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[en] Highlights: • Cellular PDZ proteins that associate with HPV16 and HPV18 E6 were purified and identified. • HPV18 E6 compared to HPV16 E6 bound additional PDZ proteins from glandular epithelia. • Several of the E6-associated cellular PDZ proteins regulate YAP1 localization. • HPV16 E6 and E7 promote the nuclear localization of YAP1; for E6 this requires PDZ protein association. HPV E6 oncoproteins associate with cellular PDZ proteins. In addition to previously identified cellular PDZ proteins, we found association of the HPV16 E6 PBM with the Dystrophin Glycoprotein Complex, LRCC1, and SLC9A3R2. HPV18 E6 had additional associations when lysates from adenomatous cell lines were used including LRPPRC, RLGAPB, EIF3A, SMC2 and 3, AMOT, AMOTL1, and ARHGEF1; some of these cellular PDZ proteins are implicated in the regulation of the YAP1 transcriptional co-activator. In keratinocytes, nuclear translocation of YAP1 was promoted by the complete HPV-16 genome, or by expression of the individual E6 or E7 oncoproteins; the activity of E6 required an intact PBM at the carboxy-terminus. This work demonstrates that E6 association with cellular PDZ proteins promotes the nuclear localization of YAP1. The ability of E6 to promote the nuclear transport of YAP1 thus identifies an E6 activity that could contribute to the transformation of cells by E6.
[en] Highlights: • In lab-adapted tier-1 Env isolates, the Env CoRbs is directly accessible to CD4-induced (CD4i) antibodies. • Inaccessibility of the Env CoRbs in tier-2 & tier-3 isolates is associated with lack of neutralization by CD4i antibodies. • Lower molecular mass CD4i antibody domains such as m36.4 can access the CoRbs. • M36.4 neutralizes some tier-2 and tier-3 Env isolates even prior to engagement to the primary receptor, CD4. • Neutralization of cell-free viruses by m36.4 is Env-specific and independent of viral subtype or tier categorization. HIV-1 virus entry into target cells requires the envelope glycoprotein (Env) to first bind the primary receptor, CD4 and subsequently the co-receptor. Antibody access to the co-receptor binding site (CoRbs) in the pre-receptor-engaged state, prior to cell attachment, remains poorly understood. Here, we have demonstrated that for tier-1 Envs, the CoRbs is directly accessible to full-length CD4-induced (CD4i) antibodies even before primary receptor engagement, indicating that on these Envs the CoRbs site is either preformed or can conformationally sample post-CD4-bound state. Tier-2 and tier-3 Envs, which are resistant to full-length CD4i antibody, are neutralized by m36.4, a lower molecular mass of CD4i-directed domain antibody. In some tier-2 and tier-3 Envs, CoRbs is accessible to m36.4 even prior to cellular attachment in an Env-specific manner independent of their tier category. These data suggest differential structural arrangements of CoRbs and varied masking of ligand access to the CoRbs in different Env isolates.
[en] Glycoproteins gB and gH/gL are required for entry of Epstein-Barr virus (EBV) into cells, but the role of each glycoprotein and how they function together to mediate fusion is unclear. Analysis of the functional homology of gB from the closely related primate gammaherpesvirus, rhesus lymphocryptovirus (Rh-LCV), showed that EBV gB could not complement Rh gB due to a species-specific dependence between gB and gL. To map domains of gB required for this interaction, we constructed a panel of EBV/Rh gB chimeric proteins. Analysis showed that insertion of Rh gB from residues 456 to 807 restored fusion function of EBV gB with Rh gH/gL, suggesting this region of gB is important for interaction with gH/gL. Split YFP bimolecular complementation (BiFC) provided evidence of an interaction between EBV gB and gH/gL. Together, our results suggest the importance of a gB-gH/gL interaction in EBV-mediated fusion with B cells requiring the region of EBV gB from 456 to 807.
[en] Highlights: • Lipid biosensors have been used to characterize HIV-1 assembly sites. • Wild type and deletion matrix HIV-1 viruses assemble at different sites. • The lipid biosensor for PI(4,5)P2 is enriched in wild type HIV-1. • The lipid biosensor for PI3P is enriched in deletion matrix HIV-1. The matrix (MA) domain of the HIV-1 precursor Gag protein (PrGag) has been shown interact with the HIV-1 envelope (Env) protein, and to direct PrGag proteins to plasma membrane (PM) assembly sites by virtue of its affinity to phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2). Unexpectedly, HIV-1 viruses with large MA deletions (ΔMA) have been shown to be conditionally infectious as long as they are matched with Env truncation mutant proteins or alternative viral glycoproteins. To characterize the interactions of wild type (WT) and ΔMA Gag proteins with PI(4,5)P2 and other acidic phospholipids, we have employed a set of lipid biosensors as probes. Our investigations showed marked differences in WT and ΔMA Gag colocalization with biosensors, effects on biosensor release, and association of biosensors with virus-like particles. These results demonstrate an alternative approach to the analysis of viral protein-lipid associations, and provide new data as to the lipid compositions of HIV-1 assembly sites.
[en] HSV-2 spread is predominantly dependent on cell-to-cell contact. However, the underlying mechanisms remain to be determined. Here we demonstrate that HSV-2 gJ, which was previously assigned no specific function, promotes HSV-2 cell-to-cell spread and syncytia formation. In the context of viral infection, knockout or knockdown of gJ impairs HSV-2 cell-to-cell spread among epithelial cells or from epithelial cells to neuronal cells, which leads to decreased virus production, whereas ectopic expression of gJ enhances virus production. Mechanistically, gJ increases the expression levels of HSV-2 proteins, and also enhances viral protein expression and replication of heterologous viruses like HIV-1 and JEV, suggesting that HSV-2 gJ likely functions as a regulator of viral protein expression and virus production. Findings in this study provide a basis for further understanding the role of gJ in HSV-2 replication.
[en] Highlights: • The V3-loop of HIV-1 gp120 contributes to Env trimer stability and viral entry. • The hydrophobic patch in the tip of the V3 loop is critical for pre-triggered Env trimer stability. • The hydrophobic patch is a conserved motif in primate immunodeficiency viruses. The V3 loop of the human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein (Env) becomes exposed after CD4 binding and contacts the coreceptor to mediate viral entry. Prior to CD4 engagement, a hydrophobic patch located at the tip of the V3 loop stabilizes the non-covalent association of gp120 with the Env trimer of HIV-1 subtype B strains. Here, we show that this conserved hydrophobic patch (amino acid residues 307, 309 and 317) contributes to gp120-trimer association in HIV-1 subtype C, HIV-2 and SIV. Changes that reduced the hydrophobicity of these V3 residues resulted in increased gp120 shedding and decreased Env-mediated cell-cell fusion and virus entry in the different primate immunodeficiency viruses tested. Thus, the hydrophobic patch is an evolutionarily conserved element in the tip of the gp120 V3 loop that plays an essential role in maintaining the stability of the pre-triggered Env trimer in diverse primate immunodeficiency viruses.
[en] Venezuelan equine encephalitis virus (VEEV) is a prototypical enveloped ssRNA virus of the family Togaviridae. To better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. These particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. Purified pre-viral nucleocapsids were spherical with a unimodal diameter distribution. The structure of one class of pre-viral nucleocapsids was determined with single particle reconstruction of cryo-electron microscopy images. These studies showed that pre-viral nucleocapsids assembled into an icosahedral structure with a capsid stoichiometry similar to the mature nucleocapsid. However, the individual capsomers were organized significantly differently within the pre-viral and mature nucleocapsids. The pre-viral nucleocapsid structure implies that nucleocapsids are highly plastic and undergo glycoprotein and/or lipid-driven rearrangements during virus self-assembly. This mechanism of self-assembly may be general for other enveloped viruses.
[en] Both mutant p53 and chemoresistance are poor prognostic factors in cancer. Many studies have examined the influence of these factors on fluoro-2-deoxy-D-glucose (FDG) incorporation. Whilst mutant p53 is associated with increased FDG incorporation, chemoresistance, especially when associated with P-glycoprotein, is associated with decreased FDG incorporation.
[en] The structural analysis of glycoproteins is a challenging endeavor and is under steadily increasing demand, but only a very limited number of labs have the expertise required to accomplish this task. This tutorial is aimed at researchers from the fields of molecular biology and biochemistry that have discovered that glycoproteins are important in their biological research and are looking for the tools to elucidate their structure. It provides brief descriptions of the major and most common analytical techniques used in glycomics and glycoproteomics analysis, including explanations of the rationales for individual steps and references to published literature containing the experimental details necessary to carry out the analyses. Glycomics includes the comprehensive study of the structure and function of the glycans expressed in a given cell or organism along with identification of all the genes that encode glycoproteins and glycosyltransferases. Glycoproteomics which is subset of both glycomics and proteomics is the identification and characterization of proteins bearing carbohydrates as posttranslational modification. This tutorial is designed to ease entry into the glycomics and glycoproteomics field for those without prior carbohydrate analysis experience.