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Viral-mediated ubiquitination impacts interactions of host proteins with viral RNA and promotes viral RNA processing

ABSTRACT: Viruses promote infection by hijacking the host ubiquitin machinery to counteract or redirect cellular processes. Adenovirus encodes two early proteins, E1B55K and E4orf6, that together co-opt a cellular ubiquitin ligase complex to overcome host defenses and promote virus production. Adenovirus mutants lacking E1B55K or E4orf6 display defects in viral RNA processing and protein production, but previously identified substrates of the ligase do not explain these phenotypes. Here we used a quantitative proteomics approach to identify substrates of E1B55K/E4orf6 that are ubiquitinated to facilitate RNA processing. While cellular proteins known as substrates of E1B55K/E4orf6 are degraded by the proteasome, we uncovered RNA-binding proteins (RBPs) as high-confidence substrates which are not decreased in overall abundance. We focused on two predominant RBPs, RALY and hnRNP-C, which we confirm are ubiquitinated without degradation. Knockdown of RALY and hnRNP-C rescued levels of viral RNA splicing, protein, and progeny production during infection with E1B55K-deleted virus. Furthermore, deletion of E1B55K resulted in increased interaction of hnRNP-C with viral RNA and attenuation of viral RNA processing. These data suggest viral-mediated ubiquitination of RALY and hnRNP-C relieves a restriction on viral RNA processing, revealing an unexpected role for non-degradative ubiquitination in the manipulation of cellular processes during virus infection.

ORGANISM(S): Homo sapiens

PROVIDER: GSE145411 | GEO | 2020/07/09

REPOSITORIES: GEO

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Project description:Viral infections are associated with extensive remodeling of the cellular proteome. Viruses encode gene products that manipulate host proteins to redirect cellular processes or subvert antiviral immune responses. One way in which host antiviral proteins antagonize viral infection is by associating with viral genomes and inhibiting essential viral processes. Adenovirus (AdV) encodes gene products from the early E4 region which are necessary for productive infection. Some cellular antiviral proteins are known to be targeted by AdV E4 gene products, resulting in their degradation or mislocalization. However, the full repertoire of host proteins manipulated by viral E4 proteins has not been defined. To identify cellular proteins and processes manipulated by viral products, we developed a global, un-biased proteomics approach to analyze changes to the host proteome during infection with Adenovirus serotype 5 (Ad5) virus. We combined quantification of total protein abundance in the proteome together with an analysis of proteins associated with viral genomes using isolation of Proteins on Nascent DNA (iPOND). By Integrating these proteomics datasets, we identified cellular factors that are degraded in an E4-dependent manner or are associated with the viral genome in the absence of E4 proteins. We further show that some identified proteins exert inhibitory effects on Ad5 infection. Our systems-level analysis reveals cellular processes that are manipulated during Ad5 infection and points to host factors counteracted by early viral proteins as they remodel the host proteome to promote efficient infection. Importance Viral infections stimulate myriad changes to the host cell proteome. As viruses harness cellular processes and counteract host defenses, they impact abundance, modifications, or localization of cellular proteins. Elucidating the dynamic changes to the cellular proteome during viral replication is integral to understanding how virus-host interactions influence the outcome of infection. Adenovirus serotype 5 (Ad5) encodes early gene products from the E4 genomic region that are known to alter host response pathways and promote replication, but the full extent of proteome modifications they mediate is not known. We use an integrated proteomics approach to quantitate protein abundance and protein associations with viral DNA during virus infection. Systems-level analysis identifies cellular proteins and processes impacted in an E4-dependent manner that could overcome potentially inhibitory host defenses. This study provides a global view of Adenovirus-mediated proteome remodeling which can serve as a model to investigate virus-host interactions of DNA viruses.

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Viral-mediated ubiquitination impacts interactions of host proteins with viral RNA and promotes viral RNA processing

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