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RNP remodeling during RNA virus replication through phosphoregulation of SR proteins revealed by metabolic labeling

ABSTRACT: Mechanisms used by positive-stranded RNA viruses to control the translation-replication switch remain largely unexplored. Using metabolic labeling coupled with quantitative proteomics analyses, we unraveled the protein composition of temporal ribonucleoprotein complexes (RNPs) isolated during early and late enterovirus A71 (EV-A71) infection. Comparing the RNP components at the eclipse and maturation phase, representing early and late infection, revealed RNP remodeling over time by exchanging nuclear with cytoplasmic proteins. Among the nuclear proteins, EV-A71 infection induced the phosphorylation and cytoplasmic re-localization of nuclear SR proteins. During early infection, phosphorylated SR proteins cofractionated with the translation machinery rather than the replication organelles. During late infection, this co-localization, as well as the EV-A71-induced phosphorylated SR proteins, was no longer detected. Inhibition of SR protein phosphorylation by the kinase inhibitors SRPKIN-1 and TG003 significantly reduced the replication of several enteroviruses. Our results demonstrate the importance of phosphoregulation of SR proteins during enterovirus replication and reveal a potential target for broad-spectrum antivirals.

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ORGANISM(S): Homo Sapiens (human) Enterovirus A71

TISSUE(S): Muscle Contraction, Cell Culture

SUBMITTER: Chih-Ching Wu

LAB HEAD: Chih-Ching Wu

PROVIDER: PXD036890 | Pride | 2025-06-04

REPOSITORIES: Pride

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Project description:During viral infections cellular gene expression is subject to rapid alterations induced by both viral and antiviral mechanisms. In this study, we applied metabolic labeling of newly transcribed RNA with 4-thiouridine (4sU-tagging) to dissect the real-time kinetics of cellular and viral transcriptional activity during lytic murine cytomegalovirus (MCMV) infection. Microarray profiling on newly transcribed RNA obtained from different times during the first six hours of MCMV infection revealed discrete functional networks of cellular genes regulated with distinct kinetics at surprising temporal resolution. Most of these were undetectable in total RNA, either due to low temporal sensitivity of standard gene expression profiling using total RNA, or due to rapid (viral) counter-regulation. Furthermore, metabolic labeling and purification of newly transcribed RNA detailed the real-time kinetics and regulation of viral gene expression in absence of any interfering virion-associated RNA. Both qRT-PCR and next-generation sequencing of newly transcribed RNA demonstrated an unexpected peak of viral gene expression during the first two hours of infection including transcription of immediate early, early and even well characterized late genes. Interestingly, this peak was subject to rapid gene silencing (independent of the multiplicity of infection) with similar transcriptional activity only reached late in infection. For some genes, e.g. m152, this resulted in massive down-regulation of transcriptional activity by 6 hours post infection, which was not even reversed late in infection. Our findings thus highlight the importance of transcriptional activity during the first few hours of CMV infection and hint at novel mechanisms governing the kinetics of viral gene expression. In conclusion, studying real-time transcriptional activity during lytic CMV infection provides intriguing new insights into the regulation of cellular and viral gene expression. In total 13 samples were examined: 3 samples of total RNA (mock, 25, 48 hours past MCMV infection), 3 samples pre-existing RNA (mock, 25, 48 hpi), and 7 samples newly-transcribed RNA (mock, 1-2, 5-6, 11-12, 18-19, 24-25, 47-48 hpi)

			Action	DRS
	20190614-SRS-1-4C-01.RAW	Raw
	20190614-SRS-1-4C-02.RAW	Raw
	20190614-SRS-1-4C-03.RAW	Raw
	20190614-SRS-1-4C-04.RAW	Raw
	20190614-SRS-1-4C-05.RAW	Raw

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RNP remodeling during RNA virus replication through phosphoregulation of SR proteins revealed by metabolic labeling

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