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Differential modification of interferon regulatory factor 3 following virus particle entry.

ABSTRACT: Viral infection elicits the activation of numerous cellular signal transduction pathways, leading to the induction of both innate and adaptive immune responses in the host. In particular, interferon regulatory factor 3 (IRF3) has been shown to be essential for the induction of an antiviral response. Current models suggest that virus replication causes phosphorylation of C-terminal serine and threonine residues on IRF3, leading to its dimerization and translocation to the nucleus, where it activates interferon. Upon entry of replication-deficient Newcastle disease virus (NDV) particles, however, we failed to detect IRF3 dimerization or hyperphosphorylation, despite robust interferon-stimulated gene (ISG) and antiviral state induction and confirmation by small interfering RNA knockdown that IRF3 is essential for this response. To further compare the effects of various viruses and their replication status on IRF3 activation and to determine the minimal posttranslational modification required for IRF3 activation, two-dimensional gel electrophoresis and native polyacrylamide gel electrophoresis were employed. However, we failed to identify a minimal posttranslational modification of IRF3 that correlated with downstream biological activity, and the extent of posttranslational modification observed on IRF3 did not correlate with the degree of subsequent ISG induction. Thus, current techniques used to detect IRF3 activation are insufficient to infer its role in mediating downstream biological response induction and should be utilized with caution.

SUBMITTER: Noyce RS

PROVIDER: S-EPMC2668492 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Differential modification of interferon regulatory factor 3 following virus particle entry.

Noyce Ryan S RS Collins Susan E SE Mossman Karen L KL

Journal of virology 20090211 9

Viral infection elicits the activation of numerous cellular signal transduction pathways, leading to the induction of both innate and adaptive immune responses in the host. In particular, interferon regulatory factor 3 (IRF3) has been shown to be essential for the induction of an antiviral response. Current models suggest that virus replication causes phosphorylation of C-terminal serine and threonine residues on IRF3, leading to its dimerization and translocation to the nucleus, where it activa ...[more]

PMID: 19211751

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Project description:Infectious rotavirus particles are triple-layered, icosahedral assemblies. The outer layer proteins, VP4 (cleaved to VP8* and VP5*) and VP7, surround a transcriptionally competent, double-layer particle (DLP), which they deliver into the cytosol. During entry of rhesus rotavirus, VP8* interacts with cell surface gangliosides, allowing engulfment into a membrane vesicle by a clathrin-independent process. Escape into the cytosol and outer-layer shedding depend on interaction of a hydrophobic surface on VP5* with the membrane bilayer and on a large-scale conformational change. We report here experiments that detect the fate of released DLPs and their efficiency in initiating RNA synthesis. By replacing the outer layer with fluorescently tagged, recombinant proteins and also tagging the DLP, we distinguished particles that have lost their outer layer and entered the cytosol (uncoated) from those still within membrane vesicles. We used fluorescent in situ hybridization with probes for nascent transcripts to determine how soon after uncoating transcription began and what fraction of the uncoated particles were active in initiating RNA synthesis. We detected RNA synthesis by uncoated particles as early as 15 min after adding virus. The uncoating efficiency was 20 to 50%; of the uncoated particles, about 10 to 15% synthesized detectable RNA. In the format of our experiments, about 10% of the added particles attached to the cell surface, giving an overall ratio of added particles to RNA-synthesizing particles of between 250:1 and 500:1, in good agreement with the ratio of particles to focus-forming units determined by infectivity assays. Thus, RNA synthesis by even a single, uncoated particle can initiate infection in a cell.IMPORTANCE The pathways by which a virus enters a cell transform its packaged genome into an active one. Contemporary fluorescence microscopy can detect individual virus particles as they enter cells, allowing us to map their multistep entry pathways. Rotaviruses, like most viruses that lack membranes of their own, disrupt or perforate the intracellular, membrane-enclosed compartment into which they become engulfed following attachment to a cell surface, in order to gain access to the cell interior. The properties of rotavirus particles make it possible to determine molecular mechanisms for these entry steps. In the work described here, we have asked the following question: what fraction of the rotavirus particles that penetrate into the cell make new viral RNA? We find that of the cell-attached particles, between 20 and 50% ultimately penetrate, and of these, about 10% make RNA. RNA synthesis by even a single virus particle can initiate a productive infection.

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Differential modification of interferon regulatory factor 3 following virus particle entry.

Publications

Differential modification of interferon regulatory factor 3 following virus particle entry.

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