Increased Ubiquitination and Other Covariant Phenotypes Attributed to a Strain- and Temperature-Dependent Defect of Reovirus Core Protein μ2

To replicate efficiently, viruses must create favorable cell conditions and overcome cell antiviral responses. We previously reported that the reovirus protein 2 from strain T1L, but not strain T3D, represses one antiviral response: alpha/beta interferon signaling. We report here that T1L, but not T3D, 2 localizes to nuclear speckles, where it forms a complex with the mRNA splicing factor SRSF2 and alters its subnuclear localization. Reovirus replicates in cytoplasmic viral factories, and there is no evidence that reovirus genomic or messenger RNAs are spliced, suggesting that T1L 2 might target splicing of cell RNAs. Indeed, RNA sequencing revealed that reovirus T1L, but not T3D, infection alters the splicing of transcripts for host genes involved in mRNA posttranscriptional modifications. Moreover, depletion of SRSF2 enhanced reovirus replication and cytopathic effect, suggesting that T1L 2 modulation of splicing benefits the virus. This provides the first report of viral antagonism of the splicing factor SRSF2 and identifies the viral protein that determines strain-specific differences in cell RNA splicing.IMPORTANCE Efficient viral replication requires that the virus create favorable cell conditions. Many viruses accomplish this by repressing specific antiviral responses. We demonstrate here that some mammalian reoviruses, RNA viruses that replicate strictly in the cytoplasm, express a protein variant that localizes to nuclear speckles, where it targets a cell mRNA splicing factor. Infection with a reovirus strain that targets this splicing factor alters splicing of cell mRNAs involved in the maturation of many other cell mRNAs. Depletion of this cell splicing factor enhances reovirus replication and cytopathic effect. Our results provide the first evidence of viral antagonism of this splicing factor and suggest that downstream consequences to the cell are global and benefit the virus.KEYWORDS RNA processing, SRSF, reovirus, splicing V iruses are obligatory intracellular pathogens that require a hospitable cell environment for their replication. However, viral infection induces cell innate responses that are antiviral. Accordingly, viruses have evolved numerous strategies to evade these responses. For example, viral infection of virtually any cell type induces a protective type I interferon (IFN-␣/␤) response, and many viruses use at least one mechanism to suppress this antiviral system (1, 2). Viral proteins that inhibit cell antiviral responses have been identified for most viruses, as have the cell proteins they target (1, 3). Not surprisingly, the most commonly identified targets are cell proteins involved in the IFN-␣/␤ response (1, 2).Mammalian reoviruses are double-stranded RNA nonenveloped viruses that replicate in membrane-associated cytoplasmic viral factories (VFs) (4, 5). Reovirus strain type 1 Lang (T1L) represses IFN-␤ signaling, while type 3 Dearing (T3D) does not (6), and this

Section: Resultsmentioning

confidence: 99%

A Cytoplasmic RNA Virus Alters the Function of the Cell Splicing Protein SRSF2

Rivera-Serrano

Fritch

Scholl

et al. 2017

“…MRV stocks (T1L, T2J, and T3D C ) were obtained from Max Nibert (Harvard Medical School, Boston, MA). The superscript C in T3D C is used to differentiate the T3D strain used in this study from a T3D strain (T3D N ) that has previously been shown to differ in M1 gene sequence, factory morphology, and 2 ubiquitination phenotype (37,43). Purified virions were prepared as described previously (12) and stored in virion buffer (150 mM NaCl, 10 mM Tris [pH 7.4], 10 mM MgCl 2 ) at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Mammalian Orthoreovirus Particles Induce and Are Recruited into Stress Granules at Early Times Postinfection

Qin

Hastings

Miller

2009

Self Cite

132

Infection with many mammalian orthoreovirus (MRV) strains results in shutoff of host, but not viral, protein synthesis via protein kinase R (PKR) activation and phosphorylation of translation initiation factor eIF2␣. Following inhibition of protein synthesis, cellular mRNAs localize to discrete structures in the cytoplasm called stress granules (SGs), where they are held in a translationally inactive state. We examined MRV-infected cells to characterize SG formation in response to MRV infection. We found that SGs formed at early times following infection (2 to 6 h postinfection) in a manner dependent on phosphorylation of eIF2␣. MRV induced SG formation in all four eIF2␣ kinase knockout cell lines, suggesting that at least two kinases are involved in induction of SGs. Inhibitors of MRV disassembly prevented MRV-induced SG formation, indicating that viral uncoating is a required step for SG formation. Neither inactivation of MRV virions by UV light nor treatment of MRV-infected cells with the translational inhibitor puromycin prevented SG formation, suggesting that viral transcription and translation are not required for SG formation. Viral cores were found to colocalize with SGs; however, cores from UV-inactivated virions did not associate with SGs, suggesting that viral core particles are recruited into SGs in a process that requires the synthesis of viral mRNA. These results demonstrate that MRV particles induce SGs in a step following viral disassembly but preceding viral mRNA transcription and that core particles are themselves recruited to SGs, suggesting that the cellular stress response may play a role in the MRV replication cycle.

“…A previous study suggested that the T3 2 protein is subject to temperature-dependent misfolding (28), which led us to consider whether straindependent differences in viral replication efficiency in MDCK cells might be influenced by the temperature of infection. To test this hypothesis, viral titers were monitored in MDCK cells (Fig.…”

Section: Ultrastructural Analysis Of Viral Inclusions In Mdck Cellsmentioning

confidence: 99%

Reovirus Replication Protein μ2 Influences Cell Tropism by Promoting Particle Assembly within Viral Inclusions

Ooms

Jerome

Dermody

et al. 2012

The double-stranded RNA virus mammalian reovirus displays broad cell, tissue, and host tropism. A critical checkpoint in the reovirus replication cycle resides within viral cytoplasmic inclusions, which are biosynthetic centers of genome multiplication and new-particle assembly. Replication of strain type 3 Dearing (T3) is arrested in Madin-Darby canine kidney (MDCK) cells at a step subsequent to inclusion development and prior to formation of genomic double-stranded RNA. This phenotype is primarily regulated by viral replication protein μ2. To understand how reovirus inclusions differ in productively and abortively infected MDCK cells, we used confocal immunofluorescence and thin-section transmission electron microscopy (TEM) to probe inclusion organization and particle morphogenesis. Although no abnormalities in inclusion morphology or viral protein localization were observed in T3-infected MDCK cells using confocal microscopy, TEM revealed markedly diminished production of mature progeny virions. T3 inclusions were less frequent and smaller than those formed by T3-T1M1, a productively replicating reovirus strain, and contained decreased numbers of complete particles. T3 replication was enhanced when cells were cultivated at 31°C, and inclusion ultrastructure at low-temperature infection more closely resembled that of a productive infection. These results indicate that particle assembly in T3-infected MDCK cells is defective, possibly due to a temperature-sensitive structural or functional property of μ2. Thus, reovirus cell tropism can be governed by interactions between viral replication proteins and the unique cell environment that modulate efficiency of particle assembly.