Search

Abstract HIV-1 envelope glycoproteins (Envs) of most primary HIV-1 strains exist in closed conformation and infrequently sample open states, limiting access to internal epitopes. Thus, immunogen design aims to mimic the closed Env conformation as preferred target for eliciting broadly neutralizing antibodies (bnAbs). Here we identify incompletely closed Env conformations of 6 out of 13 transmitted/founder (T/F) strains that are sensitive to antibodies that recognize internal epitopes typically exposed on open Envs. A 3.6 Å cryo-electron microscopy structure of unliganded, incompletely closed T/F Envs (1059-SOSIP) reveals protomer motion that increased sampling of states with incompletely closed trimer apex. We reconstruct de novo the post-transmission evolutionary pathway of a second T/F. Evolved viruses exhibit increased Env resistance to cold, soluble CD4 and 19b, all of which correlate with closing of the adapted Env trimer. Lastly, we show that the ultra-broad N6 bnAb efficiently recognizes different Env conformations and exhibits improved antiviral breadth against VRC01-resistant Envs isolated during the first-in-humans antibody-mediated-prevention trial.

ABSTRACTThe envelope glycoprotein (Env) trimer on the surface of human immunodeficiency virus type I (HIV-1) mediates viral entry into host CD4+ T cells and is the sole target of neutralizing antibodies. Broadly neutralizing antibodies (bnAbs) that target gp120 V3-glycan of HIV-1 Env trimer are potent and block the entry of diverse HIV-1 strains. Most V3-glycan bnAbs interact, to a different extent, with a glycan attached to N332, but Asn at this position is not absolutely conserved or required for HIV-1 entry based on the prevalence of N332 in different circulating HIV-1 strains from diverse clades. Here, we studied the effects of amino acid changes at position 332 of HIV-1AD8 Envs on HIV-1 sensitivity to antibodies, cold exposure, and soluble CD4. We further investigated how these changes affect Env function and HIV-1 infectivity in vitro. Our results suggest robust tolerability of HIV-1AD8 Env N332 to changes, with specific changes that resulted in extended exposure of gp120 V3 loop, which is typically concealed in most primary HIV-1 isolates. Viral evolution leading to Asn at position 332 of HIVAD8 Envs is supported by the selection advantage of high levels of cell–cell fusion, transmission, and infectivity with high levels of cell surface expression and slightly higher gp120 shedding than most N332 variants. Thus, tolerance of HIV-1AD8 Envs to different amino acids at position 332 provides increased flexibility to respond to changing conditions/environments and evade the immune system. Modeling studies of the distance between N332 glycan and specific bnAbs were in agreement with N332 glycan dependency on bnAb neutralization. Overall, our studies provide insights into the contribution of specific amino acids at position 332 to Env antigenicity, stability on ice, and conformational states.IMPORTANCEGlycan attached to amino acid asparagine at position 332 of HIV-1 envelope glycoproteins is a main target of a subset of broadly neutralizing antibodies that block HIV-1 infection. Here, we defined the contribution of different amino acids at this position to Env antigenicity, stability on ice, and conformational states.

Abstract An effective human immunodeficiency virus type I (HIV-1) vaccine that robustly elicits broadly neutralizing antibodies (bnAbs) against HIV-1 envelope glycoproteins (Envs) to block viral entry is still not available. Thus, identifying triggers for elicitation of different types of anti-HIV-1 Env antibodies by vaccination could provide further guidance for immunogen design and vaccine development. Here, we studied the immune response to HIV-1 Env immunogens in rabbits. We show that sequential immunizations with conformation-specific Env immunogens can elicit low titer but broad neutralization responses against heterologous, neutralization-resistant (tier 2/3) transmitted/founder (T/F) HIV-1 strains. More importantly, an mRNA vaccine candidate that could mediate the presentation of a cytoplasmic tail-deleted (ΔCT) HIV-1AD8 Env immunogen on virus-like particles significantly increased the neutralization response. This strategy shifted the type of elicited antibodies, decreasing the level of binding to soluble Envs while significantly increasing their overall viral neutralization activity. The breadth and potency of neutralizing response against heterologous, T/F HIV-1 strains significantly increased in a subset of rabbits. Efficient neutralization activity was associated with high cellular immune responses specific to HIV-1 Envs. These results help to understand the immune response to different immunization schemes and will allow developing new approaches to selectively manipulate the type of humoral immune response by specific vaccination.

ABSTRACTHIV-1 can efficiently replicate by direct transmission from infected to uninfected CD4+ T cells at confined local sites designated virological synapses (VSs). VSs are formed by cell junctions between HIV-1 envelope glycoproteins (Envs) on an infected cell and CD4 on an uninfected cell. These sites facilitate highly efficient viral transmission and contribute to HIV-1 evasion from neutralizing antibodies, but accurate quantification of the efficiency of cell-cell transmission is still challenging. Here, we developed an ultrasensitive HIV-1 cell-to-cell transmission assay that triggers the expression of the nanoluciferase (nluc) gene in target cells upon transmission and after reverse transcription of the HIV-1 RNA genome. The assay is based on the insertion of the nluc gene in an antisense orientation into the HIV-1 provirus. Nluc expression is blocked in virus-producing cells because the nluc gene contains an intron that can be efficiently spliced out only when mRNA is transcribed from the opposite (sense) strand. Thus, only sense mRNA that is spliced and subsequently reverse transcribed during transmission to target cells will support Nluc expression. Assay optimization resulted in a very low background, >99% splicing efficiency, high sensitivity, and wide dynamic range for the detection of cell-cell transmission in T cell lines and primary CD4+ T cells. The optimized reporter vector detects cell-cell transmission using single-round viral vectors and HIV-1 molecular clones, which provide viral proteins of different HIV-1 strains, and reproducibly measures the sensitivity of HIV-1 transmission to antibody neutralization in vitro. This assay will contribute to understanding the fundamental mechanisms of HIV-1 cell-to-cell transmission, allow evaluation of pre-existing or acquired HIV-1 resistance in clinical trials, and can be adapted to study other retroviruses.IMPORTANCEHIV-1 can efficiently transmit from one cell to another but accurate quantification of this mode of transmission is still challenging. Here, we developed an ultrasensitive assay to measure HIV-1 transmission between cells and to evaluate HIV-1 escape from broadly neutralizing antibodies in primary human T cells. This assay will contribute to understanding the fundamental mechanisms of HIV-1 cell-to-cell transmission, allow evaluation of pre-existing or acquired HIV-1 resistance in clinical trials, and can be adapted to study the biology of other retroviruses.

Summary: Activated-to-memory transitioning CD4+ T cells display elevated expression of the HIV-1 co-receptor CCR5 and are more prone to HIV-1 latent infection. Here, we show that p53-regulated miRNA-103 downmodulates CCR5 levels in CD4+ T lymphocytes. We reveal that miRNA-103 mimics, as well as Nutlin-3, an inhibitor of Mdm2-mediated p53 degradation, decrease CCR5-dependent HIV-1 infection. Using a dual-reporter virus, we subsequently validate that in transitioning CD4+ T cells, Nutlin-3 treatment decreases the frequency of both productively and latently infected cells via upregulation of miRNA-103. Importantly, we provide evidence that CD4+ T cells from HIV-1 elite controllers express less CCR5 than those from antiretroviral therapy-naïve progressors, an effect linked to a significant increase in miRNA-103 levels. By contributing to the control of CCR5 expression in CD4+ T cells, miRNA-103 is likely to play a key role in countering the establishment of latent HIV-1 reservoirs in vivo.

ABSTRACT Infection of rhesus macaques with simian-human immunodeficiency viruses (SHIVs) is the preferred model system for vaccine development because SHIVs encode human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Envs)—a key target of HIV-1 neutralizing antibodies. Since the goal of vaccines is to prevent new infections, SHIVs encoding circulating HIV-1 Env are desired as challenge viruses. Development of such biologically relevant SHIVs has been challenging, as they fail to infect rhesus macaques, mainly because most circulating HIV-1 Envs do not use rhesus CD4 (rhCD4) receptor for viral entry. Most primary HIV-1 Envs exist in a closed conformation and occasionally transit to a downstream, open conformation through an obligate intermediate conformation. Here, we provide genetic evidence that open Env conformations can overcome the rhCD4 entry barrier and increase replication of SHIVs in rhesus lymphocytes. Consistent with prior studies, we found that circulating HIV-1 Envs do not use rhCD4 efficiently for viral entry. However, by using HIV-1 Envs with single amino acid substitutions that alter their conformational state, we found that transitions to intermediate and open Env conformations allow usage of physiological levels of rhCD4 for viral entry. We engineered these single amino acid substitutions in the transmitted/founder HIV-1BG505 Envs encoded by SHIV-BG505 and found that open Env conformation enhances SHIV replication in rhesus lymphocytes. Lastly, CD4-mediated SHIV pulldown, sensitivity to soluble CD4, and fusogenicity assays indicated that open Env conformation promotes efficient rhCD4 binding and viral-host membrane fusion. These findings identify the conformational state of HIV-1 Env as a major determinant for rhCD4 usage, viral fusion, and SHIV replication. IMPORTANCE Rhesus macaques are a critical animal model for preclinical testing of HIV-1 vaccine and prevention approaches. However, HIV-1 does not replicate in rhesus macaques, and thus, chimeric simian-human immunodeficiency viruses (SHIVs), which encode HIV-1 envelope glycoproteins (Envs), are used as surrogate challenge viruses to infect rhesus macaques for modeling HIV-1 infection. Development of SHIVs encoding Envs from clinically relevant, circulating HIV-1 variants has been extremely challenging, as such SHIVs replicate poorly, if at all, in rhesus lymphocytes. This is most probably because many circulating HIV-1 Envs do not use rhesus CD4 efficiently for viral entry. In this study, we identified conformational state of HIV-1 envelope as a key determinant for rhesus CD4 usage, viral-host membrane fusion, and SHIV replication in rhesus lymphocytes.

Temsavir, a compound that inhibits HIV entry by binding envelope (Env), is currently in clinical development. Here, Lai et al. identify a more than 10-fold improved compound and, using lattice engineering, obtain crystal structures that give insights into improved inhibition between small molecules and Env.

SERINC5 incorporates into HIV-1 particles and inhibits the ability of Env glycoprotein to mediate virus-cell fusion. SERINC5-resistance maps to Env, with primary isolates generally showing greater resistance than laboratory-adapted strains. Here, we examined a relationship between the inhibition of HIV-1 infectivity and the rate of Env inactivation using a panel of SERINC5-resistant and -sensitive HIV-1 Envs. SERINC5 incorporation into pseudoviruses resulted in a faster inactivation of sensitive compared to resistant Env strains. A correlation between fold reduction in infectivity and the rate of inactivation was also observed for multiple Env mutants known to stabilize and destabilize the closed Env structure. Unexpectedly, most mutations disfavoring the closed Env conformation rendered HIV-1 less sensitive to SERINC5. In contrast, functional inactivation of SERINC5-containing viruses was significantly accelerated in the presence of a CD4-mimetic compound, suggesting that CD4 binding sensitizes Env to SERINC5. Using a small molecule inhibitor that selectively targets the closed Env structure, we found that, surprisingly, SERINC5 increases the potency of this compound against a laboratory-adapted Env which prefers a partially open conformation, indicating that SERINC5 may stabilize the closed trimeric Env structure. Our results reveal a complex effect of SERINC5 on Env conformational dynamics that promotes Env inactivation and is likely responsible for the observed restriction phenotype.

Summary: HIV-1 envelope glycoproteins (Envs) bind to CD4 receptor and CCR5/CXCR4 coreceptor and mediate viral entry (Feng et al., 1996; Herschhorn et al., 2016, 2017; Kwong et al., 1998). HIV-1 Envs are the sole target of neutralizing antibodies and a main focus of vaccine development (Flemming et al., 2018). Here, we provide a step-by-step protocol to measure Env sensitivity to ligands, cold, and small molecules, as well as to study viral infectivity and to dissect parameters affecting HIV-1 Env function.For complete details on the use and execution of this protocol, please refer to Harris et al. (2020).

Summary: We describe the production of single-cycle (sc) and replication-competent recombinant vesicular stomatitis viruses (rcVSVs) displaying heterologous envelope glycoproteins (Envs) on their surface. We prepare scVSVs by transiently expressing HIV-1 Envs or SARS-CoV-2 spike followed by infection of the cells with scVSV particles, which do not carry the vsv-g gene. To prepare rcVSVs, we replace the vsv-g with a specific env-encoding gene, transfect cells with multiple plasmids for production of the genomic RNA and viral proteins, and rescue replication-competent viruses.

ABSTRACT Lentiviral DNA integration favors transcriptionally active chromatin. We previously showed that the interaction of human immunodeficiency virus type 1 (HIV-1) capsid with cleavage and polyadenylation specificity factor 6 (CPSF6) localizes viral preintegration complexes (PICs) to nuclear speckles for integration into transcriptionally active speckle-associated domains (SPADs). In the absence of the capsid-CPSF6 interaction, PICs uncharacteristically accumulate at the nuclear periphery and target heterochromatic lamina-associated domains (LADs) for integration. The integrase-binding protein lens epithelium-derived growth factor (LEDGF)/p75 in contrast to CPSF6 predominantly functions to direct HIV-1 integration to interior regions of transcription units. Though CPSF6 and LEDGF/p75 can reportedly interact with the capsid and integrase proteins of both primate and nonprimate lentiviruses, the extents to which these different viruses target SPADs versus LADs, as well as their dependencies on CPSF6 and LEDGF/p75 for integration targeting, are largely unknown. Here, we mapped 5,489,157 primate and nonprimate lentiviral integration sites in HEK293T and Jurkat T cells as well as derivative cells that were knocked out or knocked down for host factor expression. Despite marked preferences of all lentiviruses to target genes for integration, nonprimate lentiviruses only marginally favored SPADs, with corresponding upticks in LAD-proximal integration. While LEDGF/p75 knockout disrupted the intragenic integration profiles of all lentiviruses similarly, CPSF6 depletion specifically counteracted SPAD integration targeting by primate lentiviruses. CPSF6 correspondingly failed to appreciably interact with nonprimate lentiviral capsids. We conclude that primate lentiviral capsid proteins evolved to interact with CPSF6 to optimize PIC localization for integration into transcriptionally active SPADs. IMPORTANCE Integration is the defining step of the retroviral life cycle and underlies the inability to cure HIV/AIDS through the use of intensified antiviral therapy. The reservoir of latent, replication-competent proviruses that forms early during HIV infection reseeds viremia when patients discontinue medication. HIV cure research is accordingly focused on the factors that guide provirus formation and associated chromatin environments that regulate transcriptional reactivation, and studies of orthologous infectious agents such as nonprimate lentiviruses can inform basic principles of HIV biology. HIV-1 utilizes the integrase-binding protein LEDGF/p75 and the capsid interactor CPSF6 to target speckle-associated domains (SPADs) for integration. However, the extent to which these two host proteins regulate integration of other lentiviruses is largely unknown. Here, we mapped millions of retroviral integration sites in cell lines that were depleted for LEDGF/p75 and/or CPSF6. Our results reveal that primate lentiviruses uniquely target SPADs for integration in a CPSF6-dependent manner.

Binding of viral envelope glycoproteins (Env) to the host cell CD4 receptor mediates HIV-1 entry. Here, the authors develop compounds that inhibit the CD4-induced conformational changes in Env and show that the gp120 β20-β21 element is a key regulator for Env transitions.

ABSTRACT Primary human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimers [(gp120/gp41)3] typically exist in a metastable closed conformation (state 1). Binding the CD4 receptor triggers Env to undergo extensive conformational changes to mediate virus entry. We identified specific gp120 residues that restrain Env in state 1. Alteration of these restraining residues destabilized state 1, allowing Env to populate a functional conformation (state 2) intermediate between state 1 and the full CD4-bound state (state 3). Increased state 2 occupancy was associated with lower energy barriers between the states. State 2 was an obligate intermediate for all transitions between state 1 and state 3. State 2-enriched Envs required lower CD4 concentrations to trigger virus entry and more efficiently infected cells expressing low levels of CD4. These Envs were resistant to several broadly neutralizing antibodies and small-molecule inhibitors. Thus, state 2 is an Env conformation on the virus entry pathway; sampling state 2 increases the adaptability of HIV-1 to different host cell receptor levels and immune environments. Our results provide new insights into the conformational regulation of HIV-1 entry. IMPORTANCE The envelope glycoproteins (Env) of HIV-1 mediate virus entry and are the sole targets of neutralizing antibodies. Understanding the way that Env promotes HIV-1 entry can expedite drug and vaccine development. By destabilizing Env, we found that it assumes an intermediate state that is functional and obligate for transitions to entry-competent conformations. Increased sampling of this state enhances the ability of HIV-1 to infect cells that express low levels of the CD4 receptor and allows the virus to evade neutralizing antibodies and small-molecule inhibitors. These findings provide new mechanistic insights into the function and inhibition of HIV-1 Env and will contribute to ongoing therapeutic and prevention efforts to combat HIV-1.

We have recently developed the first microscopy-based strategy that enables simultaneous multiplex detection of viral RNA (vRNA), viral DNA (vDNA), and viral protein. Here, we used this approach to study the kinetics of latency reactivation in cells infected with the human immunodeficiency virus (HIV). We showed the transcription of nascent vRNA from individual latently integrated and reactivated vDNA sites appearing earlier than viral protein. We further demonstrated that this method can be used to quantitatively assess the efficacy of a variety of latency reactivating agents. Finally, this microscopy-based strategy was augmented with a flow-cytometry-based approach, enabling the detection of transcriptional reactivation of large numbers of latently infected cells. Hence, these approaches are shown to be suitable for qualitative and quantitative studies of HIV-1 latency and reactivation.

The original version of this Article contained an error in the spelling of the author Amos B. Smith, III, which was incorrectly given as Amos B. SmithIII. This has now been corrected in both the PDF and HTML versions of the Article.

Protection from human immunodeficiency virus (HIV) acquisition will likely require an effective vaccine that elicits antibodies against the HIV‐1 envelope glycoproteins (Envs), which are the sole target of neutralizing antibodies and a main focus of vaccine development. Adjuvants have been widely used to augment the magnitude and longevity of the adaptive immune responses to immunizations with HIV‐1 Envs and to guide the development of specific immune responses. Here, we review the adjuvants that have been used in combination with HIV‐1 Envs in several preclinical and human clinical trials in recent years. We summarize the interactions between the HIV‐1 Envs and adjuvants, and highlight the routes of vaccine administration for various formulations. We then discuss the use of combinations of different adjuvants, the potential effect of adjuvants on the elicitation of antibodies enriched in somatic hypermutation and containing long complementarity‐determining region 3 of the antibody heavy chain, and the elicitation of non‐neutralizing antibodies. [ABSTRACT FROM AUTHOR]