprotein (Env) spikes on AIDS retroviruses initiate infection of host cells and are therefore targets for vaccine development. Though crystal structures for partial Env subunits are known, the structure and distribution of native Env spikes on virions is obscure. We applied cryoelectron microscopy tomography to define ultrastructural details of spikes. Virions of wild-type human immunodeficiency virus 1 (HIV-1) and a mutant simian immunodeficiency virus (SIV) had approximately 14 and approximately 73 spikes per particle, respectively, with some clustering of HIV-1 spikes. Three-dimensional averaging showed that the surface glycoprotein (gp120) 'head' of each subunit of the trimeric SIV spike contains a primary mass, with two secondary lobes. The transmembrane glycoprotein 'stalk' of each trimer is composed of three independent legs that project obliquely from the trimer head, tripod-like. Reconciling available atomic structures with the three-dimensional whole spike density map yields insights into the orientation of Env spike structural elements and possible structural bases of their functions.