Viral-mediated ubiquitination impacts interactions of host proteins with viral RNA and promotes viral RNA processing
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ABSTRACT:
PROVIDER: PRJNA607121 | ENA |
REPOSITORIES: ENA
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Project description: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.
2020-07-09 | GSE145411 | GEO
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Project description:RNA silencing is a post-transcriptional gene-silencing mechanism mediated by microRNAs (miRNAs). However, the regulatory mechanism of RNA silencing during viral infection is unclear. TAR RNA-binding protein (TRBP) is an enhancer of RNA silencing that induces miRNA maturation by interacting with the ribonuclease Dicer. TRBP interacts with a virus sensor protein, laboratory of genetics and physiology 2 (LGP2), in the early stage of viral infection of human cells. Next, it induces apoptosis by inhibiting the maturation of miRNAs, thereby upregulating the expression of apoptosis regulatory genes. In this study, we show that TRBP undergoes a functional conversion in the late stage of viral infection. Viral infection resulted in the activation of caspases that proteolytically processed TRBP into two fragments. The N-terminal fragment did not interact with Dicer but interacted with type I interferon (IFN) signaling modulators, such as protein kinase R (PKR) and LGP2, and induced ER stress. The end results were irreversible apoptosis and suppression of IFN signaling. Our results demonstrate that the processing of TRBP enhances apoptosis, reducing IFN signaling during viral infection.
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Project description:RNA silencing is a post-transcriptional gene-silencing mechanism mediated by microRNAs (miRNAs). However, the regulatory mechanism of RNA silencing during viral infection is unclear. TAR RNA-binding protein (TRBP) is an enhancer of RNA silencing that induces miRNA maturation by interacting with the ribonuclease Dicer. TRBP interacts with a virus sensor protein, laboratory of genetics and physiology 2 (LGP2), in the early stage of viral infection of human cells. Next, it induces apoptosis by inhibiting the maturation of miRNAs, thereby upregulating the expression of apoptosis regulatory genes. In this study, we show that TRBP undergoes a functional conversion in the late stage of viral infection. Viral infection resulted in the activation of caspases that proteolytically processed TRBP into two fragments. The N-terminal fragment did not interact with Dicer but interacted with type I interferon (IFN) signaling modulators, such as protein kinase R (PKR) and LGP2, and induced ER stress. The end results were irreversible apoptosis and suppression of IFN signaling. Our results demonstrate that the processing of TRBP enhances apoptosis, reducing IFN signaling during viral infection.
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Project description:Anti-viral innate immunity represents the first line of defense against invading viruses and is key to control viral infections, including the pandemic SARS-CoV-2. Body temperature is an omnipresent variable but was so far neglected when addressing host defense mechanisms and susceptibility to SARS-CoV-2 infection. Here we show that increasing temperature in a 1.5°C window, between 36.5°C and 38°C, strongly increases anti-viral immunity. We show that alternative splicing coupled to nonsense-mediated decay decreases STAT2 expression in colder conditions and suggest that increased STAT2 expression at elevated temperature induces the expression of diverse anti-viral genes. This cascade is activated in a remarkably narrow temperature range below febrile temperature, which reflects individual, circadian and age-dependent body temperature variation. Accordingly, decreased body temperature with ageing correlates with reduced expression of anti-viral genes in older individuals. Using cell culture and in vivo models, we show that higher body temperature indeed reduces SARS-CoV-2 replication, which likely impacts on the different vulnerability of children versus seniors towards severe SARS-CoV-2 infection. Altogether, our data reveal a molecular mechanism, from temperature sensing and pre-mRNA processing to an in vivo phenotype connecting body temperature variation with SARS-CoV-2 replication, thus providing a new paradigm for the regulation of anti-viral innate immunity.
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Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 specifically binds to the 5?-end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RNA-binding protein and direct SND1 interaction partner, is covalently linked to the 5'-ends of positive and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.
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2023-12-10 | GSE249455 | GEO
Project description:The coronavirus disease 2019 (COVID-19) pandemic has devastated global health. Identifying key host factors essential for SARS-CoV-2 RNA replication is expected to unravel cellular targets for the development of broad-spectrum antiviral drugs which have been quested for the preparedness of future viral outbreaks. Here, we have identified host proteins that associate with non-structural protein 12 (nsp12), the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 using a mass spectrometry (MS)-based proteomic approach. Among the candidate factors, CDK2 (Cyclin-dependent kinase 2), a member of cyclin-dependent kinases, interacts with nsp12 and causes its phosphorylation at T20, thus facilitating the assembly of the RdRp complex consisting of nsp12, nsp7 and nsp8 and promoting efficient synthesis of viral RNA. The crucial role of CDK2 in viral RdRp function is further supported by our observation that CDK2 inhibitors potently impair viral RNA synthesis and SARS-CoV-2 infection. Taken together, we have discovered CDK2 as a key host factor of SARS-CoV-2 RdRp complex, thus serving a promising target for the development of SARS-CoV-2 RdRp inhibitors.
2022-09-28 | PXD037068 | iProX
Project description:Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5ʹ end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5ʹ ends of positive and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.
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