Cryo-ET of Env on intact HIV virions reveals structural variation and positioning on the Gag lattice.

HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here, we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1-Å sub-tomogram-averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers, and a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.

An amphipathic sequence in the cytoplasmic tail of HIV-1 Env alters cell tropism and modulates viral receptor specificity.

The human immunodeficiency virus type 1 (HIV-1) 92UG046 Env protein, obtained from a CD4-independent HIV-1 primary isolate (Zerhouni et al., 2004), has the ability to initiate an infection in HeLa cells expressing CD4 when carrying the full-length (FL) Env, but uses CD8 molecules for receptor-mediated entry when carrying a truncated Env (CT84). To determine whether a specific length or structure in the cytoplasmic tail (CT) is responsible for this alteration of tropism, we compared a series of Env constructs with different CT truncations and the presence or absence of an amphipathic alpha- helical sequence. We found that truncated constructs containing the alpha-helical LLP-2 structure in their CT domains conferred a switch from CD4 to CD8 tropism. The results support the conclusion that the structure of the CT domain can play an important role in determining receptor specificity.

Temsavir Modulates HIV-1 Envelope Conformation by Decreasing Its Proteolytic Cleavage.

HIV-1 envelope glycoproteins (Envs) mediate viral entry and represent a target of choice for small molecule inhibitors. One of them, temsavir (BMS-626529) prevents the interaction of the host cell receptor CD4 with Env by binding the pocket under the ß20-ß21 loop of the Env subunit gp120. Along with its capacity to prevent viral entry, temsavir stabilizes Env in its "closed" conformation. We recently reported that temsavir affects glycosylation, proteolytic processing, and overall conformation of Env. Here, we extend these results to a panel of primary Envs and infectious molecular clones (IMCs), where we observe a heterogeneous impact on Env cleavage and conformation. Our results suggest that the effect of temsavir on Env conformation is associated with its capacity to decrease Env processing. Indeed, we found that the effect of temsavir on Env processing affects the recognition of HIV-1-infected cells by broadly neutralizing antibodies and correlates with their capacity to mediate antibody-dependent cellular cytotoxicity (ADCC).

Temsavir blocks the immunomodulatory activities of HIV-1 soluble gp120.

While HIV-1-mediated CD4 downregulation protects infected cells from antibody-dependent cellular cytotoxicity (ADCC), shed gp120 binds to CD4 on uninfected bystander CD4+ T cells, sensitizing them to ADCC mediated by HIV+ plasma. Soluble gp120-CD4 interaction on multiple immune cells also triggers a cytokine burst. The small molecule temsavir acts as an HIV-1 attachment inhibitor by preventing envelope glycoprotein (Env)-CD4 interaction and alters the overall antigenicity of Env by affecting its processing and glycosylation. Here we show that temsavir also blocks the immunomodulatory activities of shed gp120. Temsavir prevents shed gp120 from interacting with uninfected bystander CD4+ cells, protecting them from ADCC responses and preventing a cytokine burst. Mechanistically, this depends on temsavir's capacity to prevent soluble gp120-CD4 interaction, to reduce gp120 shedding, and to alter gp120 antigenicity. This suggests that the clinical benefits provided by temsavir could extend beyond blocking viral entry.

Temsavir Treatment of HIV-1-Infected Cells Decreases Envelope Glycoprotein Recognition by Broadly Neutralizing Antibodies.

The heavily glycosylated HIV-1 envelope glycoprotein (Env) is the sole viral antigen present at the surface of virions and infected cells, representing the main target for antibody responses. The FDA-approved small molecule temsavir acts as an HIV-1 attachment inhibitor by preventing Env-CD4 interaction. This molecule also stabilizes Env in a prefusion "closed" conformation that is preferentially targeted by several broadly neutralizing antibodies (bNAbs). A recent study showed that an analog of temsavir (BMS-377806) affects the cleavage and addition of complex glycans on Env. In this study, we investigated the impact of temsavir on the overall glycosylation, proteolytic cleavage, cell surface expression, and antigenicity of Env. We found that temsavir impacts Env glycosylation and processing at physiological concentrations. This significantly alters the capacity of several bNAbs to recognize Env present on virions and HIV-1-infected cells. Temsavir treatment also reduces the capacity of bNAbs to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC). Consequently, the impact of temsavir on Env glycosylation and antigenicity should be considered for the development of new antibody-based approaches in temsavir-treated individuals. IMPORTANCE FDA-approved fostemsavir, the prodrug for the active moiety small molecule temsavir (GSK 2616713 [formally BMS-626529]), acts as an attachment inhibitor by targeting the HIV-1 envelope (Env) and preventing CD4 interaction. Temsavir also stabilizes Env in its "closed," functional state 1 conformation, which represents an ideal target for broadly neutralizing antibodies (bNAbs). Since these antibodies recognize conformation-dependent epitopes composed of or adjacent to glycans, we evaluated the impact of temsavir treatment on overall Env glycosylation and its influence on bNAb recognition. Our results showed an alteration of Env glycosylation and cleavage by temsavir at physiological concentrations. This significantly modifies the overall antigenicity of Env and therefore reduces the capacity of bNAbs to recognize and eliminate HIV-1-infected cells by ADCC. These findings provide important information for the design of immunotherapies aimed at targeting the viral reservoir in temsavir-treated individuals.

HIV-1 Envelope Glycoproteins Proteolytic Cleavage Protects Infected Cells from ADCC Mediated by Plasma from Infected Individuals.

The HIV-1 envelope glycoprotein (Env) is synthesized in the endoplasmic reticulum as a trimeric gp160 precursor, which requires proteolytic cleavage by a cellular furin protease to mediate virus-cell fusion. Env is conformationally flexible but controls its transition from the unbound "closed" conformation (State 1) to downstream CD4-bound conformations (States 2/3), which are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the premature "opening" of Env which exposes highly conserved epitopes recognized by non-neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the "closed" conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC responses.

Myomedin scaffold variants targeted to 10E8 HIV-1 broadly neutralizing antibody mimic gp41 epitope and elicit HIV-1 virus-neutralizing sera in mice.

One of the proposed strategies for the development of a more efficient HIV-1 vaccine is based on the identification of proteins binding to a paratope of chosen broadly neutralizing antibody (bNAb) that will mimic cognate HIV-1 Env (glyco)protein epitope and could be used as potent immunogens for induction of protective virus-neutralizing antibodies in the immunized individuals. To verify this "non-cognate ligand" concept, we developed a highly complex combinatorial library designed on a scaffold of human myomesin-1 protein domain and selected proteins called Myomedins specifically binding to variable regions of HIV-1 broadly neutralizing antibody 10E8. Immunization of mice with these Myomedin variants elicited the production of HIV-1 Env-specific antibodies. Hyperimmune sera bound to Env pseudotyped viruses and weakly/moderately neutralized 54% of tested clade A, B, C, and AE pseudotyped viruses variants in vitro. These results demonstrate that Myomedin variants have the potential to mimic Env epitopes and could be used as potential HIV-1 vaccine components.

A computational model for predicting transmembrane regions of retroviruses.

Transmembrane region (TR) is a conserved region of transmembrane (TM) subunit in envelope (env) glycoprotein of retrovirus. Evidences have shown that TR is responsible for anchoring the env glycoprotein on the lipid bilayer and substitution of the TR for a covalently linked lipid anchor abrogates fusion. However, universal software could not achieve sufficient accuracy as TM in env also has several motifs such as signal peptide, fusion peptide and immunosuppressive domain composed largely of hydrophobic residues. In this paper, a support vector machine-based (SVM) model is proposed to identify TRs in retroviruses. Firstly, physicochemical and evolutionary information properties were extracted as original features. And then, the feature importance was analyzed by minimum Redundancy Maximum Relevance (mRMR) feature selection criterion. Our model achieved an Sn of 0.955, Sp of 0.998, ACC of 0.995, MCC of 0.954 using 10-fold cross-validation on the training dataset. These results suggest that the proposed model can be used to predict TRs in non-annotation retroviruses and 11917, 3344, 2, 289 and 6 new putative TRs were found in HERV, HIV, HTLV, SIV, MLV, respectively.

Effects of modification of the HIV-1 Env cytoplasmic tail on immunogenicity of VLP vaccines.

We investigated the effects on assembly and antigenic properties of specific modifications of the transmembrane spanning (TMS) and cytoplasmic tail (CT) domains of HIV-1 Env from a transmitted/founder (T/F) ZM53 Env glycoprotein. A construct containing a short version of the TMS domain derived from the mouse mammary tumor virus (MMTV) Env with or without a GCN4 trimerization sequence in the CT exhibited the highest levels of incorporation into VLPs and induced the highest titers of anti-Env IgG immune responses in a VLP context. Sera from guinea pigs immunized by VLPs with high Env content, and containing the CT trimerization sequence, had increased neutralization activity and antibody avidity. A cross-clade prime-boost regimen with clade B SF162 or clade C ZM53 Env DNA priming and boosting with VLPs containing modified ZM53 Env further enhanced these immune responses. The modified VLPs demonstrate improved potential as HIV-1 vaccine antigens.