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The envelope glycoprotein (Env) of retroviruses, such as the Feline leukemia virus (FeLV), is the main target of neutralizing humoral response, and therefore, a promising vaccine candidate, despite its reported poor immunogenicity. The incorporation of mutations that stabilize analogous proteins from other viruses in their prefusion conformation (e.g., HIV Env, SARS-CoV-2 S, or RSV F glycoproteins) has improved their capability to induce neutralizing protective immune responses. Therefore, we have stabilized the FeLV Env protein following a strategy based on the incorporation of a disulfide bond and an Ile/Pro mutation (SOSIP) previously used to generate soluble HIV Env trimers. We have characterized this SOSIP-FeLV Env in its soluble form and as a transmembrane protein present at high density on the surface of FeLV Gag-based VLPs. Furthermore, we have tested its immunogenicity in DNA-immunization assays in C57BL/6 mice. Low anti-FeLV Env responses were detected in SOSIP-FeLV soluble protein-immunized animals; however, unexpectedly no responses were detected in the animals immunized with SOSIP-FeLV Gag-based VLPs. In contrast, high humoral response against FeLV Gag was observed in the animals immunized with control Gag VLPs lacking SOSIP-FeLV Env, while this response was significantly impaired when the VLPs incorporated SOSIP-FeLV Env. Our data suggest that FeLV Env can be stabilized as a soluble protein and can be expressed in high-density VLPs. However, when formulated as a DNA vaccine, SOSIP-FeLV Env remains poorly immunogenic, a limitation that must be overcome to develop an effective FeLV vaccine. [ABSTRACT FROM AUTHOR]

After the onset of the AIDS pandemic, HIV-1 (genus Lentivirus) became the predominant model for studying retrovirus Env glycoproteins and their role in entry. However, HIV Env is an inadequate model for understanding entry of viruses in the Alpharetrovirus, Gammaretrovirus and Deltaretrovirus genera. For example, oncogenic model system viruses such as Rous sarcoma virus (RSV, Alpharetrovirus), murine leukemia virus (MLV, Gammaretrovirus) and human T-cell leukemia viruses (HTLV-I and HTLV-II, Deltaretrovirus) encode Envs that are structurally and functionally distinct from HIV Env. We refer to these as Gamma-type Envs. Gamma-type Envs are probably the most widespread retroviral Envs in nature. They are found in exogenous and endogenous retroviruses representing a broad spectrum of vertebrate hosts including amphibians, birds, reptiles, mammals and fish. In endogenous form, gamma-type Envs have been evolutionarily coopted numerous times, most notably as placental syncytins (e.g., human SYNC1 and SYNC2). Remarkably, gamma-type Envs are also found outside of the Retroviridae. Gp2 proteins of filoviruses (e.g., Ebolavirus) and snake arenaviruses in the genus Reptarenavirus are gamma-type Env homologs, products of ancient recombination events involving viruses of different Baltimore classes. Distinctive hallmarks of gamma-type Envs include a labile disulfide bond linking the surface and transmembrane subunits, a multi-stage attachment and fusion mechanism, a highly conserved (but poorly understood) "immunosuppressive domain", and activation by the viral protease during virion maturation. Here, we synthesize work from diverse retrovirus model systems to illustrate these distinctive properties and to highlight avenues for further exploration of gamma-type Env structure and function. [ABSTRACT FROM AUTHOR]

The use of broadly neutralizing antibodies (bNAbs) is a promising approach to HIV-1 treatment. In this work, we evaluate the neutralizing activity of the following HIV-1 bNAbs: VCR07-523, N6, PGDM1400, CAP256-VRC26.25, 10-1074, PGT128, 10E8, and DH511.11P, which are directed to different Env surface epitopes. We used the global panel of HIV-1 pseudoviruses to analyze the bNAbs' potency and chose the most potent ones. To achieve maximum neutralization breadth and minimum IC50 concentration, the most effective antibodies were tested in double and triple combinations. Among the doubles, the combinations of N6+PGDM1400 and N6+PGT128 with IC50 ≤ 0.3 µg/mL proved to be the most effective. The most effective triple combination was N6+PGDM1400+PGT128. Our data demonstrate that this combination neutralizes pseudoviruses of the global HIV-1 panel with IC50 ≤ 0.11 µg/mL and IC80 ≤ 0.25 µg/mL. [ABSTRACT FROM AUTHOR]

Autophagy is a lysosomal-dependent degradative process essential for maintaining cellular homeostasis, and is a key player in innate and adaptive immune responses to intracellular pathogens such as human immunodeficiency virus type 1 (HIV-1). In HIV-1 target cells, autophagy mechanisms can (i) selectively direct viral proteins and viruses for degradation; (ii) participate in the processing and presentation of viral-derived antigens through major histocompatibility complexes; and (iii) contribute to interferon production in response to HIV-1 infection. As a consequence, HIV-1 has evolved different strategies to finely regulate the autophagy pathway to favor its replication and dissemination. HIV-1 notably encodes accessory genes encoding Tat, Nef and Vpu proteins, which are able to perturb and hijack canonical and non-canonical autophagy mechanisms. This review outlines the current knowledge on the complex interplay between autophagy and HIV-1 replication cycle, providing an overview of the autophagy-mediated molecular processes deployed both by infected cells to combat the virus and by HIV-1 to evade antiviral response. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV) infections lead to a progressive loss of CD4 T cells primarily via the process of apoptosis. With a limited number of infected cells and vastly disproportionate apoptosis in HIV infected patients, it is believed that apoptosis of uninfected bystander cells plays a significant role in this process. Disease progression in HIV infected individuals is highly variable suggesting that both host and viral factors may influence HIV mediated apoptosis. Amongst the viral factors, the role of Envelope (Env) glycoprotein in bystander apoptosis is well documented. Recent evidence on the variability in apoptosis induction by primary patient derived Envs underscores the role of Env glycoprotein in HIV disease. Amongst the host factors, the role of C-C Chemokine Receptor type 5 (CCR5), a coreceptor for HIV Env, is also becoming increasingly evident. Polymorphisms in the CCR5 gene and promoter affect CCR5 cell surface expression and correlate with both apoptosis and CD4 loss. Finally, chronic immune activation in HIV infections induces multiple defects in the immune system and has recently been shown to accelerate HIV Env mediated CD4 apoptosis. Consequently, those factors that affect CCR5 expression and/or immune activation in turn indirectly regulate HIV mediated apoptosis making this phenomenon both complex and multifactorial. This review explores the complex role of various host and viral factors in determining HIV mediated bystander apoptosis. [ABSTRACT FROM AUTHOR]

BST-2 or tetherin is a host cell restriction factor that prevents the budding of enveloped viruses at the cell surface, thus impairing the viral spread. Several countermeasures to evade this antiviral factor have been positively selected in retroviruses: the human immunodeficiency virus type 2 (HIV-2) relies on the envelope glycoprotein (Env) to overcome BST-2 restriction. The Env gp36 ectodomain seems involved in this anti-tetherin activity, however residues and regions interacting with BST-2 are not clearly defined. Among 32 HIV-2 ROD Env mutants tested, we demonstrated that the asparagine residue at position 659 located in the gp36 ectodomain is mandatory to exert the anti-tetherin function. Viral release assays in cell lines expressing BST-2 showed a loss of viral release ability for the HIV-2 N659D mutant virus compared to the HIV-2 wild type virus. In bst-2 inactivated H9 cells, those differences were lost. Subtilisin treatment of infected cells demonstrated that the N659D mutant was more tethered at the cell surface. Förster resonance energy transfer (FRET) experiments confirmed a direct molecular link between Env and BST-2 and highlighted an inability of the mutant to bind BST-2. We also tested a virus presenting a truncation of 109 amino acids at the C-terminal part of Env, a cytoplasmic tail partial deletion that is spontaneously selected in vitro. Interestingly, viral release assays and FRET experiments indicated that a full Env cytoplasmic tail was essential in BST-2 antagonism. In HIV-2 infected cells, an efficient Env-mediated antagonism of BST-2 is operated through an intermolecular link involving the asparagine 659 residue as well as the C-terminal part of the cytoplasmic tail. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus type 1 (HIV-1) transmission takes place primarily through cell-cell contacts known as virological synapses. Formation of these transient adhesions between infected and uninfected cells can lead to transmission of viral particles followed by separation of the cells. Alternatively, the cells can fuse, thus forming a syncytium. Tetraspanins, small scaffolding proteins that are enriched in HIV-1 virions and actively recruited to viral assembly sites, have been found to negatively regulate HIV-1 Env-induced cell-cell fusion. How these transmembrane proteins inhibit membrane fusion, however, is currently not known. As a first step towards elucidating the mechanism of fusion repression by tetraspanins, e.g., CD9 and CD63, we sought to identify the stage of the fusion process during which they operate. Using a chemical epistasis approach, four fusion inhibitors were employed in tandem with CD9 overexpression. Cells overexpressing CD9 were found to be sensitized to inhibitors targeting the pre-hairpin and hemifusion intermediates, while they were desensitized to an inhibitor of the pore expansion stage. Together with the results of a microscopy-based dye transfer assay, which revealed CD9- and CD63-induced hemifusion arrest, our investigations strongly suggest that tetraspanins block HIV-1-induced cell-cell fusion at the transition from hemifusion to pore opening. [ABSTRACT FROM AUTHOR]

Apoptosis of uninfected bystander cells is a key element of HIV pathogenesis and believed to be the driving force behind the selective depletion of CD4+ T cells leading to immunodeficiency. While several viral proteins have been implicated in this process the complex interaction between Env glycoprotein expressed on the surface of infected cells and the receptor and co-receptor expressing bystander cells has been proposed as a major mechanism. HIV-1 utilizes CD4 as the primary receptor for entry into cells; however, it is the viral co-receptor usage that greatly influences CD4 decline and progression to AIDS. This phenomenon is relatively simple for X4 viruses, which arise later during the course of the disease, are considered to be highly fusogenic, and cause a rapid CD4+ T cell decline. However, in contrast, R5 viruses in general have a greater transmissibility, are encountered early during the disease and have a lesser pathogenic potential than the former. The above generalization gets complicated in numerous situations where R5 viruses persist throughout the disease and are capable of causing a rigorous CD4+ T cell decline. This review will discuss the multiple factors that are reported to influence HIV induced bystander apoptosis and pathogenesis including Env glycoprotein phenotype, virus tropism, disease stage, co-receptor expression on CD4+ T cells, immune activation and therapies targeting the viral envelope [ABSTRACT FROM AUTHOR]

The initial step in retroviral infection involves specific interactions between viral envelope proteins (Env) and specific receptors on the surface of target cells. For many years, little was known about the entry receptors for HTLV-1. During this time, however, functional domains of the HTLV-1 Env were identified by analyzing the effects of neutralizing antibodies and specific mutations in Env on HTLV-1 infectivity. More recent studies have revealed that HTLV-1 infectivity involves interactions with three different molecules: heparan sulfate proteoglycans (HSPG), the VEGF-165 receptor Neuropilin 1 (NRP-1) and glucose transporter type 1 (GLUT1). Here, we revisit previously published data on the functional domains of Env in regard to the recent knowledge acquired about this multi-receptor complex. We also discuss the similarities and differences between HTLV-1 and other deltaretroviruses in regards to receptor usage. [ABSTRACT FROM AUTHOR]

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a lung cancer in sheep known as ovine pulmonary adenocarcinoma (OPA). The disease has been identified around the world in several breeds of sheep and goats, and JSRV infection typically has a serious impact on affected flocks. In addition, studies on OPA are an excellent model for human lung carcinogenesis. A unique feature of JSRV is that its envelope (Env) protein functions as an oncogene. The JSRV Env-induced transformation or oncogenesis has been studied in a variety of cell systems and in animal models. Moreover, JSRV studies have provided insights into retroviral genomic RNA export/expression mechanisms. JSRV encodes a trans-acting factor (Rej) within the env gene necessary for the synthesis of Gag protein from unspliced viral RNA. This review summarizes research pertaining to JSRV-induced pathogenesis, Env transformation, and other aspects of JSRV biology. [ABSTRACT FROM AUTHOR]

Gene overprinting occurs when point mutations within a genomic region with an existing coding sequence create a new one in another reading frame. This process is quite frequent in viral genomes either to maximize the amount of information that they encode or in response to strong selective pressure. The most frequent scenario involves two different reading frames in the same DNA strand (sense overlap). Much less frequent are cases of overlapping genes that are encoded on opposite DNA strands (antisense overlap). One such example is the antisense ORF, asp in the minus strand of the HIV-1 genome overlapping the env gene. The asp gene is highly conserved in pandemic HIV-1 strains of group M, and it is absent in non-pandemic HIV-1 groups, HIV-2, and lentiviruses infecting non-human primates, suggesting that the ~190-amino acid protein that is expressed from this gene (ASP) may play a role in virus spread. While the function of ASP in the virus life cycle remains to be elucidated, mounting evidence from several research groups indicates that ASP is expressed in vivo. There are two alternative hypotheses that could be envisioned to explain the origin of the asp ORF. On one hand, asp may have originally been present in the ancestor of contemporary lentiviruses, and subsequently lost in all descendants except for most HIV-1 strains of group M due to selective advantage. Alternatively, the asp ORF may have originated very recently with the emergence of group M HIV-1 strains from SIVcpz. Here, we used a combination of computational and statistical approaches to study the genomic region of env in primate lentiviruses to shed light on the origin, structure, and sequence evolution of the asp ORF. The results emerging from our studies support the hypothesis of a recent de novo addition of the antisense ORF to the HIV-1 genome through a process that entailed progressive removal of existing internal stop codons from SIV strains to HIV-1 strains of group M, and fine tuning of the codon sequence in env that reduced the chances of new stop codons occurring in asp. Altogether, the study supports the notion that the HIV-1 asp gene encodes an accessory protein, providing a selective advantage to the virus. [ABSTRACT FROM AUTHOR]

The HIV-1 envelope (Env) is an essential determinant of viral infectivity, tropism and spread between T cells. Lentiviral Env contain an unusually long 150 amino acid cytoplasmic tail (EnvCT), but the function of the EnvCT and many conserved domains within it remain largely uncharacterised. Here, we identified a highly conserved tryptophan motif at position 757 (W757) in the LLP-2 alpha helix of the EnvCT as a key determinant for HIV-1 replication and spread between T cells. Alanine substitution at this position potently inhibited HIV-1 cell–cell spread (the dominant mode of HIV-1 dissemination) by preventing recruitment of Env and Gag to sites of cell–cell contact, inhibiting virological synapse (VS) formation and spreading infection. Single-molecule tracking and super-resolution imaging showed that mutation of W757 dysregulates Env diffusion in the plasma membrane and increases Env mobility. Further analysis of Env function revealed that W757 is also required for Env fusion and infectivity, which together with reduced VS formation, result in a potent defect in viral spread. Notably, W757 lies within a region of the EnvCT recently shown to act as a supporting baseplate for Env. Our data support a model in which W757 plays a key role in regulating Env biology, modulating its temporal and spatial recruitment to virus assembly sites and regulating the inherent fusogenicity of the Env ectodomain, thereby supporting efficient HIV-1 replication and spread. [ABSTRACT FROM AUTHOR]

HIV-1 infection is enhanced by cell–cell adhesions between infected and uninfected T cells called virological synapses (VS). VS are initiated by the interactions of cell-surface HIV-1 envelope glycoprotein (Env) and CD4 on target cells and act as sites of viral assembly and viral transfer between cells. To study the process that recruits and retains HIV-1 Env at the VS, a replication-competent HIV-1 clone carrying an Env-sfGFP fusion protein was designed to enable live tracking of Env within infected cells. Combined use of surface pulse-labeling of Env and fluorescence recovery after photobleaching (FRAP) studies, enabled the visualization of the targeted accumulation and sustained recycling of Env between endocytic compartments (EC) and the VS. We observed dynamic exchange of Env at the VS, while the viral structural protein, Gag, was largely immobile at the VS. The disparate exchange rates of Gag and Env at the synapse support that the trafficking and/or retention of a majority of Env towards the VS is not maintained by entrapment by a Gag lattice or immobilization by binding to CD4 on the target cell. A FRAP study of an Env endocytosis mutant showed that recycling is not required for accumulation at the VS, but is required for the rapid exchange of Env at the VS. We conclude that the mechanism of Env accumulation at the VS and incorporation into nascent particles involves continuous internalization and targeted secretion rather than irreversible interactions with the budding virus, but that this recycling is largely dispensable for VS formation and viral transfer across the VS. [ABSTRACT FROM AUTHOR]

To minimize immune responses against infected cells, HIV-1 has evolved different mechanisms to limit the surface expression of its envelope glycoproteins (Env). Recent observations suggest that the binding of certain broadly neutralizing antibodies (bNAbs) targeting the 'closed' conformation of Env induces its internalization. On the other hand, non-neutralizing antibodies (nNAbs) that preferentially target Env in its 'open' conformation, remain bound to Env on the cell surface for longer periods of time. In this study, we attempt to better understand the underlying mechanisms behind the differential rates of antibody-mediated Env internalization. We demonstrate that 'forcing' open Env using CD4 mimetics allows for nNAb binding and results in similar rates of Env internalization as those observed upon the bNAb binding. Moreover, we can identify distinct populations of Env that are differentially targeted by Abs that mediate faster rates of internalization, suggesting that the mechanism of antibody-induced Env internalization partially depends on the localization of Env on the cell surface. [ABSTRACT FROM AUTHOR]

HIV-1 vaccine research has obtained an enormous boost since the discovery of many broadly neutralizing antibodies (bnAbs) targeting all accessible sites on the HIV-1 envelope glycoprotein (Env). This in turn facilitated high-resolution structures of the Env glycoprotein in complex with bnAbs. Here we focus on gp41, its highly conserved heptad repeat region 1 (HR1), the fusion peptide (FP) and the membrane-proximal external region (MPER). Notably, the broadest neutralizing antibodies target MPER. Both gp41 HR1 and MPER are only fully accessible once receptor-induced conformational changes have taken place, although some studies suggest access to MPER in the close to native Env conformation. We summarize the data on the structure and function of neutralizing antibodies targeting gp41 HR1, FP and MPER and we review their access to Env and their complex formation with gp41 HR1, MPER peptides and FP within native Env. We further discuss MPER bnAb binding to lipids and the role of somatic mutations in recognizing a bipartite epitope composed of the conserved MPER sequence and membrane components. The problematic of gp41 HR1 access and MPER bnAb auto- and polyreactivity is developed in the light of inducing such antibodies by vaccination. [ABSTRACT FROM AUTHOR]

HIV infects several cell types in the body, including CD4+ T cells and macrophages. Here we review the role of macrophages in HIV infection and describe complex interactions between viral proteins and host defenses in these cells. Macrophages exist in many forms throughout the body, where they play numerous roles in healthy and diseased states. They express pattern-recognition receptors (PRRs) that bind viral, bacterial, fungal, and parasitic pathogens, making them both a key player in innate immunity and a potential target of infection by pathogens, including HIV. Among these PRRs is mannose receptor, a macrophage-specific protein that binds oligosaccharides, restricts HIV replication, and is downregulated by the HIV accessory protein Vpr. Vpr significantly enhances infection in vivo, but the mechanism by which this occurs is controversial. It is well established that Vpr alters the expression of numerous host proteins by using its co-factor DCAF1, a component of the DCAF1–DDB1–CUL4 ubiquitin ligase complex. The host proteins targeted by Vpr and their role in viral replication are described in detail. We also discuss the structure and function of the viral protein Env, which is stabilized by Vpr in macrophages. Overall, this literature review provides an updated understanding of the contributions of macrophages and Vpr to HIV pathogenesis. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV) infection constitutes a major health and social issue worldwide. HIV infects cells by fusing its envelope with the target cell plasma membrane. This process is mediated by the viral Env glycoprotein and depends on the envelope lipid composition. Fluorescent microscopy has been employed to investigate the envelope properties, and the processes of viral assembly and fusion, but the application of this technique to the study of HIV is still limited by a number of factors, such as the small size of HIV virions or the difficulty to label the envelope components. Here, we review fluorescence imaging studies of the envelope lipids and proteins, focusing on labelling strategies and model systems. [ABSTRACT FROM AUTHOR]

The HIV-1 entry-route is a matter of ongoing controversy, and there is evidence for fusion either at the cell surface or from within endosomes. A recent report demonstrated that isoform 4 of nuclear receptor coactivator 7 (NCOA7iso4) interacts with endolysosomal vacuolar-type H+-ATPase (V-ATPase), increasing lytic activity and thereby severely affecting the entry of vesicular stomatitis virus glycoprotein (VSV-G)-mediated, but not HIV-Env-mediated, entry and infection. As basal expression of NCOA7iso4 is low in the absence of type-1 interferons, its overexpression is a novel tool to study viral entry. [ABSTRACT FROM AUTHOR]

Although effective antibody-based vaccines have been developed against multiple viruses, such approaches have so far failed for the human immunodeficiency virus type 1 (HIV-1). Despite the success of anti-retroviral therapy (ART) that has turned HIV-1 infection into a chronic disease and has reduced the number of new infections worldwide, a vaccine against HIV-1 is still urgently needed. We discuss here the major reasons for the failure of “classical” vaccine approaches, which are mostly due to the biological properties of the virus itself. HIV-1 has developed multiple mechanisms of immune escape, which also account for vaccine failure. So far, no vaccine candidate has been able to induce broadly neutralizing antibodies (bnAbs) against primary patient viruses from different clades. However, such antibodies were identified in a subset of patients during chronic infection and were shown to protect from infection in animal models and to reduce viremia in first clinical trials. Their detailed characterization has guided structure-based reverse vaccinology approaches to design better HIV-1 envelope (Env) immunogens. Furthermore, conserved Env epitopes have been identified, which are promising candidates in view of clinical applications. Together with new vector-based technologies, considerable progress has been achieved in recent years towards the development of an effective antibody-based HIV-1 vaccine. [ABSTRACT FROM AUTHOR]