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Decoying the cap- mRNA degradation system by a double-stranded RNA virus and poly(A)- mRNA surveillance by a yeast
D C Masison1, A Blanc, J C Ribas
1Section on Genetics of Simple Eukaryotes, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA.
Abstract:
The major coat protein of the L-A double-stranded RNA virus of Saccharomyces cerevisiae covalently binds m7 GMP from 5' capped mRNAs in vitro. We show that this cap binding also occurs in vivo and that, while this activity is required for expression of viral information (killer toxin mRNA level and toxin production) in a wild-type strain, this requirement is suppressed by deletion of SKI1/XRN1/SEP1. We propose that the virus creates decapped cellular mRNAs to decoy the 5'-->3' exoribonuclease specific for cap- RNA encoded by XRN1. The SKI2 antiviral gene represses the copy numbers of the L-A and L-BC viruses and the 20S RNA replicon, apparently by specifically blocking translation of viral RNA. We show that SKI2, SKI3, and SKI8 inhibit translation of electroporated luciferase and beta-glucuronidase mRNAs in vivo, but only if they lack the 3' poly(A) structure. Thus, L-A decoys the SKI1/XRN1/SEP1 exonuclease directed at 5' uncapped ends, but translation of the L-A poly(A)- mRNA is repressed by Ski2,3,8p. The SKI2-SKI3-SKI8 system is more effective against cap+ poly(A)- mRNA, suggesting a (nonessential) role in blocking translation of fragmented cellular mRNAs.
Insights
The L-A virus coats cap-protected mRNA to evade cellular defenses. Deleting SKI1/XRN1/SEP1 suppresses this, while the Ski2,3,8p complex inhibits viral mRNA lacking a poly(A) tail.
Area of Science:
- Virology
- Molecular Biology
- Yeast Genetics
Background:
- The L-A virus in Saccharomyces cerevisiae relies on its major coat protein for replication.
- Viral gene expression is often regulated by host cell machinery, including mRNA capping and degradation pathways.
Purpose of the Study:
- To investigate the in vivo role of L-A virus cap-binding activity.
- To elucidate the mechanisms by which host factors (SKI genes) regulate viral RNA and protein synthesis.
Main Methods:
- In vivo cap-binding assays using Saccharomyces cerevisiae.
- Analysis of viral gene expression (killer toxin) in wild-type and mutant yeast strains.
- Investigating the effect of SKI gene deletions on viral RNA and protein levels.
- Electroporation of capped and uncapped reporter mRNAs to assess translation inhibition by SKI proteins.
Main Results:
- L-A virus coat protein binds 5' capped mRNAs in vivo, essential for viral expression.
- Deletion of SKI1/XRN1/SEP1 suppresses the requirement for L-A cap-binding, suggesting decoy of 5'-->3' exoribonuclease.
- The SKI2-SKI3-SKI8 complex inhibits translation of viral RNA, particularly uncapped or poly(A)-deficient mRNAs.
- SKI2, SKI3, and SKI8 inhibit translation of electroporated reporter mRNAs lacking a 3' poly(A) structure.
Conclusions:
- The L-A virus employs a strategy to decoy the host SKI1/XRN1/SEP1 exonuclease by creating decapped cellular mRNAs.
- Host antiviral factors Ski2,3,8p repress L-A viral mRNA translation, especially those lacking a poly(A) tail.
- The SKI2-SKI3-SKI8 system may also play a role in degrading fragmented cellular mRNAs.