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Requirements for Human Respiratory Syncytial Virus Glycoproteins in Assembly and Egress from Infected Cells.

ABSTRACT: Human respiratory syncytial virus (HRSV) is an enveloped RNA virus that assembles and buds from the plasma membrane of infected cells. The ribonucleoprotein complex (RNP) must associate with the viral matrix protein and glycoproteins to form newly infectious particles prior to budding. The viral proteins involved in HRSV assembly and egress are mostly unexplored. We investigated whether the glycoproteins of HRSV were involved in the late stages of viral replication by utilizing recombinant viruses where each individual glycoprotein gene was deleted and replaced with a reporter gene to maintain wild-type levels of gene expression. These engineered viruses allowed us to study the roles of the glycoproteins in assembly and budding in the context of infectious virus. Microscopy data showed that the F glycoprotein was involved in the localization of the glycoproteins with the other viral proteins at the plasma membrane. Biochemical analyses showed that deletion of the F and G proteins affected incorporation of the other viral proteins into budded virions. However, efficient viral release was unaffected by the deletion of any of the glycoproteins individually or in concert. These studies attribute a novel role to the F and G proteins in viral protein localization and assembly.

SUBMITTER: Batonick M

PROVIDER: S-EPMC3175114 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Respiratory syncytial virus (RSV) is a mucosa-restricted virus that is a leading cause of epidemic respiratory tract infections in children. In epithelial cells, RSV replication activates nuclear translocation of the inducible transcription factor nuclear factor kappaB (NF-kappaB) through proteolysis of its cytoplasmic inhibitor, IkappaB. In spite of a putative role in mediating virus-inducible gene expression, the spectrum of NF-kappaB-dependent genes induced by RSV infection has not yet been determined. To address this, we developed a tightly regulated cell system expressing a nondegradable, epitope-tagged IkappaBalpha isoform (Flag-IkappaBalpha Mut) whose expression could be controlled by exogenous addition of nontoxic concentrations of doxycycline. Flag-IkappaBalpha Mut expression potently inhibited IkappaBalpha proteolysis, NF-kappaB binding, and NF-kappaB-dependent gene transcription in cells stimulated with the prototypical NF-kappaB-activating cytokine tumor necrosis factor alpha (TNF-alpha) and in response to RSV infection. High-density oligonucleotide microarrays were then used to profile constitutive and RSV-induced gene expression in the absence or presence of Flag-IkappaBalpha Mut. Comparison of these profiles revealed 380 genes whose expression was significantly changed by the dominant-negative NF-kappaB. Of these, 236 genes were constitutive (not RSV regulated), and surprisingly, only 144 genes were RSV regulated, representing numerically approximately 10% of the total population of RSV-inducible genes at this time point. Hierarchical clustering of the 144 RSV- and Flag-IkappaBalpha Mut-regulated genes identified two discrete gene clusters. The first group had high constitutive expression, and its expression levels fell in response to RSV infection. In this group, constitutive mRNA expression was increased by Flag-IkappaBalpha Mut expression, and the RSV-induced decrease in expression was partly inhibited. In the second group, constitutive expression was very low (or undetectable) and, after RSV infection, expression levels strongly increased. In this group, NF-kappaB was required for RSV-inducible expression because Flag-IkappaBalpha Mut expression blocked their induction by RSV. This latter cluster includes chemokines, transcriptional regulators, intracellular proteins regulating translation and proteolysis, and secreted proteins (complement components and growth factor regulators). These data suggest that NF-kappaB action induces global cellular responses after viral infection.

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Requirements for Human Respiratory Syncytial Virus Glycoproteins in Assembly and Egress from Infected Cells.

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