RESUMO
Glycoprotein L (gL), which complexes with gH, is a conserved herpesvirus protein that is essential for Epstein-Barr virus (EBV) entry into host cells. The gH/gL complex has a conserved role in entry among herpesviruses, yet the mechanism is not clear. To gain a better understanding of the role of gL in EBV-mediated fusion, chimeric proteins were made using rhesus lymphocryptovirus (Rh-LCV) gL (Rh gL), which shares a high sequence homology with EBV gL but does not complement EBV gL in mediating fusion with B cells. A reduction in fusion activity was observed with chimeric gL proteins that contained the amino terminus of Rh gL, although they retained their ability to process and transport gH/gL to the cell surface. Amino acids not conserved within this region in EBV gL when compared to Rh gL were further analyzed, with the results mapping residues 54 and 94 as being functionally important for EBV-mediated fusion. All chimeras and mutants displayed levels of cell surface expression similar to that of wild-type gL and interacted with gH and gp42. Our data also suggest that the role of gL involves the activation or recruitment of gB with the gH/gL complex, as we found that reduced fusion of Rh gL, EBV/Rh-LCV chimeras, and gL point mutants could be restored by replacing EBV gB with Rh gB. These observations demonstrate a distinction between the role of gL in the processing and trafficking of gH to the cell surface and a posttrafficking role in cell-cell fusion.
Assuntos
Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Lymphocryptovirus/fisiologia , Fusão de Membrana , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus , Sequência de Aminoácidos , Animais , Linfócitos B/metabolismo , Linfócitos B/virologia , Células CHO , Fusão Celular , Cricetinae , Cricetulus , Infecções por Vírus Epstein-Barr/fisiopatologia , Herpesvirus Humano 4/química , Herpesvirus Humano 4/genética , Humanos , Lymphocryptovirus/química , Lymphocryptovirus/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Human 2',5'-oligoadenylate synthetase 3 (OAS3) exerts antiviral effect against alphaviruses including Chikungunya virus (CHIKV) by inhibiting viral RNA accumulation. Here, we identified a CHIKV variant exhibiting a remarkable resistance to the antiviral action of OAS3 in human epithelial HeLa cells. Using a molecular clone of CHIKV with Renilla luciferase inserted as a reporter gene in the non-structural region, we demonstrated that a single glutamine-to-lysine amino acid change at position 166 of the envelope E2 glycoprotein restores CHIKV replication in OAS3 expressing HeLa cells. Viral entry assays showed that CHIKV with a lysine at position E2-166 was more efficient at entering the replicative pathway. The E2-E166K substitution promotes a greater efficiency of CHIKV replication in human myoblasts leading to severe apoptosis through a more robust activation of the PKR pathway. These observations provide a new insight into the role of E2 into the pathogenicity of CHIKV in human cells.
Assuntos
2',5'-Oligoadenilato Sintetase/biossíntese , Vírus Chikungunya/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Internalização do Vírus , 2',5'-Oligoadenilato Sintetase/imunologia , Animais , Apoptose , Fusão Gênica Artificial , Vírus Chikungunya/genética , Vírus Chikungunya/crescimento & desenvolvimento , Vírus Chikungunya/imunologia , Genes Reporter , Células HeLa , Humanos , Luciferases/análise , Luciferases/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mioblastos/fisiologia , Mioblastos/virologia , Renilla/enzimologiaRESUMO
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.
Assuntos
Herpesvirus Humano 4/fisiologia , Lymphocryptovirus/metabolismo , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus , Sequência de Aminoácidos , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/química , Herpesvirus Humano 4/genética , Lymphocryptovirus/química , Lymphocryptovirus/fisiologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Proteínas do Envelope Viral/genética , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Nectin-1 is an immunoglobulin (Ig)-like entry receptor for herpes simplex virus (HSV). Like other nectins, nectin-1 forms dimers and mediates cell adhesion through interactions with other nectins. We constructed a second-domain deletion mutant of nectin-1 (nectin-1-Delta2) to examine the role of the second Ig-like domain in HSV entry. Nectin-1-Delta2 exhibited a severely reduced ability to mediate HSV entry and accumulated in the endoplasmic reticulum but retained the ability to interact with its HSV ligand, gD. The failure of nectin-1-Delta2 to mediate HSV entry probably resulted from its failure to be transported to a membrane targeted by HSV for viral entry.
Assuntos
Moléculas de Adesão Celular/metabolismo , Processamento de Proteína Pós-Traducional , Receptores Virais/metabolismo , Simplexvirus/fisiologia , Animais , Células CHO , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Cricetinae , Retículo Endoplasmático/química , Nectinas , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Virais/química , Receptores Virais/genética , Deleção de Sequência , Proteínas do Envelope Viral/metabolismoRESUMO
Effects of gemcitabine (Gemzar) on immune cells were examined in pancreas cancer patients to determine whether it was immunosuppressive, or potentially could be combined with vaccines or other immunotherapy to enhance patient's responses to their tumors. Blood was obtained at five time-points, before therapy, 3-4 days after initial gemcitabine infusion and immediately preceding three additional weekly infusions. Effects on T-cell subsets, B-cells, myeloid dendritic cell precursors, antigen presenting cells (APC), activated/memory, and naive cells were examined. Functional activity was measured by intracellular staining for cytokines before and after T-cell activation, and by interferon gamma production in EliSpot responses to tumor presentation. Although absolute lymphocyte counts decreased with the initial treatment with gemcitabine infusion, the counts stabilized during subsequent treatments, then returned within normal ranges seven days after the fourth treatment so that the absolute lymphocyte count no longer differed significantly from that prior to treatment. These effects on absolute lymphocyte counts were mirrored by statistically significant decreases in absolute numbers of CD3 and CD20 lymphocytes during these time periods. The proportions of T and B-cells, however did not change significantly with therapy, although significance changes were observed in some specialized subsets. A decrease in the proportions of the major BDCA-1+, CD1b myeloid dendritic cell subset and a reciprocal increase in the minor BDCA-3+ dendritic cell subsets resulted at 3-4 days, then their levels returned to normal. No significant changes in percentages of CD86 and CD80 APCs or CD4+, CD25+ T-cells were documented. Increased percentages of CD3+, CD45RO+ memory lymphocytes reached significance at day 7, then declined to statistically significant decrease at days 14 and 21 after the second and third infusions, respectively. Immune T-cells were functional in pancreas cancer patients treated with gemcitabine. The data suggest that gemcitabine therapy may decrease memory T-cells and promote naive T-cell activation. We conclude that gemcitabine therapy (1) is not immunosuppressive and (2) may enhance responses to specific vaccines or immunotherapy administered to activate or support immune responses directed toward driving effector immunity to cancer cells.