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A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins
Published on: May 29, 2016
Cut site preference allows influenza A virus PA-X to discriminate between host and viral mRNAs
Lea Gaucherand1,2,3, Amrita Iyer2, Isabel Gilabert2,4
1Program in Molecular Microbiology, Tufts University Graduate School of Biomedical Sciences, Boston, MA, USA.
Abstract:
Many viruses block host gene expression to take over the infected cell. This process, termed host shutoff, is thought to promote viral replication by preventing antiviral responses and redirecting cellular resources to viral processes. Several viruses from divergent families accomplish host shutoff through RNA degradation by endoribonucleases. However, viruses also need to ensure expression of their own genes. The influenza A virus endoribonuclease PA-X solves this problem by sparing viral mRNAs and some host RNAs necessary for viral replication. To understand how PA-X distinguishes between RNAs, we characterized PA-X cut sites transcriptome-wide using 5' rapid amplification of complementary DNA ends coupled to high-throughput sequencing. This analysis, along with RNA structure predictions and validation experiments using reporters, shows that PA-Xs from multiple influenza strains preferentially cleave RNAs at GCUG tetramers in hairpin loops. Importantly, GCUG tetramers are enriched in the human but not the influenza transcriptome. Moreover, optimal PA-X cut sites inserted in the influenza A virus genome are quickly selected against during viral replication in cells. This finding suggests that PA-X evolved these cleavage characteristics to preferentially target host over viral mRNAs in a manner reminiscent of cellular self versus non-self discrimination.
Insights
Influenza A virus PA-X endonuclease degrades host RNA to aid viral replication. It preferentially cleaves GCUG tetramers in RNA hairpin loops, distinguishing host from viral genetic material.
Area of Science:
- Virology
- Molecular Biology
- Gene Expression
Background:
- Viruses often inhibit host gene expression (host shutoff) to facilitate replication.
- RNA degradation by endoribonucleases is a common mechanism for host shutoff.
- Influenza A virus PA-X endonuclease spares viral RNA while degrading host RNA.
Purpose of the Study:
- To elucidate the RNA sequence and structural determinants of PA-X cleavage.
- To understand how PA-X distinguishes between host and viral RNAs.
Main Methods:
- Transcriptome-wide characterization of PA-X cleavage sites using 5' rapid amplification of complementary DNA ends coupled to high-throughput sequencing (5' RACE-Seq).
- RNA structure prediction and reporter assays to validate cleavage preferences.
- In vivo selection experiments within the influenza A virus genome.
Main Results:
- PA-X preferentially cleaves RNAs at GCUG tetramers located within hairpin loops.
- GCUG tetramers are significantly enriched in the human transcriptome compared to the influenza virus transcriptome.
- Influenza A virus rapidly selects against optimal PA-X cut sites when introduced into its genome.
Conclusions:
- PA-X exhibits sequence and structural preferences for RNA cleavage, targeting GCUG tetramers in hairpin loops.
- These preferences enable PA-X to discriminate against viral RNA, sparing it from degradation.
- The cleavage characteristics of PA-X suggest an evolutionary mechanism for targeting host mRNA over viral mRNA, akin to self/non-self discrimination.
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