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Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P2 depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P2 depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P2 depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P2 depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain. IMPORTANCE Human immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions. [ABSTRACT FROM AUTHOR]

The reasons that lead some individuals living with the Human T Lymphotropic Virus 1 (HTLV‐1) to develop HAM/TSP are still unclear. To better understand the viral genetic factors that may be associated with the development of HAM/TSP, this study aims to evaluate the impact of HTLV‐1 genome mutations on the development of this disease through a systematic review. This review followed the PRISMA guidelines and was registered in the PROSPERO database. The search for articles was performed in PMC, PubMed, Lilacs, SciELO, and Embase databases using the following search descriptors: HTLV‐1, HAM/TSP, mutation, polymorphism, genetic variation, and sequenc*. From the 1,929 articles found in the search, 20 were selected according to the pre‐defined inclusion and exclusion criteria. A total of 619 HAM/TSP cases were compared with 555 AC controls. The mutations possibly related to the disease progression were detected in hbz (R119Q), tax (A7959V), ORF‐I (R88K, P86S, S69G, P45L, L40F, C39R, CR9Y), and gp46 (V247I, N93D, S72G) genetic regions. The data collected and analyzed here indicate that mutations in the HTLV‐1 genome could play an important role in the chronic inflammatory state and may be related to the development of HAM/TSP. [ABSTRACT FROM AUTHOR]

The HIV-1 envelope glycoprotein (Env) is the sole neutralizing determinant on the surface of the virus. The Env gp120 and gp41 subunits mediate receptor binding and membrane fusion and are generated from the gp160 precursor by cellular furins. This cleavage event is required for viral entry. One approach to generate HIV-1 neutralizing antibodies following immunization is to express membranebound Env anchored on the cell-surface by genetic means using the natural HIV gp41 transmembrane (TM) spanning domain. To simplify the process of Env trimer membrane expression we sought to remove the need for Env precursor cleavage while maintaining native-like conformation following genetic expression. To accomplish these objectives, we selected our previously developed 'native flexibly linked' (NFL) stabilized soluble trimers that are both near-native in conformation and cleavage-independent. We genetically fused the NFL construct to the HIV TM domain by using a short linker or by restoring the native membrane external proximal region, absent in soluble trimers, to express the full HIV Env ectodomain on the plasma membrane. Both forms of cellsurface NFL trimers, without and with the MPER, displayed favorable antigenic profiles by flow cytometry when expressed from plasmid DNA or mRNA. These results were consistent with the presence of well-ordered cell surface native-like trimeric Env, a necessary requirement to generate neutralizing antibodies by vaccination. Inoculation of rabbits with mRNA lipid nanoparticles (LNP) expressing membrane-bound stabilized HIV Env NFL trimers generated tier 2 neutralizing antibody serum titers in immunized animals. Multiple inoculations of mRNA LNPs generated similar neutralizing antibody titers compared to immunizations of matched NFL soluble proteins in adjuvant. Given the recent success of mRNA vaccines to prevent severe COVID, these are important developments for genetic expression of native-like HIV Env trimers in animals and potentially in humans. [ABSTRACT FROM AUTHOR]

Previously obtained highly immunogenic Env-VLPs ensure overcoming the natural resistance of HIV-1 surface proteins associated with their low level of incorporation and inaccessibility of conserved epitopes to induce neutralizing antibodies. We also adopted this technology to modify Env trimers of the ZM53(T/F) strain to produce Env-VLPs by recombinant vaccinia viruses (rVVs). For VLP production, rVVs expressing Env, Gag-Pol (HIV-1/SIV), and the cowpox virus hr gene, which overcomes the restriction of vaccinia virus replication in CHO cells, were used. The CHO Lec1 engineered cell line lacking GlcNAc-TI was used for generating VLPs with Env proteins containing a cytoplasmic (CT) domain affecting the surface subunit (SU) conformation. This has created the opportunity to modulate the glycan composition, and refine the conditions for their production, and optimize approaches to overcoming HIV-1 resistance associated with abundant glycosylation. [ABSTRACT FROM AUTHOR]

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]

Much attention has been focused on the possibility that cytoplasmic proteins and RNA may be conveyed between cells in extracellular vesicles (EVs) and tunneling nanotube (TNT) structures. Here, we set up two quantitative delivery reporters to study cargo transfer between cells. We found that EVs are internalized by reporter cells but do not efficiently deliver functional Cas9 protein to the nucleus. In contrast, donor and acceptor cells co-cultured to permit cell contact resulted in a highly effective transfer. Among our tested donor and acceptor cell pairs, HEK293T and MDA-MB-231 recorded optimal intercellular transfer. Depolymerization of F-actin greatly decreased Cas9 transfer, whereas inhibitors of endocytosis or knockdown of genes implicated in this process had little effect on transfer. Imaging results suggest that intercellular transfer of cargos occurred through open-ended membrane tubular connections. In contrast, cultures consisting only of HEK293T cells form close-ended tubular connections ineffective in cargo transfer. Depletion of human endogenous fusogens, syncytins, especially syncytin-2 in MDA-MB-231 cells, significantly reduced Cas9 transfer. Full-length mouse syncytin, but not truncated mutants, rescued the effect of depletion of human syncytins on Cas9 transfer. Mouse syncytin overexpression in HEK293T cells partially facilitated Cas9 transfer among HEK293T cells. These findings suggest that syncytin may serve as the fusogen responsible for the formation of an open-ended connection between cells.

The development of HIV prophylactic vaccines is facing an impasse, since all phase IIb/III clinical trials were halted in 2023 without demonstrating efficacy. Thus, the field is in need of developing novel immunogens and vaccination strategies that induce broadly neutralising antibodies together with potent Fc-dependent effector functions, as well as protective cross-reactive CD4+ and CD8+ T cell responses. Nucleic acid vaccines, particularly mRNA vaccines, have been one of the major groundbreaking advances in the current decade. Nucleic acid vaccines may help recalibrate the HIV vaccine field towards the use of delivery systems that allow the proper expression of immunogens as a sole antigen (i.e., membrane-bound trimeric envelope glycoproteins) or even to be displayed in a multiantigen platform that will be synthesised by the host. In this review, we will summarise how the multiple HIV vaccine strategies pursued in the last 40 years of HIV research have driven current vaccine development, which are the most relevant immunogens identified so far to induce balanced adaptive immune responses, and how they can benefit from the acceptance of nucleic acid vaccines in the market by reducing the limitations of previous delivery systems. The incorporation of nucleic acid vaccines into the current heterogeneous repertoire of vaccine platforms may represent an invaluable opportunity to reignite the fight against HIV. [ABSTRACT FROM AUTHOR]

The HIV-1 envelope glycoprotein (Env) is the sole neutralizing determinant on the surface of the virus. The Env gp120 and gp41 subunits mediate receptor binding and membrane fusion and are generated from the gp160 precursor by cellular furins. This cleavage event is required for viral entry. One approach to generate HIV-1 neutralizing antibodies following immunization is to express membrane-bound Env anchored on the cell-surface by genetic means using the natural HIV gp41 transmembrane (TM) spanning domain. To simplify the process of Env trimer membrane expression we sought to remove the need for Env precursor cleavage while maintaining native-like conformation following genetic expression. To accomplish these objectives, we selected our previously developed ‘native flexibly linked’ (NFL) stabilized soluble trimers that are both near-native in conformation and cleavage-independent. We genetically fused the NFL construct to the HIV TM domain by using a short linker or by restoring the native membrane external proximal region, absent in soluble trimers, to express the full HIV Env ectodomain on the plasma membrane. Both forms of cell-surface NFL trimers, without and with the MPER, displayed favorable antigenic profiles by flow cytometry when expressed from plasmid DNA or mRNA. These results were consistent with the presence of well-ordered cell surface native-like trimeric Env, a necessary requirement to generate neutralizing antibodies by vaccination. Inoculation of rabbits with mRNA lipid nanoparticles (LNP) expressing membrane-bound stabilized HIV Env NFL trimers generated tier 2 neutralizing antibody serum titers in immunized animals. Multiple inoculations of mRNA LNPs generated similar neutralizing antibody titers compared to immunizations of matched NFL soluble proteins in adjuvant. Given the recent success of mRNA vaccines to prevent severe COVID, these are important developments for genetic expression of native-like HIV Env trimers in animals and potentially in humans.

CD4-mimetic compounds (CD4mcs) bind the human immunodeficiency virus (HIV-1) gp120 exterior envelope glycoprotein (Env) and compete for binding to CD4, the host receptor. CD4mcs prematurely trigger conformational changes in Env similar to those induced by CD4, leading to transient activation of infectivity followed by irreversible virus inactivation. Natural HIV-1 variants exhibit a wide range of susceptibilities to CD4mc inhibition, only a small fraction of which can be explained by variation in the gp120 Phe-43 cavity/vestibule where CD4mcs bind. Here, we study Envs from the resistant HIV-1BG505 and the more sensitive HIV-1191955_A4 clade A strains. The major determinant of the relative sensitivity of the HIV-1191955_A4 Env to CD4mcs mapped to a single residue change (F317Y) in the tip of the gp120 V3 variable loop. In the Envs of several HIV-1 strains, replacement of the more prevalent Phe 317 with a tyrosine residue increased virus sensitivity to multiple CD4mcs. Tryptophan substitutions at residues 317 and 316 resulted in increases and decreases, respectively, in sensitivity to CD4mcs. Some of the gp120 V3 changes increased virus sensitivity to inactivation by both CD4mc and cold exposure, phenotypes indicative of increased Env triggerability. Infection of CD4-negative cells expressing the CCR5 coreceptor by these Env variants was triggered more efficiently by CD4mcs. For the panel of studied HIV-1 Envs, resistance to the CD4mcs was associated with decreased ability to support virus entry. These studies illustrate how variation in gp120 outside the CD4mc binding site can influence the sensitivity of natural HIV-1 strains to inhibition by these compounds. [ABSTRACT FROM AUTHOR]

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.

The jaagsiekte sheep retrovirus (JSRV), belonging to the betaretrovirus genus of the retroviridae family, includes both exogenous and endogenous jaagsiekte sheep retroviruses (exJSRV and enJSRV, respectively). At the proviral genome level, exJSRV and enJSRV strains have a high degree of similarity with their main variation regions being the LTR, gag, and env genes. In this study, for the first time, we investigated and compared the distribution of CpG islands between these enJSRV and exJSRV strains. Specifically, we analyzed a total of 42 fulllength JSRV genomic sequences obtained from the GenBank® database to identify CpG islands in the exJSRV and enJSRV genomes using the MethPrimer software. Our results showed that the CpG islands in the two JSRV strains were mainly distributed in the LTR, gag, and env genes. In exJSRVs, 66.66% (6/9), 33.33% (3/9), and 100% (9/9) of the sequences presented at least one CpG island in LTR, gag, env genes, respectively, and for enJSRVs, 84.84% (28/33), 57.57% (19/33), and 96.96% (32/33) of the sequences presented at least one CpG island in the LTR, gag, and env genes. These findings suggested that the distribution, length, and genetic traits of CpG islands were different for the exJSRV and enJSRV strains. In future, it would be necessary to demonstrate the biological significance of CpG islands within these genes in exJSRV and enJSRV genomes. This will enhance understanding regarding the potential role of CpG islands in epigenetic regulation. [ABSTRACT FROM AUTHOR]

The role of vaccine-induced anti-V2 Abs was tested in three protection experiments in rhesus macaques. In an experiment using immunogens similar to those in the RV144 vaccine trial (Anti-envelope [Env]), nine rhesus macaques were coimmunized with gp16092TH023 DNA and SIV gag and gp120A244 and gp120MN proteins. In two V2-focused experiments (Anti-V2 and Anti-V2 Mucosal), nine macaques in each group were immunized with V1V292TH023 DNA, V1V2A244 and V1V2CasaeA2 proteins, and cyclic V2CaseA2 peptide. DNA and protein immunogens, formulated in Adjuplex, were given at 0, 4, 12, and 20 weeks, followed by intrarectal SHIVBaL.P4 challenges. Peak plasma viral loads (PVL) of 106-107 copies/ml developed in all nine sham controls. Overall, PVL was undetectable in one third of immunized macaques, and two animals tightly controlled the virus with the Anti-V2 Mucosal vaccine strategy. In the Anti-Env study, Abs that captured or neutralized SHIVBaL.P4 inversely correlated with PVL. Conversely, no correlation with PVL was found in the Anti-V2 experiments with nonneutralizing plasma Abs that only captured virus weakly. Titers of Abs against eight V1V2 scaffolds and cyclic V2 peptides were comparable between controllers and noncontrollers as were Ab-dependent cellular cytotoxicity and Ab-dependent cell-mediated virus inhibition activities against SHIV-infected target cells and phagocytosis of gp120-coated beads. The Anti-Env experiment supports the role of vaccine-elicited neutralizing and nonneutralizing Abs in control of PVL. However, the two V2-focused experiments did not support a role for nonneutralizing V2 Abs alone in controlling PVL, as neither Ab-dependent cellular cytotoxicity, Ab-dependent cell-mediated virus inhibition, nor phagocytosis correlated inversely with heterologous SHIVBaL.P4 infection.

Abstract Background Avian leukosis virus (ALV) is an infectious retrovirus, that mainly causes various forms of tumours, immunosuppression, a decreased egg production rate and slow weight gain in poultry. ALV consists of 11 subgroups, A–K, among which ALV-K is an emerging subgroup that has become prevalent in the past 10 years. Most ALV-K isolates showed weak replication ability and pathogenicity. In this study, the weak replication ability of ALV-K was explored from the perspective of the interaction between ALV-K gp85 and the Tva receptor. Methods Fourteen soluble recombinant ALV-A/K gp85 chimeric proteins were constructed by substituting the sequence difference regions (hr1, hr2 and vr3) of the ALV-A gp85 protein with the skeleton ALV-K gp85 protein for co-IP and competitive blocking tests. Results The binding capacity of ALV-K gp85 to Tva was significantly weaker than that of ALV-A gp85 (P

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]

HIV-1 does not persistently infect macaques due in part to restriction by several macaque host factors. This has been partially circumvented by generating chimeric SIV/HIV-1 viruses (SHIVs) that encode SIV antagonist of known restriction factors. However, most SHIVs replicate poorly in macaques unless they are further adapted in culture and/or macaques (adapted SHIVs). Therefore, development of SHIVs encoding HIV-1 sequences derived directly from infected humans without adaptation (unadapted SHIVs) has been challenging. In contrast to the adapted SHIVs, the unadapted SHIVs have lower replication kinetics in macaque lymphocytes and are sensitive to type-1 interferon (IFN). The HIV-1 Envelope (Env) in the chimeric virus determines both the reduced replication and the IFN-sensitivity differences. There is limited information on macaque restriction factors that specifically limit replication of the more biologically relevant, unadapted SHIV variants. In order to identify the IFN-induced host factor(s) that could contribute to the inhibition of SHIVs in macaque lymphocytes, we measured IFN-induced gene expression in immortalized pig-tailed macaque (Ptm) lymphocytes using RNA-Seq. We found 147 genes that were significantly upregulated upon IFN treatment in Ptm lymphocytes and 31/147 were identified as genes that encode transmembrane helices and thus are likely present in membranes where interaction with viral Env is plausible. Within this group of upregulated genes with putative membrane-localized proteins, we identified several interferon-induced transmembrane protein (IFITM) genes, including several previously uncharacterized Ptm IFITM3-related genes. An evolutionary genomic analysis of these genes suggests the genes are IFITM3 duplications not found in humans that are both within the IFITM locus and also dispersed elsewhere in the Ptm genome. We observed that Ptm IFITMs are generally packaged at higher levels in unadapted SHIVs when compared to adapted SHIVs. CRISPR/Cas9-mediated knockout of Ptm IFITMs showed that depletion of IFITMs partially rescues the IFN sensitivity of unadapted SHIV. Moreover, we found that the depletion of IFITMs also increased replication of unadapted SHIV in the absence of IFN treatment, suggesting that Ptm IFITMs are likely important host factors that limit replication of unadapted SHIVs. In conclusion, this study shows that Ptm IFITMs selectively restrict replication of unadapted SHIVs. These findings suggest that restriction factors including IFITMs vary in their potency against different SHIV variants and may play a role in selecting for viruses that adapt to species-specific restriction factors.

The HIV Research for Prevention (HIVR4P) conference is dedicated to advancing HIV prevention research, responding to a growing consensus that effective and durable prevention will require a combination of approaches as well as unprecedented collaboration among scientists, practitioners, and community workers from different fields and geographic areas. The conference theme in 2018, "From Research to Impact," acknowledged an increasing focus on translation of promising research findings into practical, accessible, and affordable HIV prevention options for those who need them worldwide. HIVR4P 2018 was held in Madrid, Spain, on 21-25 October, with >1,400 participants from 52 countries around the globe, representing all aspects of HIV prevention research and implementation. The program included 137 oral and 610 poster presentations. This article presents a brief summary of highlights from the conference. More detailed information, complete abstracts as well as webcasts and daily Rapporteur summaries may be found on the conference website.

Feline leukemia virus (FeLV) is horizontally transmitted among cats and causes a variety of hematopoietic disorders. Five subgroups of FeLV, A to D and T, each with distinct receptor usages, have been described. Recently, we identified a new FeLV Env (TG35-2) gene from a pseudotyped virus that does not belong to any known subgroup. FeLV-A is the primary virus from which other subgroups have emerged via mutation or recombination of the subgroup A env gene. Retrovirus entry into cells is mediated by the interaction of envelope protein (Env) with specific cell surface receptors. Here, phenotypic screening of a human/hamster radiation hybrid panel identified SLC19A1, a feline reduced folate carrier (RFC) and potential receptor for TG35-2-phenotypic virus. RFC is a multipass transmembrane protein. Feline and human RFC cDNAs conferred susceptibility to TG35-2-pseudotyped virus when introduced into nonpermissive cells but did not render these cells permissive to other FeLV subgroups or feline endogenous retrovirus. Moreover, human cells with genomic deletion of RFC were nonpermissive for TG35-2-pseudotyped virus infection, but the introduction of feline and human cDNAs rendered them permissive. Mutation analysis of FeLV Env demonstrated that amino acid substitutions within variable region A altered the specificity of the Env-receptor interaction. We isolated and reconstructed the full-length infectious TG35-2-phenotypic provirus from a naturally FeLV-infected cat, from which the FeLV Env (TG35-2) gene was previously isolated, and compared the replication of the virus in hematopoietic cell lines with that of FeLV-A 61E by measuring the viral RNA copy numbers. These results provide a tool for further investigation of FeLV infectious disease.IMPORTANCE Feline leukemia virus (FeLV) is a member of the genus Gammaretrovirus, which causes malignant diseases in cats. The most prevalent FeLV among cats is FeLV subgroup A (FeLV-A), and specific binding of FeLV-A Env to its viral receptor, thiamine transporter feTHTR1, is the first step of infection. In infected cats, novel variants of FeLV with altered receptor specificity for viral entry have emerged by mutation or recombination of the env gene. A novel FeLV variant arose from a subtle mutation of FeLV-A Env, which altered the specific interaction of the virus with its receptor. RFC, a folate transporter, is a potential receptor for the novel FeLV variant. The perturbation of specific retrovirus-receptor interactions under selective pressure by the host results in the emergence of novel viruses.

Capture of retroviral envelope genes from endogenous retroviruses has played a role in the evolution of mammals, with evidence for the involvement of these genes in the formation of the maternofetal interface of the placenta. It has been shown that the diversity of captured genes is likely to be responsible for the diversity of placental structures, ranging from poorly invasive (epitheliochorial) to highly invasive (hemochorial), with an intermediate state (endotheliochorial) as found in carnivorans. The latter recapitulate part of this evolution, with the hyena being the sole carnivoran with a hemochorial placenta. In this study, we performed RNA sequencing on hyena placental transcripts and searched for endogenous retroviral envelope genes that have been captured specifically in the Hyaenidae clade and are not found in any other carnivoran. We identified an envelope gene that is expressed in the placenta at the level of the maternofetal interface, as evidenced by in situ hybridization/immunohistochemistry. The gene entry is coincidental with the emergence of the Hyaenidae clade 30 million years ago (Mya), being found at the same genomic locus in all 4 extant hyena species. Its coding sequence has further been maintained during all of Hyaenidae evolution. It is not found in any of the 30 other carnivorans-both Felidae and Canidae-that we screened. This envelope protein does not disclose any fusogenic activity in ex vivo assays, at variance with the syncytin-Car1 gene, which is found in all carnivorans, including the hyena, in which it is still present, transcriptionally active in the placenta, and fusogenic. Together, the present results illustrate the permanent renewal of placenta-specific genes by retroviral capture and de facto provide a candidate gene for the endotheliochorial to hemochorial transition of Hyaenidae among carnivorans.IMPORTANCE The placenta is the most diverse organ among mammals, due in part to stochastic capture of retroviral envelope genes. In carnivorans, capture of syncytin-Car1 took place 80 Mya. It is fusogenic, expressed at the syncytialized placental maternofetal interface, and conserved among all carnivorans, consistent with their shared endotheliochorial placenta. Hyenas are a remarkable exception, with a highly invasive hemochorial placenta, as found in humans, where disruption of maternal blood vessels results in maternal blood bathing the syncytial maternofetal interface. In this study, we identified a retroviral envelope gene capture and exaptation that took place about 30 Mya and is coincident with the emergence of the Hyaenidae, being conserved in all extant hyena species. It is expressed at the maternofetal interface in addition to the shared syncytin-Car1 gene. This new env gene, not present in any other carnivoran, is a likely candidate to be responsible for the specific structure of the hyena placenta.

Although it is now possible to produce recombinant HIV envelope glycoproteins (Envs) with epitopes recognized by the 5-6 major classes of broadly neutralizing antibodies (bNAbs), these have failed to consistently stimulate the formation of bNAbs in immunized animals or humans. In an effort to identify new immunogens better able to elicit bNAbs, we are studying Envs derived from rare individuals who possess bNAbs and are able to control their infection without the need for anti-retroviral drugs (elite supressors or ES), hypothesizing that in at least some people the antibodies may mediate durable virus control. Because virus evolution in people with the ES only phenotype was reported to be limited, we reasoned the Env proteins recovered from these individuals may more closely resemble the Envs that gave rise to bNAbs compared to the highly diverse viruses isolated from normal progressors. Using a phenotypic assay, we screened 25 controllers and identified two for more detailed investigation. In this study, we examined 20 clade B proviral sequences isolated from an African American woman, who had the rare bNAb/ES phenotype. Phylogenetic analysis of proviral envelope sequences demonstrated low genetic diversity. Envelope proteins were unusual in that most possessed two extra cysteines within an elongated V1 region. In this report, we examine the impact of the extra cysteines on the binding to bNAbs, virus infectivity, and sensitivity to neutralization. These data suggest structural motifs in V1 can affect infectivity, and that rare viruses may be prevented from developing escape.

Vibration analysis for conditional preventive maintenance is an essential tool for the industry. The vibration signals sensored, collected and analyzed can provide information about the state of an induction motor. Appropriate processing of these vibratory signals leads to define a normal or abnormal state of the whole rotating machinery, or in particular, one of its components. The main objective of this paper is to propose a method for automatic monitoring of bearing components condition of an induction motor. The proposed method is based on two approaches with one based on signal processing using the Hilbert spectral envelope and the other approach uses machine learning based on random forests. The Hilbert spectral envelope allows the extraction of frequency characteristics that are considered as new features entering the classifier. The frequencies chosen as features are determined from a proportional variation of their amplitudes with the variation of the load torque and the fault diameter. Furthermore, a random forest-based classifier can validate the effectiveness of extracted frequency characteristics as novel features to deal with bearing fault detection while automatically locating the faulty component with a classification rate of 99.94%. The results obtained with the proposed method have been validated experimentally using a test rig.

Aim: West Nile encephalitis caused by infection with the West Nile virus (WNV) is endemic in many regions of the world and is a global public health threat. The aim of this report was to develop a method using colorimetry‐based reverse‐transcription loop‐mediated isothermal amplification (cRT‐LAMP) and RT‐LAMP combined with lateral‐flow dipstick (LFD) for rapidly detecting WNV in low‐infrastructure settings. Methods and Results: The primers for the cRT‐LAMP and RT‐LAMP‐LFD assays were designed based on env gene of the WNV. Primers concentration, temperature and time were optimized for cRT‐LAMP and RT‐LAMP‐LFD. The diagnostic performance of the cRT‐LAMP and RT‐LAMP‐LFD assays was evaluated using human serum samples from 110 patients who were clinically suspected to be infected with WNV. The RT‐LAMP was performed in a heating block at 63°C for 40 min. The LAMP amplicons were visible in the lateral‐flow dipstick within 5 min. The detection limit of the developed cRT‐LAMP and RT‐LAMP‐LFD assays was 10 copies and this assay showed a high degree of specificity for WNV. Compared with quantitative real‐time RT‐PCR assay, the kappa value of cRT‐LAMP and RT‐LAMP‐LFD were 0.970. Conclusions: These results showed that the newly developed WNV‐specific cRT‐LAMP and RT‐LAMP‐LFD assays can be employed as an alternative method for screening of WN‐suspected human samples. The results revealed that the assay could potentially identify the virus without interference from human serum samples. Collectively, all results revealed that cRT‐LAMP and RT‐LAMP‐LFD assays offer a suitable field‐based diagnosis of WNV. Significance and Impact of the Study: The cRT‐LAMP and LAMP‐LFD platform for the detection of WNV is rapid, accurate and simple‐to‐perform. Our present method has not only a short turnaround time but also avoided cross‐contamination problem. Moreover, the use of simple lateral flow dipsticks broadens its application potential for the point‐of‐care use in resource‐limited settings during outbreak situations. To the best of our knowledge, this is the first report for the development of cRT‐LAMP and LAMP‐LFD assays for rapid, simple, specific and sensitive detection of WNV using human clinical samples and EvaGreen dye. [ABSTRACT FROM AUTHOR]

[Display omitted] Selective targeting of germline B cells with specifically designed germline-targeting HIV-1 envelope immunogens (GT-Env) is considered a feasible vaccination strategy to elicit broadly neutralizing antibodies (bnAbs). BnAbs are extremely valuable because they neutralize genetically distant viral strains at the same time. To overcome its inherently low affinity to germline B cells, the aim of the study was to present GT-Env via different immobilization strategies densely arrayed on the surface of nanoparticles. We engineered a prefusion-stabilized GT-Env trimer with affinity to VRC01 germline B cells using a bioinformatics-supported design approach. Distinct glycan modifications and amino acid substitutions yielded a GT-Env trimer which bound to the receptor with a K D of 11.5 µM. Silica nanoparticles with 200 nm diameter (SiNPs) were used for the multivalent display of the novel GT-Env with a 15 nm mean centre-to-centre spacing either by site-specific, covalent conjugation or at random, non-specific adsorption. Oriented, covalent GT-Env conjugation revealed better binding of structure dependent bnAbs as compared to non-specifically adsorbed GT-Env. In addition, GT-Env covalently attached activated a B cell line expressing the germline VRC01 receptor at an EC 50 value in the nanomolar range (4 nM), while soluble GT-Env required 1,000-fold higher concentrations to induce signalling. The significantly lower GT-Env concentration was likely required due to avidity effects, which were in the picomolar range. Thus, low affinity antigens may particularly benefit from a particulate and multivalent delivery. In future, SiNPs are ideal to be modified in a modular design with various GT-Env variants that target different stages of germline and bnAb precursor B cells. [ABSTRACT FROM AUTHOR]

The HIV-1 envelope glycoprotein (Env) contains a long cytoplasmic tail harboring highly conserved motifs that direct Env trafficking and incorporation into virions and promote efficient virus spread. The cellular trafficking factor Rab11a family interacting protein 1C (FIP1C) has been implicated in the directed trafficking of Env to sites of viral assembly. In this study, we confirm that small interfering RNA (siRNA)-mediated depletion of FIP1C in HeLa cells modestly reduces Env incorporation into virions. To determine whether FIP1C is required for Env incorporation and HIV-1 replication in physiologically relevant cells, CRISPR-Cas9 technology was used to knock out the expression of this protein in several human T-cell lines—Jurkat E6.1, SupT1, and H9—and in primary human CD4+ T cells. FIP1C knockout caused modest reductions in Env incorporation in SupT1 cells but did not inhibit virus replication in SupT1 or Jurkat E6.1 T cells. In H9 cells, FIP1C knockout caused a cell density-dependent defect in virus replication. In primary CD4+ T cells, FIP1C knockout had no effect on HIV-1 replication. Furthermore, human T-cell leukemia virus type 1 (HTLV-1)-transformed cell lines that are permissive for HIV-1 replication do not express FIP1C. Mutation of an aromatic motif in the Env cytoplasmic tail (Y795W) implicated in FIP1C-mediated Env incorporation impaired virus replication independently of FIP1C expression in SupT1, Jurkat E6.1, H9, and primary T cells. Together, these results indicate that while FIP1C may contribute to HIV-1 Env incorporation in some contexts, additional and potentially redundant host factors are likely required for Env incorporation and virus dissemination in T cells. IMPORTANCE The incorporation of the HIV-1 envelope (Env) glycoproteins, gp120 and gp4+, into virus particles is critical for virus infectivity. gp4+ contains a long cytoplasmic tail that has been proposed to interact with host cell factors, including the trafficking factor Rab11a family interacting protein 1C (FIP1C). To investigate the role of FIP1C in relevant cell types—human T-cell lines and primary CD4+ T cells— we used CRISPR-Cas9 to knock out FIP1C expression and examined the effect on HIV-1 Env incorporation and virus replication. We observed that in two of the T-cell lines examined (Jurkat E6.1 and SupT1) and in primary CD4+ T cells, FIP1C knockout did not disrupt HIV-1 replication, whereas FIP1C knockout reduced Env expression and delayed replication in H9 cells. The results indicate that while FIP1C may contribute to Env incorporation in some cell lines, it is not an essential factor for efficient HIV-1 replication in primary CD4+ T cells. [ABSTRACT FROM AUTHOR]

The HIV-1 envelope glycoprotein (Env) plays crucial role in viral infection by facilitating viral attachment to host cells and inducing fusion of the virus with the host cell membrane. This fusion allows the HIV-1 viral genome to enter the target cell then triggering various stages of the viral life cycle. The native Env directly interacts with the main receptor CD4 and the co-receptor (CCR5 or CXCR4) in human cell membrane then induces membrane fusion. The elucidation of the structure of Env with CD4 and co-receptors in different HIV-1 subtypes is essential for the understanding of the mechanism of virus entry. Here we report the Cryo-EM structure of the CD4-bound HIV-1 heterotrimeric Env from Asia prevalent CRF07_BC CH119 strain. In this structure, the binding of three CD4 molecules with Env induced extensively conformational changes in gp120, resulting in the transformation of the Env from close state to intermediate open state. Additionally, the conformational shift of V1/V2 loops of the heterotrimeric Env allosterically expose the V3 loop and promoting the further interactions with co-receptor CCR5 or CXCR4. These findings not only illustrate the structural complexity and plasticity of HIV-1 Env but also give new insights how the biological trimeric Env initialize the immune recognition and membrane fusion. • The purification of CD4-bound HIV-1 trimeric Env from CRF07_BC CH119. • Cryo-EM structure of HIV-1 trimeric Env in complex with CD4. • The allosterically exposure of V1/V2/V3 dynamic loops upon CD4 binding. • Heterotrimeric Env transformed from close to intermediate open state. [ABSTRACT FROM AUTHOR]

Avian leukosis virus subgroup J (ALV-J) generally induces hemangioma, myeloid leukosis, and immunosuppression in chickens, causing significant poultry industry economic losses worldwide. The unusual env gene of ALV-J, with low homology to other subgroups of ALVs, is associated with its unique pathogenesis. However, the exact molecular basis for the pathogenesis and oncogenesis of ALV-J is still not fully understood. In this study, ALV-J infection and the overexpression of Env could efficiently downregulate the phosphorylation of SHP-2 (pSHP-2) in vitro and in vivo. The membrane-spanning domain (MSD) in Env Gp37 was the functional domain responsible for pSHP-2 downregulation. Moreover, the overexpression of SHP-2 could effectively promote the replication of ALV-J, whereas knockout or allosteric inhibition of SHP-2 could inhibit ALV-J replication. In addition, the knockout of endogenous chicken SHP-2 could significantly increase the proliferation ability of DF-1 cells. All these data demonstrate that SHP-2 dephosphorylated by ALV-J Env could efficiently promote ALV-J replication, highlighting the important role of SHP-2 in the pathogenesis of ALV-J and providing a new target for developing antiviral drugs against ALV-J.

Retroviral envelope (Env) proteins have long been recognized to exhibit immunosuppressive properties, which affect the CD8+ T-cell response to an infection but also to immunization. Interestingly, we previously showed in the Friend murine leukemia virus (F-MuLV) model that the surface Env protein gp70 also plays a role in immunosuppression, in addition to the immunosuppressive function attributed to the transmembrane Env protein. We now demonstrate that immunization with F-MuLV Env leads to a significant increase in interleukin-10 (IL-10)-producing CD4+ T cells and that the induction of CD8+ T-cell responses in the presence of Env is rescued if the capacity of CD4+ T cells to produce IL-10 is abrogated, indicating a mechanistic role of IL-10-producing CD4+ T cells in mediating the Env-induced suppression of CD8+ T-cell responses in Env co-immunization. We found that CD8+ T-cell responses against different immunogens are not all equally affected. On the other hand, suppression of immunity was observed not only in co-immunization experiments but also for immune control of subcutaneous tumor growth after an Env immunization. Finally, we show that suppression of CD8+ T cells by the surface Env protein is observed not only for Friend MuLV Env but also for the Env proteins of other gamma retroviruses. Taken together, our results show that IL-10-producing CD4+ T cells mechanistically underlie the Env-mediated suppression of CD8+ T-cell responses and suggest the presence of an immunosuppressive motif in the surface Env protein of gamma retroviruses.

The expression of genes is altered in various diseases and is responsible for the disease's initiation, progression and pathology. Several other genes, predominantly inactivated, may become activated in a given condition and contribute to the initiation and progression of the disease. Similarly, human endogenous viruses (HERVs) are an incomplete, non-productive and inactive viral sequence present in the heterochromatin of the human genome, and are often referred to as junk DNA. HERVs were inserted into the host genome millions of years ago. However, they were silenced due to multiple mutations and recombination that occurred over time. However, their expression is increased in cancers due to either epigenetic or transcriptional dysregulation. Some of the HERVs having intact open reading frames have been reported to express virus-like particles, functional peptides and proteins involved in tumorigenesis. To summarize, there is involvement of different HERVs in the initiation and progression of several cancers. The present review aims to provide concise information on HERV and its involvement in the initiation and progression of multiple types of cancer. [ABSTRACT FROM AUTHOR]

Elicitation of broadly neutralizing antibodies (bnAbs) is a goal of vaccine design as a strategy for targeting highly divergent strains of HIV-1. Current HIV-1 vaccine design efforts seek to elicit bnAbs by first eliciting their precursors through prime-boost regimens. This requires an understanding of the co-evolution between viruses and antibodies. Towards this goal, we have analyzed two cooperating antibodies, DH475 and DH272, which exerted pressure on the HIV population in an infected donor, called CH848, to evolve in such a way that it became sensitive to the V3-glycan supersite DH270 bnAb lineage. We obtained a 2.90Å crystal structure of DH475 in complex with the Man9 glycan and a negative stain EM model of DH272 in complex with the HIV-1 spike trimer, Env. Coupled with additional modeling studies and biochemical data, our studies reveal that DH475 contacts a V3- and V4-glycan dependent epitope accessible on an open or shed Env and that DH272 makes critical contacts with the V1V2 and V3 loops on HIV-1 Env. Using these data, we suggest a prime-boost regimen that may facilitate the initiation of DH270-like bnAb precursors [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]

Background: Avian leukosis virus (ALV) is an infectious retrovirus, that mainly causes various forms of tumours, immunosuppression, a decreased egg production rate and slow weight gain in poultry. ALV consists of 11 subgroups, A–K, among which ALV-K is an emerging subgroup that has become prevalent in the past 10 years. Most ALV-K isolates showed weak replication ability and pathogenicity. In this study, the weak replication ability of ALV-K was explored from the perspective of the interaction between ALV-K gp85 and the Tva receptor. Methods: Fourteen soluble recombinant ALV-A/K gp85 chimeric proteins were constructed by substituting the sequence difference regions (hr1, hr2 and vr3) of the ALV-A gp85 protein with the skeleton ALV-K gp85 protein for co-IP and competitive blocking tests. Results: The binding capacity of ALV-K gp85 to Tva was significantly weaker than that of ALV-A gp85 (P < 0.05) and the key amino acid sites 199–205, 269, 319, 321 and 324 of ALV-K env contributed to the weaker replication capacity of ALV-K than ALV-A. Conclusions: This is the first study to reveal the molecular factors of the weak replication ability of ALV-K from the perspective of the interaction of ALV-K gp85 to Tva, providing a basis for further elucidation of the infection mechanism of ALV-K. [ABSTRACT FROM AUTHOR]

HIV-1 envelope glycoprotein (Env) conformation substantially impacts antibody-dependent cellular cytotoxicity (ADCC). Envs from primary HIV-1 isolates adopt a prefusion "closed" conformation, which is targeted by broadly neutralizing antibodies (bnAbs). CD4 binding drives Env into more "open" conformations, which are recognized by non-neutralizing Abs (nnAbs). To better understand Env-Ab and Env-CD4 interaction in CD4+ T cells infected with HIV-1, we simultaneously measured antibody binding and HIV-1 mRNA expression using multiparametric flow cytometry and RNA flow fluorescent in situ hybridization (FISH) techniques. We observed that env mRNA is almost exclusively expressed by HIV-1 productively infected cells that already downmodulated CD4. This suggests that CD4 downmodulation precedes env mRNA expression. Consequently, productively infected cells express "closed" Envs on their surface, which renders them resistant to nnAbs. Cells recognized by nnAbs were all env mRNA negative, indicating Ab binding through shed gp120 or virions attached to their surface. Consistent with these findings, treatment of HIV-1-infected humanized mice with the ADCC-mediating nnAb A32 failed to lower viral replication or reduce the size of the viral reservoir. These findings confirm the resistance of productively infected CD4+ T cells to nnAbs-mediated ADCC and question the rationale of immunotherapy approaches using this strategy., Importance: Antibody-dependent cellular cytotoxicity (ADCC) represents an effective immune response for clearing virally infected cells, making ADCC-mediating antibodies promising therapeutic candidates for HIV-1 cure strategies. Broadly neutralizing antibodies (bNAbs) target epitopes present on the native "closed" envelope glycoprotein (Env), while non-neutralizing antibodies (nnAbs) recognize epitopes exposed upon Env-CD4 interaction. Here, we provide evidence that env mRNA is predominantly expressed by productively infected cells that have already downmodulated cell-surface CD4. This indicates that CD4 downmodulation by HIV-1 precedes Env expression, making productively infected cells resistant to ADCC mediated by nnAbs but sensitive to those mediated by bnAbs. These findings offer critical insights for the development of immunotherapy-based strategies aimed at targeting and eliminating productively infected cells in people living with HIV.

CD4-mimetics (CD4mcs) are small molecule compounds that mimic the interaction of the CD4 receptor with HIV-1 envelope glycoproteins (Env). Env from primary viruses normally samples a "closed" conformation that occludes epitopes recognized by CD4-induced (CD4i) non-neutralizing antibodies (nnAbs). CD4mcs induce conformational changes on Env resulting in the exposure of these otherwise inaccessible epitopes. Here, we evaluated the capacity of plasma from a cohort of 50 people living with HIV to recognize HIV-1-infected cells and eliminate them by antibody-dependent cellular cytotoxicity (ADCC) in the presence of a potent indoline CD4mc. We observed a marked heterogeneity among plasma samples. By measuring the levels of different families of CD4i Abs, we found that the levels of anti-cluster A, anti-coreceptor binding site, and anti-gp41 cluster I antibodies are responsible for plasma-mediated ADCC in the presence of CD4mc., Importance: There are several reasons that make it difficult to target the HIV reservoir. One of them is the capacity of infected cells to prevent the recognition of HIV-1 envelope glycoproteins (Env) by commonly elicited antibodies in people living with HIV. Small CD4-mimetic compounds expose otherwise occluded Env epitopes, thus enabling their recognition by non-neutralizing antibodies (nnAbs). A better understanding of the contribution of these antibodies to eliminate infected cells in the presence of CD4mc could lead to the development of therapeutic cure strategies.

The HIV-1 envelope glycoprotein (Env) is expressed at the surface of infected cells and as such can be targeted by non-neutralizing antibodies (nnAbs) that mediate antibody-dependent cellular cytotoxicity (ADCC). Previous single-molecule Förster resonance energy transfer (smFRET) studies demonstrated that Env from clinical isolates predominantly adopt a "closed" conformation (State 1), which is resistant to nnAbs. After interacting with the cellular receptor CD4, the conformational equilibrium of Env shifts toward States 2 and 3, exposing the coreceptor binding site (CoRBS) and permitting binding of antibodies targeting this site. We showed that the binding of anti-CoRBS Abs enables the engagement of other nnAbs that target the cluster A epitopes on Env. Anti-cluster A nnAbs stabilize an asymmetric Env conformation, State 2A, and have potent ADCC activity. CRF01&#95;AE strains were suggested to be intrinsically susceptible to ADCC mediated by nnAbs. This may be due to the presence of a histidine at position 375, known to shift Env towards more "open" conformations. In this work, through adaptation of an established smFRET imaging approach, we report that the conformational dynamics of native, unliganded HIV-1 CRF01&#95;AE Env indicates frequent sampling of the State 2A conformation. This is in striking contrast with Envs from clades A and B, for example HIV-1 JR-FL , which do not transition to State 2A in the absence of ligands. These findings inform on the conformational dynamics of HIV-1 CRF01&#95;AE Env, which are relevant for structure-based design of both synthetic inhibitors of receptor binding, and enhancers of ADCC as therapeutic alternatives., Competing Interests: Conflicts of Interest The authors declare no competing interests.

Self-amplifying mRNA (saRNA) represents a promising platform for nucleic acid delivery of vaccine immunogens. Unlike plasmid DNA, saRNA does not require entry into the nucleus of target cells for expression, having the capacity to drive higher protein expression compared to mRNA as it replicates within the cytoplasm. In this study, we examined the potential of stabilized native-like HIV-1 Envelope glycoprotein (Env) trimers to elicit immune responses when delivered by saRNA polyplexes (PLXs), assembled with linear polyethylenimine. We showed that Venezuelan equine encephalitis virus (VEEV) saRNA induces a stronger humoral immune response to the encoded transgene compared to Semliki Forest virus saRNA. Moreover, we characterized the immunogenicity of the soluble and membrane-bound ConSOSL.UFO Env design in mice and showed a faster humoral kinetic and an immunoglobulin G (IgG)2a skew using a membrane-bound design. The immune response generated by PLX VEEV saRNA encoding the membrane-bound Env was then evaluated in larger animal models including macaques, in which low doses induced high IgG responses. Our data demonstrated that the VEEV saRNA PLX nanoparticle formulation represents a suitable platform for the delivery of stabilized HIV-1 Env and has the potential to be used in a variety of vaccine regimens.

Elicitation of broadly neutralizing antibodies (bnAbs) is a goal of vaccine design as a strategy for targeting highly divergent strains of HIV-1. Current HIV-1 vaccine design efforts seek to elicit bnAbs by first eliciting their precursors through prime-boost regimens. This requires an understanding of the co-evolution between viruses and antibodies. Towards this goal, we have analyzed two cooperating antibodies, DH475 and DH272, which exerted pressure on the HIV population in an infected donor, called CH848, to evolve in such a way that it became sensitive to the V3-glycan supersite DH270 bnAb lineage. We obtained a 2.90Å crystal structure of DH475 in complex with the Man9 glycan and a negative stain EM model of DH272 in complex with the HIV-1 spike trimer, Env. Coupled with additional modeling studies and biochemical data, our studies reveal that DH475 contacts a V3- and V4-glycan dependent epitope accessible on an open or shed Env and that DH272 makes critical contacts with the V1V2 and V3 loops on HIV-1 Env. Using these data, we suggest a prime-boost regimen that may facilitate the initiation of DH270-like bnAb precursors.

Production of infectious HIV-1 particles requires incorporation of the viral envelope glycoprotein (Env) at the plasma membrane (PM) of infected CD41 T cells. Env trafficking to the PM exposes viral epitopes that can be exploited by the host immune system; however, HIV-1 can evade this response by endocytosis of excess Env from the PM. The fate of Env after internalization remains unclear, with evidence suggesting several different vesicular trafficking steps may be involved, including recycling pathways. To date, there have been very few studies documenting the trafficking pathways of native Env in infected T cells. Furthermore, it remains unclear whether there are T-cell-specific endosomal pathways regulating the fate of endocytic Env. Here, we use a pulse-labeling approach with a monovalent anti-Env Fab probe to characterize the trafficking of internalized Env within infected CD41 T-cell lines, together with CRISPR/Cas9-mediated endogenous protein tagging, to assess the role of host cell Rab GTPases in Env trafficking. We show that endocytosed Env traffics to Rab141 compartments that possess hallmarks of late endosomes and lysosomes. We also demonstrate that Env can recycle back to the PM, although we find that recycling does not occur at high rates when compared to the model recycling protein transferrin. These results help to resolve open questions about the fate and relevance of endocytosed Env in HIV-infected cells and suggest a novel role for Rab14 in a cell-type-specific late-endosomal/lysosomal trafficking pathway in T cells. IMPORTANCE HIV-1 envelope glycoprotein (Env) evades immune neutralization through many mechanisms. One immune evasion strategy may result from the internalization of excess surface-exposed Env to prevent antibody-dependent cellular cytotoxicity or neutralization. Characterization of the fate of endocytosed Env is critical to understand which vesicular pathways could be targeted to promote display of Env epitopes to the immune system. In this study, we characterize the endocytic fate of native Env, expressed from infected human T-cell lines. We demonstrate that Env is rapidly trafficked to a late-endosome/lysosome-like compartment and can be recycled to the cell surface for incorporation into virus assembly sites. This study implicates a novel intracellular compartment, marked by host-cell Rab14 GTPases, for the sequestration of Env. Therapeutic approaches aimed at mobilizing this intracellular pool of Env could lead to stronger immune control of HIV-1 infection via antibody-dependent cell-mediated cytotoxicity. [ABSTRACT FROM AUTHOR]

BackgroundInvestigations into which human immunodeficiency virus type 1 (HIV-1) sequence features may be selected for transmission during sexual exposure have been hampered by the small number of characterized transmission pairs in individual studies.MethodsTo boost statistical power to detect differences in glycosylation, length, and electrical charge in the HIV-1 V1-V4 coding region, we reanalyzed all available 2485 env sequences derived from 114 subjects representing 58 transmission pairs from previous studies using mixed-effects linear regression and an approach to approximate the unobserved transmitted virus.ResultsThe recipient partner had a shorter V1-V4 region and fewer potential N-linked glycosylation sites (PNGS) than sequences from the source partner. We also detected a trend toward more PNGS and lower isoelectric points in transmitted sequences with source partner and the evolutionary tendency to shorten V1-V4 sequences, reduce the number of PNGS, and lower isoelectric points in the recipient following transmission.ConclusionsBy using all available well-characterized env sequences from transmission pairs via sexual exposure, we were able to identify several important virologic factors that may be important in the development of biomedical preventive interventions.

The HIV-1 Rev protein activates nuclear export of unspliced and partially spliced viral RNA transcripts, which encode the viral genome and the genes encoding viral structural proteins, by binding to and oligomerizing on the Rev Response Element (RRE). The human DEAD-box protein 1 (DDX1) enhances the RNA export activity of Rev through an unknown mechanism. Using a single-molecule assembly assay and various DDX1 mutants, we show that DDX1 acts through the RRE RNA to specifically accelerate the nucleation step of the Rev-RRE assembly process. Single-molecule Förster resonance energy transfer (smFRET) experiments using donor-labeled Rev and acceptor-labeled DDX1 show that both proteins can associate with a single RRE molecule. However, simultaneous interaction is only observed in a subset of binding events and does not explain the extent to which DDX1 promotes the nucleation step of Rev-RRE assembly. Together, these results are consistent with a model wherein DDX1 acts as an RNA chaperone, remodeling the RRE into a conformation that is pre-organized to bind the first Rev monomer, thereby promoting the overall Rev-RRE assembly process.

Overlapping coding regions balance selective forces between multiple genes. One possible division of nucleotide sequence is that the predominant selective force on a particular nucleotide can be attributed to just one gene. While this arrangement has been observed in regions in which one gene is structured and the other is disordered, we sought to explore how overlapping genes balance constraints when both protein products are structured over the same sequence. We use a combination of sequence analysis, functional assays, and selection experiments to examine an overlapped region in HIV-1 that encodes helical regions in both Env and Rev. We find that functional segregation occurs even in this overlap, with each protein spacing its functional residues in a manner that allows a mutable non-binding face of one helix to encode important functional residues on a charged face in the other helix. Additionally, our experiments reveal novel and critical functional residues in Env and have implications for the therapeutic targeting of HIV-1.

SIRE1 is an active and relatively high copy-number retroelement belongs to the Tyl/Copia long terminal repeat (LTR) retrotransposon superfamily. Distinctive SIRE1 elements (ENV and GAG) distributions in barley genome were observed by using fluorescent in situ hybridization (FISH). We performed PCR to obtain tetramethylrhodamine-dUTP (TRITC)-labelled probes. Localizations of SIRE1 ENV and GAG domains were demonstrated under confocal microscope on Hordeum vulgare L. cv. Hasat root preparations. Our results revealed the distributions of SIRE1 elements ENV and GAG in barley genome. These results may provide to uncover the organization of SIRE retrotransposon pattern in barley genome.

[Display omitted] Immunization is a straightforward concept but remains for some pathogens like HIV-1 a challenge. Thus, new approaches towards increasing the efficacy of vaccines are required to turn the tide. There is increasing evidence that antigen exposure over several days to weeks induces a much stronger and more sustained immune response compared to traditional bolus injection, which usually leads to antigen elimination from the body within a couple of days. Therefore, we developed a poly(ethylene) glycol (PEG) hydrogel platform to investigate the principal feasibility of a sustained release of antigens to mimic natural infection kinetics. Eight-and four-armed PEG macromonomers of different MWs (10, 20, and 40 kDa) were end-group functionalized to allow for hydrogel formation via covalent cross-linking. An HIV-1 envelope (Env) antigen in its trimeric (Env tri) or monomeric (Env mono) form was applied. The soluble Env antigen was compared to a formulation of Env attached to silica nanoparticles (Env-SiNPs). The latter are known to have a higher immunogenicity compared to their soluble counterparts. Hydrogels were tunable regarding the rheological behavior allowing for different degradation times and release timeframes of Env-SiNPs over two to up to 50 days. Affinity measurements of the VCR01 antibody which specifically recognizes the CD4 binding site of Env, revealed that neither the integrity nor the functionality of Env mono -SiNPs (K d = 2.1 ± 0.9 nM) and Env tri -SiNPs (K d = 1.5 ± 1.3 nM), respectively, were impaired after release from the hydrogel (K d before release: 2.1 ± 0.1 and 7.8 ± 5.3 nM, respectively). Finally, soluble Env and Env-SiNPs which are two physico-chemically distinct compounds, were co-delivered and shown to be sequentially released from one hydrogel which could be beneficial in terms of heterologous immunization or single dose vaccination. In summary, this study presents a tunable, versatile applicable, and effective delivery platform that could improve vaccination effectiveness also for other infectious diseases than HIV-1. [ABSTRACT FROM AUTHOR]

Vibration analysis for conditional preventive maintenance is an essential tool for the industry. The vibration signals sensored, collected and analyzed can provide information about the state of an induction motor. Appropriate processing of these vibratory signals leads to define a normal or abnormal state of the whole rotating machinery, or in particular, one of its components. The main objective of this paper is to propose a method for automatic monitoring of bearing components condition of an induction motor. The proposed method is based on two approaches with one based on signal processing using the Hilbert spectral envelope and the other approach uses machine learning based on random forests. The Hilbert spectral envelope allows the extraction of frequency characteristics that are considered as new features entering the classifier. The frequencies chosen as features are determined from a proportional variation of their amplitudes with the variation of the load torque and the fault diameter. Furthermore, a random forest-based classifier can validate the effectiveness of extracted frequency characteristics as novel features to deal with bearing fault detection while automatically locating the faulty component with a classification rate of 99.94%. The results obtained with the proposed method have been validated experimentally using a test rig. [ABSTRACT FROM AUTHOR]

The HIV-1 Nef and Vpu accessory proteins are known to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) responses by limiting exposure of CD4-induced (CD4+) envelope (Env) epitopes at the cell surface. Although both proteins target the host receptor CD4 for degradation, the extent of their functional redundancy is unknown. Here, we developed an intracellular staining technique that permits the intracellular detection of both Nef and Vpu in primary CD41 T cells by flow cytometry. Using this method, we show that the combined expression of Nef and Vpu predicts the susceptibility of HIV-1-infected primary CD4+ T cells to ADCC by HIV1 plasma. We also show that Vpu cannot compensate for the absence of Nef, thus providing an explanation for why some infectious molecular clones that carry a LucR reporter gene upstream of Nef render infected cells more susceptible to ADCC responses. Our method thus represents a new tool to dissect the biological activity of Nef and Vpu in the context of other host and viral proteins within single infected CD4+ T cells. [ABSTRACT FROM AUTHOR]

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Background: Breast cancer is one of the most common malignancies in women worldwide. Global Cancer Observatory (GLOBOCAN) data for 2019 show that about two million women are diagnosed with breast cancer each year. Human endogenous retroviruses (HERV) can be involved in the development of various types of cancer. This study aimed to investigate the expression of the Env gene in the tissues of patients with breast cancer. Methods: The present case-control study was performed on 42 women with breast cancer at the age range of 40-49 years admitted to Alzahra hospital in Isfahan City and Ayatollah Kashani hospital in Shahrekord City, Iran, who underwent surgery from October 2018 to April 2019. Tumor tissues as cases and tumor margins as controls were used for RNA extraction and cDNA synthesis. Env gene expression was assessed by relative quantitation using designed primers and real-time polymerase chain reaction (PCR). Statistical analyses including Kruskal–Wallis and Wilcoxon tests were performed using SPSS software. Findings: No significant difference was observed between Env gene expression in cancerous and noncancerous tissues. Furthermore, the Env gene expression did not show a significant relationship with the stage and grade of the tumor. Conclusion: Unlike previous studies, in the present study, no differences were found between the Env gene in tumor state and normal tumor margin tissue. This result may be due to different geographical and racial conditions or due to inherent heterogeneity in tumors. In any case, more research is needed in this area.

ABSTRACT The HIV Env glycoprotein is the surface glycoprotein responsible for viral entry into CD4+ immune cells. During infection, Env also serves as a primary target for antibody responses, which are robust but unable to control virus replication. Immune evasion by HIV-1 Env appears to employ complex mechanisms to regulate what antigenic states are presented to the immune system. Immunodominant features appear to be distinct from epitopes that interfere with Env functions in mediating infection. Further, cell-cell transmission studies indicate that vulnerable conformational states are additionally hidden from recognition on infected cells, even though the presence of Env at the cell surface is required for viral infection through the virological synapse. Cell-cell infection studies support that Env on infected cells is presented in distinct conformations from that on virus particles. Here we review data regarding the regulation of conformational states of Env and assess how regulated sorting of Env within the infected cell may underlie mechanisms to distinguish Env on the surface of virus particles versus Env on the surface of infected cells. These mechanisms may allow infected cells to avoid opsonization, providing cell-to-cell infection by HIV with a selective advantage during evolution within an infected individual. Understanding how distinct Env conformations are presented on cells versus viruses may be essential to designing effective vaccine approaches and therapeutic strategies to clear infected cell reservoirs.

The recombinant ALVAC vaccine coupled with the monomeric gp120/alum protein have decreased the risk of HIV and SIV acquisition. Ab responses to the V1/V2 regions have correlated with a decreased risk of virus acquisition in both humans and macaques. We hypothesized that the breadth and functional profile of Abs induced by an ALVAC/envelope protein regimen could be improved by substituting the monomeric gp120 boost, with the full-length single-chain (FLSC) protein. FLSC is a CD4-gp120 fusion immunogen that exposes cryptic gp120 epitopes to the immune system. We compared the immunogenicity and relative efficiency of an ALVAC-SIV vaccine boosted either with bivalent FLSC proteins or with monomeric gp120 in alum. FLSC was superior to monomeric gp120 in directing Abs to the C3 α2 helix, the V5 loop, and the V3 region that contains the putative CCR5 binding site. In addition, FLSC boosting elicited significantly higher binding Abs to V2 and increased both the Ab-dependent cellular cytotoxicity activity and the breadth of neutralizing Abs. However, the FLSC vaccine regimen demonstrated only a trend in vaccine efficacy, whereas the monomeric gp120 regimen significantly decreased the risk of SIVmac251 acquisition. In both vaccine regimens, anti-V2 Abs correlated with a decreased risk of virus acquisition but differed with regard to systemic or mucosal origin. In the FLSC regimen, serum Abs to V2 correlated, whereas in the monomeric gp120 regimen, V2 Abs in rectal secretions, the site of viral challenge, were associated with efficacy.