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International audience; 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.

West Nile virus (WNV) is a mosquito-borne virus of public health importance. Currently, there is no FDA approved vaccine available against WNV infection in humans. Therefore, the early diagnosis of the WNV infection is important for epidemiologic control and timely clinical management in areas where multiple Flaviviruses are endemic. The present study aimed to develop reverse transcription polymerase spiral reaction (RT-PSR) assay that rapidly and accurately detects the envelope (env) gene of WNV. RT-PSR assay was optimized at 63°C for 60 min using real-time turbidimeter or visual detection by the addition of SYBR Green I dye. The standard curve for RT-PSR assay was generated using the 10-fold serial dilutions of in vitro transcribed WNV RNA. To determine the detection limit of RT-PSR assay, an amplified product of conventional RT-PCR was in vitro transcribed as per standard protocol. The detection limit of the newly developed RT-PSR assay was compared with that of conventional RT-PCR and CDC reported TaqMan real-time RT-PCR using a serial 10-fold dilution of IVT WNV RNA. The detection limit of RT-PSR was found to be 1 RNA copy, which is 100-fold higher than that of conventional RT-PCR (100 copies). This suggests that RT-PSR assay is a valuable diagnostic tool for rapid and real-time detection of WNV in acute-phase serum samples. The assay was validated with a panel of 107 WNV suspected human clinical samples with signs of acute posterior uveitis and onset of febrile illness. Out of 107 samples, 30 were found positive by RT-PSR assay. The specificities of the selected primer sets were established by the absence of cross-reactivity with other closely related members viruses of the Flaviviruses, Alphaviruses, and Morbilliviruses groups. No cross-reactivity was observed with other viruses. To best of our knowledge, this is the first report describing the RT-PSR assay for the detection of RNA virus (WNV) in clinical samples. RT-PSR is a high throughput method and more than 30 reactions can be run at once in real-time turbidimeter. PSR assay has potential to be used for a rapid screening of large number of clinical samples in endemic areas during an outbreak.

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.

Background The binding of HIV-1 Envelope glycoproteins (Env) to host receptor CD4 exposes vulnerable conserved epitopes within the co-receptor binding site (CoRBS) which are required for the engagement of either CCR5 or CXCR4 co-receptor to allow HIV-1 entry. Antibodies against this region have been implicated in the protection against HIV acquisition in non-human primate (NHP) challenge studies and found to act synergistically with antibodies of other specificities to deliver effective Fc-mediated effector function against HIV-1-infected cells. Here, we describe the structure and function of N12-i2, an antibody isolated from an HIV-1-infected individual, and show how the unique structural features of this antibody allow for its effective Env recognition and Fc-mediated effector function. Results N12-i2 binds within the CoRBS utilizing two adjacent sulfo-tyrosines (TYS) for binding, one of which binds to a previously unknown TYS binding pocket formed by gp120 residues of high sequence conservation among HIV-1 strains. Structural alignment with gp120 in complex with the co-receptor CCR5 indicates that the new pocket corresponds to TYS at position 15 of CCR5. In addition, structure-function analysis of N12-i2 and other CoRBS-specific antibodies indicates a link between modes of antibody binding within the CoRBS and Fc-mediated effector activities. The efficiency of antibody-dependent cellular cytotoxicity (ADCC) correlated with both the level of antibody binding and the mode of antibody attachment to the epitope region, specifically with the way the Fc region was oriented relative to the target cell surface. Antibodies with poor Fc access mediated the poorest ADCC whereas those with their Fc region readily accessible for interaction with effector cells mediated the most potent ADCC. Conclusion Our data identify a previously unknown binding site for TYS within the assembled CoRBS of the HIV-1 virus. In addition, our combined structural-modeling-functional analyses provide new insights into mechanisms of Fc-effector function of antibodies against HIV-1, in particular, how antibody binding to Env antigen affects the efficiency of ADCC response.

HIV-1 encodes an envelope glycoprotein complex (Env) containing a long cytoplasmic tail (CT) harboring trafficking motifs implicated in Env incorporation into virions. Although the requirement for the Env CT in viral transmission is known, the precise mechanism by which Env is incorporated into nascent virions and localizes to the virological synapse remains poorly defined. To further elucidate the mechanism of Env trafficking, we examined three HIV-1 strains: the lab-adapted clade B strain, NL4-3, and a transmitted/founder (T/F) clade C virus, K3016, and a T/F clade B virus, CH077. The HIV-1 Env CT contains two invariant trafficking motifs: tyrosine endocytosis motif, Y712SPL, and C-terminal dileucine motif, LL855. Virion Env incorporation analysis revealed that Y712SPL is necessary for efficient Env incorporation, while LL855 is dispensable. Spreading infection kinetics were analyzed in various T-cell lines and primary human PBMCs; the results indicated that both endocytic motifs contribute to efficient viral spread in culture. Analysis of Env localization to the T-cell uropod, the portion of the plasma membrane that forms a virological synapse with uninfected cells, was found to be dependent on the Env CT and the Y712SPL motif. Cell-to-cell and cell-free transmission assays using T cells infected with HIV-1 bearing Y712A or LL855AA Env CT mutations are ongoing to establish a role for these motifs in both modes of viral transmission. These studies will significantly enhance our understanding of Env trafficking and viral transmission, providing insights into viral Env–host interactions in physiologically relevant cells.

The Phe43 cavity of HIV-1 envelope glycoproteins (Env) is an attractive druggable target. New promising compounds, including small CD4 mimetics (CD4mc), were shown to insert deeply into this cavity. Here, we identify a new network of residues that helps to shape this highly conserved CD4 binding pocket and characterize the structural determinants responsible for Env sensitivity to small CD4 mimetics., The HIV-1 envelope glycoproteins (Env) undergo conformational changes upon interaction of the gp120 exterior glycoprotein with the CD4 receptor. The gp120 inner domain topological layers facilitate the transition of Env to the CD4-bound conformation. CD4 engages gp120 by introducing its phenylalanine 43 (Phe43) in a cavity (“the Phe43 cavity”) located at the interface between the inner and outer gp120 domains. Small CD4-mimetic compounds (CD4mc) can bind within the Phe43 cavity and trigger conformational changes similar to those induced by CD4. Crystal structures of CD4mc in complex with a modified CRF01_AE gp120 core revealed the importance of these gp120 inner domain layers in stabilizing the Phe43 cavity and shaping the CD4 binding site. Our studies reveal a complex interplay between the gp120 inner domain and the Phe43 cavity and generate useful information for the development of more-potent CD4mc.

Miura, Tomoyuki/0000-0002-1956-2180 WOS: 000524306100042 PubMed: 32138199 A tier 2 SHIV-MK38 strain was obtained after two in vivo passages of tier 1 SHIV-MK1. SHIV-MK38#818, cloned from the MK38 strain, was neutralisation-resistant, like the parental MK38 strain, to SHIV-infected monkey plasma (MP), HIV-1-infected human pooled plasma (HPP), and KD247 monoclonal antibody (mAb) (anti-V3 gp120 env). We investigated the mechanisms underlying the resistance of #818, specifically the amino acid substitutions that confer resistance to MK1. We introduced amino acid substitutions in the MK1 envelope by in vitro mutagenesis and then compared the neutralisation resistance to MP, HPP, and KD247 mAb with #818 in a neutralisation assay using TZM-bl cells. We selected 11 substitutions in the V1, V2, C2, V4, C4, and V5 regions based on the alignment of env of MK1 and #818. The neutralisation resistance of the mutant MK1s with 7 of 11 substitutions in the V1, C2, C4, and V5 regions did not change significantly. These substitutions did not alter any negative charges or N-glycans. The substitutions N169D and K187E, which added negative charges, and S190N in the V2 region of gp120 and A389T in V4, which created sites for N-glycan, conferred high neutralisation resistance. The combinations N169D+K187E, N169D+S190N, and N169D+A389T resulted in MK1 neutralisation resistance close to that of #818. The combinations without 169D were neutralisation-sensitive. Therefore, N169D is the most important substitution for neutralisation resistance. This study demonstrated that although the V3 region sequences of #818 and MK1 are the same, V3 binding antibodies cannot neutralise #818 pseudovirus. Instead, mutations in the V2 and V4 regions inhibit the neutralisation of anti-V3 antibodies. We hypothesised that 169D and 190N altered the MK1 Env conformation so that the V3 region is buried. Therefore, the V2 region may block KD247 from binding to the tip of the V3 region. Research on HIV/AIDS grant from The Ministry of Health, Labour and Welfare of JapanMinistry of Health, Labour and Welfare, Japan; Japan Society for the Promotion of ScienceMinistry of Education, Culture, Sports, Science and Technology, Japan (MEXT)Japan Society for the Promotion of Science [16H04682]; Japan Agency for Medical Research and Development (AMED)Japan Agency for Medical Research and Development (AMED) [JP18fk0410011, JP18fk0410002, JP18fk0410013] This work was supported by a Research on HIV/AIDS grant from The Ministry of Health, Labour and Welfare of Japan, by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (Grant No. 16H04682), and by a grant from the Japan Agency for Medical Research and Development (AMED) (Grants no. JP18fk0410011, JP18fk0410002, and JP18fk0410013).

International audience; The HIV-1 Env protein is exposed at the surface of virions and infected cells. Env fluctuates between different closed and open structural states and these conformations influence both viral infectivity and sensitivity to antibody binding and neutralization. We established a flow virometry assay to visualize Env proteins at the surface of human immunodeficiency virus type 1 (HIV-1) virions. The assay is performed on ultracentrifuged fluorescent viral particles that are stained with a panel of broadly neutralizing antibodies (bNAbs) and nonneutralizing antibodies (nnAbs) that probe different epitopes of Env. We used this assay to compare Env at the surface of producer cells and viral particles and to analyze the effect of Nef, CD4, and SERINC5 on Env accessibility to antibodies. We studied the laboratory-adapted strain NL4-3 and two transmitted/founder viruses, THRO and CH058. We confirm that antibody accessibility varies between viral strains and show that Nef, CD4, and SERINC5 additively impact Env conformations. We further demonstrate that the Env accessibility profile on virions is globally similar to that observed on HIV-1-infected cells, with some noticeable differences. For instance, nnAbs bind to virions more efficiently than to producer cells, likely reflecting changes in Env conformational states on mature viral particles. This test complements other techniques and provides a convenient and simple tool for quantifying and probing the structure of Env at the virion surface and to analyze the impact of viral and cellular proteins on these parameters.IMPORTANCE HIV-1 Env conformation is one of the key parameters determining viral infectivity. The flow virometry-based assay developed in this study allows for the characterization of proteins incorporated in HIV-1 particles. We studied the conformation of HIV-1 Env and the impact that the viral protein Nef and the cellular proteins CD4 and SERINC5 have on Env accessibility to antibodies. Our assay permitted us to highlight some noticeable differences in the conformation of Env between producer cells and viral particles. It contributes to a better understanding of the actual composition of HIV-1 particles.

[Euskaraz] Giza Immunoeskasiaren Birusaren (GIBaren) infekzioa mundu osoan zehar zabaldutako Hartutako Immuno Eskasiaren Sindromearen (HIESaren) kausa da. GIBaren mintzean jatorri birikoko proteina bakarra adierazten da: gainazal glikoproteina (Env). Env bi azpiunitatez osatutako trimero gisa txertatzen da mintzean: mintz kanpoko gp120 proteina eta gp41 transmintz proteina. GIBaren gp41 proteinak, Lentivirus generoko gainontzeko birus gehienek ez bezala, domeinu zitoplasmatiko (DZ) luzea du; gainera, GIBaren andui ezberdinetan sekuentzia sendoki kontserbatzen da. Honek, DZak GIBaren bizi-zikloan funtzio gakoa izan dezakeela iradokitzen du. Gure taldean burututako ikerketen emaitzek, Env proteinak kolesterolarekin elkar eragiteko gp41aren DZa nahitaezko eragilea dela frogatu dute, baita elkarrekintza hori Env proteina guztien taldekatzea (clustering-a) emateko beharrezkoa dela ere; gainera, Env azaleko glikoproteinen multzokatze hau ezinbestekoa da birusak infekzio gaitasuna eskuratu dezan. Palmitoilazioak proteinak, Env barne, kolesterolean aberatsak diren domeinuetara, baltsa lipidikoetara alegia, bideratzen dituenaren ebidentzia dago. Hala ere, ikerketa horiek detergenteekiko mintz erresistenteen (DRM, Detergent Resistant Membrane) metodoetan oinarrituta daude eta teknika honen mugak direla eta, ez dute zuzenki frogatzen baltsa lipidikoetarako asoziazioa. Izan ere, DRM erauzketa baldintzek lipido-proteina asoziazio berriak sortaraz ditzakete. Hala, lan honen helburua, gp41 proteinaren DZko palmitoilazioak Env-kolesterol elkarrekintzan daukan papera in vivo aztertzea da, klik kimika metodoaren eta lipido fotoaktibagarrien erabilerarekin. Esperimentuak gauzatzeko GIB-1NL4-3 C762S mutantea erabili da, zeinak 762. aminoazido posizioan zisteina serina ordezkapena duen eta beraz, palmitoilazioa galtzen duen. Ikerkuntza lan honek DZaren palmitoilazioa Env-kolesterol elkarrekintzaren erantzule ez dela frogatzen du, interakzioa DZaren eta mintzeko kolesterolaren artean zuzenean ematen delarik. [English] The Human Immunodeficiency Virus (HIV) infection is the cause of the worldwide spread Acquired Immune Deficiency Syndrome (AIDS). HIV expresses a single viral glycoprotein in the viral membrane: envelope (Env). This glycoprotein is formed by a trimer formed by two subunits, a surface gp120 protein and a transmembrane gp41 protein. In HIV, the cytoplasmic domain (CTD) sequence of the gp41 protein is uncommonly long among other lentiviruses and highly conserved within HIV serotypes, suggesting possible essential function in HIV´s life cycle. Preliminary results from the group show that the CTD is responsible for protein-cholesterol interaction and that such interaction is necessary for Env clustering within viral membrane. Moreover, clustering of Env has been proved to be a prerequisite for viral infection. Palmitoylation is thought to be one of the responsible factors for targeting membrane proteins, including Env, to cholesterol rich domains, commonly called, lipid rafts. However, studies were conducted by using DRM (Detergent Resistant Membrane) method which presents limitations in the establishment of a reliable direct association between proteins and lipid rafts. Since DRM extraction conditions can form new lipid-protein assemblies, the objective of this study was to clarify the role of palmitoylation in the interaction of the gp41 CTD-cholesterol in vivo by new techniques, such as, Click chemistry and radioactive photoactivable lipids. Experiments were performed with the HIV-1NL4-3 C762S mutant, which presents the substitution of the cysteine residue to serine in the 762nd position of the CTD, and therefore, it lacks of palmytoilation. The results of this scientific study demonstrate that the interaction of Env protein to cholesterol through the CTD is not due to palmitoylation, but direct domain-viral membrane interaction. [ES] La infección por el virus de la inmunodeficiencia humana (VIH) es la causa de la propagación mundial del síndrome de inmunodeficiencia adquirida (SIDA). El VIH expresa una sola glicoproteína viral en la membrana viral: envoltura (Env). Esta glicoproteína está formada por un trímero formado por dos subunidades, una proteína gp120 de superficie y una proteína gp41 transmembrana. En el VIH, la secuencia del dominio citoplasmático (CTD) de la proteína gp41 es poco común entre otros lentivirus y está muy conservada en los serotipos del VIH, lo que sugiere una posible función esencial en el ciclo de vida del VIH. Los resultados preliminares del grupo muestran que el CTD es responsable de la interacción proteína-colesterol y que dicha interacción es necesaria para la agrupación de Env dentro de la membrana viral. Además, se ha demostrado que la agrupación de Env es un requisito previo para la infección viral. Se cree que la palmitoilación es uno de los factores responsables de dirigir las proteínas de membrana, incluida Env, a dominios ricos en colesterol, comúnmente llamados balsas lipídicas. Sin embargo, los estudios se realizaron utilizando el método DRM (Detergent Resistant Membrane) que presenta limitaciones en el establecimiento de una asociación directa confiable entre proteínas y balsas lipídicas. Dado que las condiciones de extracción de DRM pueden formar nuevos ensamblajes lípido-proteína, el objetivo de este estudio fue aclarar el papel de la palmitoilación en la interacción de la gp41 CTD-colesterol in vivo mediante nuevas técnicas, como la química Click y los lípidos fotoactivables radiactivos. Se realizaron experimentos con el mutante HIV-1NL4-3 C762S, que presenta la sustitución del residuo cisteína por serina en la posición 762 del CTD, y por tanto, carece de palmitoilación. Los resultados de este estudio científico demuestran que la interacción de la proteína Env con el colesterol a través del CTD no se debe a la palmitoilación, sino a la interacción dominio directo-membrana viral.

The viral envelope glycoprotein, known as “Env” in Retroviridae, is found on the virion surface and facilitates virus entry into cells by mediating cell attachment and fusion. Env is a major structural component of retroviruses and is targeted by all arms of the immune response, including adaptive and innate immunity. Less is known about how cell-intrinsic immunity prevents retrovirus replication at the level of individual cells. Here, we show that cellular IFITM3 and IFITM2 inhibit the fusion potential of retroviral virions by inhibiting Env protein via a two-pronged mechanism. IFITM proteins inhibit Env abundance in cells and also impair its function when levels are low. The posttranslational block of retroviral Env function by IFITM proteins is likely to impede both exogenous and endogenous retrovirus replication. In support of a relevant role for IFITM3 in retrovirus control, the retroviral accessory protein glycoGag counteracts IFITM3 function to promote virus infectivity., Interferon-induced transmembrane (IFITM) proteins are encoded by many vertebrate species and exhibit antiviral activities against a wide range of viruses. IFITM3, when present in virus-producing cells, reduces the fusion potential of HIV-1 virions, but the mechanism is poorly understood. To define the breadth and mechanistic basis for the antiviral activity of IFITM3, we took advantage of a murine leukemia virus (MLV)-based pseudotyping system. By carefully controlling amounts of IFITM3 and envelope protein (Env) in virus-producing cells, we found that IFITM3 potently inhibits MLV infectivity when Env levels are limiting. Loss of infectivity was associated with defective proteolytic processing of Env and lysosomal degradation of the Env precursor. Ecotropic and xenotropic variants of MLV Env, as well as HIV-1 Env and vesicular stomatitis virus glycoprotein (VSV-G), are sensitive to IFITM3, whereas Ebola glycoprotein is resistant, suggesting that IFITM3 selectively inactivates certain viral glycoproteins. Furthermore, endogenous IFITM3 in human and murine cells negatively regulates MLV Env abundance. However, we found that the negative impact of IFITM3 on virion infectivity is greater than its impact on decreasing Env incorporation, suggesting that IFITM3 may impair Env function, as well as reduce the amount of Env in virions. Finally, we demonstrate that loss of virion infectivity mediated by IFITM3 is reversed by the expression of glycoGag, a murine retrovirus accessory protein previously shown to antagonize the antiviral activity of SERINC proteins. Overall, we show that IFITM3 impairs virion infectivity by regulating Env quantity and function but that enhanced Env expression and glycoGag confer viral resistance to IFITM3.

HERV-K(HML-2) ist das jüngste und am besten erhaltene humane endogene Retrovirus. Menschliche Zellen, die diese Viren alle im Genom tragen sind in der Lage funktionelle virale Hüllproteine zu exprimieren. Diese Proteine können andere Retroviren pseudotypisieren und ihren Zelltropismus dadurch verändern. Wichtige Charakteristika des Eintrittprozesses, der durch das Hüllprotein von HERV-K(HML-2) vermittelt wird, sind noch unbekannt und wurden in dieser Arbeit mit Hilfe von pseudotypisierten lentiviralen Reporterviren analysiert. Als HERV-K(HML-2)-Hüllprotein wurde eine zuvor rekonstituierte Sequenz von HERV-K113 verwendet. Im Fokus der Untersuchungen stand die Identifizierung der Domäne des Hüllproteins, die an den noch unbekannten Rezeptor bindet. Dafür wurde durch einen in silico-Vergleich mit Sequenzen verwandter β-Retroviren zunächst eine putative Rezeptorbindestelle gefunden. Durch gezielte Mutagenese wurde dieser Bereich mehrfach modifiziert. Die mutierten Hüllproteine waren nicht mehr in der Lage, den Zelleintritt der Viren zu gewährleisten oder verminderten ihn erheblich, was in Übereinstimmung mit einer Rezeptorbindungsfunktion dieser Proteinregion steht. Nachfolgend wurde der Versuch unternommen, durch Austausch der gefundenen Rezeptorbindedomäne von HERV-K(HML-2) gegen die des nahen verwandten MMTV, den Virustropismus zu verändern. Die chimären Proteine wurden jedoch nicht in Retroviren eingebaut. In einem weiteren Ansatz wurden lösliche Versionen der rezeptorbindenden SU-Einheit des HERV-K(HML-2)-Hüllproteins als Fusionsprotein mit einer C-terminalen Immunglobulindomäne hergestellt. Als Kontrolle wurden homologe Proteine mit Hüllproteinen anderer Retroviren kloniert und augereinigt. In einer Reihe von Experimenten konnte die spezifische Bindung dieser Proteine an einen zellulären Rezeptor in permissiven Zellen nachgewiesen. In einem proof-of principle Experiment wurde dann die Möglichkeit zur Kopräzipitation des unbekannten Rezeptors von HERV-K(HML-2) mit Hilfe dieser Fusionsproteine gezeigt. Im Zuge der Arbeit wurde der Rezeptor noch nicht isoliert. Die Herstellung und erfolgreiche Charkterisierung der SU-Ig-Fusionsproteine leistet dazu aber eine sehr aussichtsreiche und wichtige Voraussetzung., HERV-K(HML-2) is the youngest and best-conserved human endogenous retrovirus. Human cells, which are carrying these viruses in their genome, can express functional viral envelope proteins. These proteins can be used for pseudotyping other retroviruses and therefore for changing their cell tropism. In the present work, important characteristics of the entry process mediated through the envelope protein of HERV-K(HML-2), which are still unknown, were analysed using pseudotyped lentiviral reporter viruses. A previously reconstituted sequence of HERV-K113 was used as an HERV-K(HML-2) envelope protein. This study focused on finding the domain of the envelope-protein that binds to the yet unknown receptor. To achieve this, an in silico comparison with protein sequences of closely related Betaretroviruses was performed. This comparison allowed a prediction of a putative receptor binding site, which then underwent site-directed mutagenesis leading to several modifications within the region. The generated envelope mutants were either unable to mediate the cell entry of the viruses or did it in a heavily depleted manner, indicating the receptor binding role of the region. In order to alter the virus tropism, we interchanged the then found receptor binding domain of HERV-K(HML-2) with that of the most closely related MMTV, but the chimeric proteins couldn’t be incorporated into the retroviruses. A further approach consisted of expressing a soluble version of the receptor binding subunit of HERV-K(HML-2) envelope protein as a fusion protein with a C-terminal immunoglobulin domain. As control, homologous proteins based on envelope proteins from other retroviruses were cloned and affinity-purified. In a series of experiments, the specific binding of the proteins on a cellular receptor in permissive cells could be demonstrated. In a proof-of principle experiment, the possibility of a precipitation of the unknown receptor of HERV-K(HML-2) by using the fusion proteins was shown. In the course of this work, the receptor has not yet been isolated. The production and successful characterization of SU-Ig- fusion proteins supply however a promising and important requirement for future attempts.

Lentiviruses harbour high genetic variability for efficient evasion from host immunity. An attenuated equine infectious anaemia (EIA) vaccine was developed decades ago in China and presented remarkably robust protection against EIA. The vaccine was recently proven to have high genomic diversity, particular in env. However, how and to what extent the high env diversity relates to immune protection remains unclear. In this study, we compared immune protections and responses of three groups of horses stimulated by the high-diversity vaccine EIAV_HD, a single molecular clone of the vaccine EIAV_LD with low env diversity, as well as a constructed vaccine strain EIAV_MD with moderate env diversity. The disparity of virus-host interactions between three env diversity-varied groups (5 horses in each group) was evaluated using clinical manifestation, pathological scores, and env-specific antibody. We found the highest titres of env antibodies (Abs) or neutralizing Abs (nAbs) in the EIAV_HD group, followed by the EIAV_MD group, and the lowest titres in the EIAV_LD group (Penv diversity-related linear relationship was observed in the clinical manifestations and pathological changes. This diversity-dependent disparity in changes between the three groups was more distinct after immunosuppression, suggesting that env diversity plays an important role in protection under low host immunocompetence. In summary, inoculation with vaccines with higher genetic diversity could present broader and more efficient protection. Our findings strongly suggest that an abundance of Env antigens are required for efficient protection against lentiviruses.

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. PC was awarded a contract from the Basque Government Postdoctoral Programme (POS_2019_2_0022). JLN acknowledges financial support by the Spanish Government (BIO2015-64421-R (MINECO/AEI/FEDER UE) and RTI2018-095624-B-C21 (MCIU/AEI/FEDER UE) Grants) and the Basque Government (IT1196-19 Grant). CE acknowledges support from the Wolfson Imaging Centre Oxford (Christoffer Lagerholm), the Wolfson Foundation (18272), the Medical Research Council (MC_UU_12010/unit programmes G0902418 and MC_UU_12025), MRC/BBSRC/ESPRC (grant number MR/K01577X/1), the Wellcome Trust (104924/14/Z/14, 100262Z/12/Z, 098274/Z/12/Z and Strategic Award 091911 (107457/Z/15/Z, Advanced Micron Bioimaging Unit)), the Deutsche Forschungsgemeinschaft (Research unit 1905, Jena Excellence Cluster “Balance of the Microverse”, project no. 3162113987 – SFB 1278 (project C05), the Newton-Katip Celebi Institutional Links grant (352333122), and internal University of Oxford funding (EPA Cephalosporin Fund and John Fell Fund).

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.

LTR-retrotransposons share a common genomic organization in which the 5′ long terminal repeat (LTR) is followed by the gag and pol genes and terminates with the 3′ LTR. Although GAG-POL-encoded proteins are considered sufficient to accomplish the LTR-retrotransposon transposition, a number of elements carrying additional open reading frames (aORF) have been described. In some cases, the presence of an aORF can be explained by a phenomenon similar to retrovirus gene transduction, but in these cases the aORFs are present in only one or a few copies. On the contrary, many elements contain aORFs, or derivatives, in all or most of their copies. These aORFs are more frequently located between pol and 3′ LTR, and they could be in sense or antisense orientation with respect to gag-pol. Sense aORFs include those encoding for ENV-like proteins, so called because they have some structural and functional similarities with retroviral ENV proteins. Antisense aORFs between pol and 3′ LTR are also relatively frequent and, for example, are present in some characterized LTR-retrotransposon families like maize Grande, rice RIRE2, or Silene Retand, although their possible roles have been not yet determined. Here, we discuss the current knowledge about these sense and antisense aORFs in plant LTR-retrotransposons, suggesting their possible origins, evolutionary relevance, and function., This work was funded by grants AGL2016-78992-R from FEDER/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación (Spain) and by the CERCA Programme of the Generalitat de Catalunya. We also acknowledge financial support from the Spanish Ministerio de Economía y Competitividad through the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016-2019 (SEV-2015-0533).

Objective Breast cancer is one of the most common health problems. It has been suggested that several risk factors, either considered as external or internal, play a critical role in the pathogenesis of breast cancer, which among them, HERV-k, has the most fundamental role. In the present study, we aimed to evaluate the role of HERV-k env, gag, rec, np9 expressions in breast cancer progression. Materials and methods We collected 40 breast cancer tissues and their normal adjacent ones. After extracting the RNA of breast samples, we evaluated the expression of HERV-k env, gag, rec, np9 by using Quantitative real-time PCR (qRT-PCR). Results The resulting data revealed that while there was a meaningful increase in the expression level of HERV-k env, gag and np9 in breast cancer tissues (P ≤ 0.01, 0.05, 0.05, respectively), we failed to find any significant elevation in the expression level of rec mRNA level. Conclusion The results of our study suggested that there is a plausible correlation between the mRNA expression level of HERV-K env, gag and np9 and the progression of breast cancer, proposing these markers as promising biomarkers to diagnose breast cancer.

Summary Potent and broadly neutralizing antibodies (bnAbs) are the hallmark of HIV-1 protection by vaccination. The membrane-proximal external region (MPER) of the HIV-1 gp41 fusion protein is targeted by the most broadly reactive HIV-1 neutralizing antibodies. Here, we examine the structural and molecular mechansims of neutralization by anti-MPER bnAb, LN01, which was isolated from lymph-node-derived germinal center B cells of an elite controller and exhibits broad neutralization breadth. LN01 engages both MPER and the transmembrane (TM) region, which together form a continuous helix in complex with LN01. The tilted TM orientation allows LN01 to interact simultaneously with the peptidic component of the MPER epitope and membrane via two specific lipid binding sites of the antibody paratope. Although LN01 carries a high load of somatic mutations, most key residues interacting with the MPER epitope and lipids are germline encoded, lending support for the LN01 epitope as a candidate for lineage-based vaccine development., Graphical Abstract, Highlights • bNAb LN01 neutralizes 92% of a 118-strain virus panel • LN01 targets the HIV-1 gp41 MPER, the TM region, and lipids • LN01-complexed MPER forms a continuous helix with TM • Most LN01 paratope residues interacting with MPER-TM and lipids are germline encoded, The gp41 membrane-proximal external region (MPER) is a highly conserved region of HIV-1 Env. Pinto et al. characterize the broadly neutralizing anti-MPER mAb LN01, which shows low autoreactivity. LN01 interacts with a complex epitope comprising MPER, the transmembrane region, and lipids, providing insights for vaccine design.

International audience; Potent and broadly neutralizing antibodies (bnAbs) are the hallmark of HIV-1 protection by vaccination. The membrane-proximal external region (MPER) of the HIV-1 gp41 fusion protein is targeted by the most broadly reactive HIV-1 neutralizing antibodies. Here, we examine the structural and molecular mechansims of neutralization by anti-MPER bnAb, LN01, which was isolated from lymph-node-derived germinal center B cells of an elite controller and exhibits broad neutralization breadth. LN01 engages both MPER and the transmembrane (TM) region, which together form a continuous helix in complex with LN01. The tilted TM orientation allows LN01 to interact simultaneously with the peptidic component of the MPER epitope and membrane via two specific lipid binding sites of the antibody paratope. Although LN01 carries a high load of somatic mutations, most key residues interacting with the MPER epitope and lipids are germline encoded, lending support for the LN01 epitope as a candidate for lineage-based vaccine development.

The analysis of patient derived HIV neutralizing antibodies (nAbs) and their target epitopes in the viral envelope (Env) protein provides important basic information for vaccine design. In this study we optimized an epitope, EC26-2A4, that is targeted by neutralizing antibodies from an elite controller (EC26) and localizes in the membrane-proximal external region from the gp41 transmembrane protein. Due to its overlap with the epitope of the first generation broadly neutralizing monoclonal Ab (mAb) 2F5 associated with autoreactivity, we first defined the minimal core epitope reacting with antibodies from EC26 plasma, but not with mAb 2F5. The optimized minimal epitope, EC26-2A4ΔM, was able to induce neutralizing antibodies in vaccinated mice. We further analyzed the frequency of antibodies against the EC26-2A4ΔM peptide in HIV-positive patient sera from a treated cohort and an untreated long-term nonprogressor (LTNP) cohort. Interestingly, 27% of the LTNP sera reacted with the peptide, whereas only 9% showed reactivity in the treated cohort. Although there was no association between the presence of antibodies against the EC26-2A4ΔM epitope and viral load or CD4 count in these patients, the CD4 nadir in the treated cohort was higher in patients positive for EC26-2A4ΔM antibodies, in particular in patients having such antibodies at an early and a late timepoint after infection.

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.