Description: Previous studies from our laboratory revealed that cellular poly(C) binding protein 2 (PCBP2) downregulates vesicular stomatitis virus (VSV) gene expression. We show here that VSV infection induces the formation of granular structures in the cytoplasm containing cellular RNA-binding proteins, including PCBP2, T-cell-restricted intracellular antigen 1 (TIA1), and TIA1-related protein (TIAR). Depletion of TIA1 via small interfering RNAs (siRNAs), but not depletion of TIAR, results in enhanced VSV growth and gene expression. The VSV-induced granules appear to be similar to the stress granules (SGs) generated in cells triggered by heat shock or oxidative stress but do not contain some of the bona fide SG markers, such as eukaryotic initiation factor 3 (eIF3) or eIF4A, or the processing body (PB) markers, such as mRNA-decapping enzyme 1A (DCP1a), and thus may not represent canonical SGs or PBs. Our results revealed that the VSV-induced granules, called SG-like structures here, contain the viral replicative proteins and RNAs. The formation and maintenance of the SG-like structures required viral replication and ongoing protein synthesis, but an intact cytoskeletal network was not necessary. These results suggest that cells respond to VSV infection by aggregating the antiviral proteins, such as PCBP2 and TIA1, to form SG-like structures. The functional significance of these SG-like structures in VSV-infected cells is currently under investigation.

Description: Respiratory syncytial virus (RSV) infects small foci of respiratory epithelial cells via infected droplets. Infection induces expression of type I and III interferons (IFNs) and proinflammatory cytokines, the balance of which may restrict viral replication and affect disease severity. We explored this balance by infecting two respiratory epithelial cell lines with low doses of recombinant RSV expressing green fluorescent protein (rgRSV). A549 cells were highly permissive, whereas BEAS-2B cells restricted infection to individual cells or small foci. After infection, A549 cells expressed higher levels of IFN-β-, IFN--, and NF-B-inducible proinflammatory cytokines. In contrast, BEAS-2B cells expressed higher levels of antiviral interferon-stimulated genes, pattern recognition receptors, and other signaling intermediaries constitutively and after infection. Transcriptome analysis revealed that constitutive expression of antiviral and proinflammatory genes predicted responses by each cell line. These two cell lines provide a model for elucidating critical mediators of local control of viral infection in respiratory epithelial cells. IMPORTANCE Airway epithelium is both the primary target of and the first defense against respiratory syncytial virus (RSV). Whether RSV replicates and spreads to adjacent epithelial cells depends on the quality of their innate immune responses. A549 and BEAS-2B are alveolar and bronchial epithelial cell lines, respectively, that are often used to study RSV infection. We show that A549 cells are permissive to RSV infection and express genes characteristic of a proinflammatory response. In contrast, BEAS-2B cells restrict infection and express genes characteristic of an antiviral response associated with expression of type I and III interferons. Transcriptome analysis of constitutive gene expression revealed patterns that may predict the response of each cell line to infection. This study suggests that restrictive and permissive cell lines may provide a model for identifying critical mediators of local control of infection and stresses the importance of the constitutive antiviral state for the response to viral challenge.

Description: In a genome-wide small interfering RNA (siRNA) screen, we recently identified the interferon (IFN)-inducible protein 35 (IFI35; also known as IFP35) as a factor required for vesicular stomatitis virus (VSV) infection. Studies reported here were conducted to further understand the role and requirement of IFI35 in VSV infection. Consistent with the siRNA screening data, we found that depletion of IFI35 led to reduced VSV replication at the level of viral gene expression. Although no direct interaction of IFI35 with the viral replication machinery was observed, we found that IFI35 negatively regulated the host innate immune response and rescued poly(I·C)-induced inhibition of VSV replication. Promoter-driven reporter gene assays demonstrated that IFI35 overexpression suppressed the activation of IFN-β and ISG56 promoters, whereas its depletion had the opposite effect. Further investigation revealed that IFI35 specifically interacted with retinoic acid-inducible gene I (RIG-I) and negatively regulated its activation through mechanisms that included (i) suppression of dephosphorylation (activation) of RIG-I and (ii) proteasome-mediated degradation of RIG-I via K48-linked ubiquitination. Overall, the results presented here suggest a novel role for IFI35 in negative regulation of RIG-I-mediated antiviral signaling, which will have implications for diseases associated with excessive immune signaling. IMPORTANCE Mammalian cells employ a variety of mechanisms, including production of interferons (IFNs), to counteract invading pathogens. In this study, we identified a novel role for a cellular protein, IFN-inducible protein 35 (IFP35/IFI35), in negatively regulating the host IFN response during vesicular stomatitis virus (VSV) infection. Specifically, we found that IFI35 inhibited activation of the RNA sensor, the retinoic acid-inducible gene I (RIG-I), leading to inhibition of IFN production and thus resulting in better replication of VSV. The identification of a cellular factor that attenuates the IFN response will have implications toward understanding inflammatory diseases in humans that have been found to be associated with defects in the regulation of host IFN production, such as systemic lupus erythematosus and psoriasis.