Site specific target binding controls RNA cleavage efficiency by the Kaposi's sarcoma-associated herpesvirus endonuclease SOX.

Aaron S Mendez,Carolin Vogt,Britt A Glaunsinger,Jens Bohne

doi:10.1093/nar/gky932

Abstract

A number of viruses remodel the cellular gene expression landscape by globally accelerating messenger RNA (mRNA) degradation. Unlike the mammalian basal mRNA decay enzymes, which largely target mRNA from the 5′ and 3′ end, viruses instead use endonucleases that cleave their targets internally. This is hypothesized to more rapidly inactivate mRNA while maintaining selective power, potentially though the use of a targeting motif(s). Yet, how mRNA endonuclease specificity is achieved in mammalian cells remains largely unresolved. Here, we reveal key features underlying the biochemical mechanism of target recognition and cleavage by the SOX endonuclease encoded by Kaposi's sarcoma-associated herpesvirus (KSHV). Using purified KSHV SOX protein, we reconstituted the cleavage reaction in vitro and reveal that SOX displays robust, sequence-specific RNA binding to residues proximal to the cleavage site, which must be presented in a particular structural context. The strength of SOX binding dictates cleavage efficiency, providing an explanation for the breadth of mRNA susceptibility observed in cells. Importantly, we establish that cleavage site specificity does not require additional cellular cofactors, as had been previously proposed. Thus, viral endonucleases may use a combination of RNA sequence and structure to capture a broad set of mRNA targets while still preserving selectivity.

Highlights

Viral infection dramatically reshapes the gene expression landscape of the host cell
Using an RNA substrate that is efficiently cleaved by SOX in cells, we revealed that specific RNA sequences within and outside of the cleavage site significantly contribute to SOX binding efficiency and target processing
The messenger RNA (mRNA) fragments resulting from the primary SOX endonucleolytic cleavage are predominantly cleared by the host 5 -3 exonuclease XRN1, while in vitro, RNA fragments are rapidly degraded by 5 -3 exonucleolytic activity intrinsic to purified SOX [9]

Summary

Introduction

Viral infection dramatically reshapes the gene expression landscape of the host cell. Suppression of host gene expression, termed host shutoff, can occur via a variety of mechanisms, but one common strategy is to accelerate degradation of mRNA [1,2,3]. This occurs during infection with DNA viruses such as alphaherpesviruses, gammaherpesvirues, and vaccinia virus, as well as with RNA viruses such as influenza A virus and SARS and MERS coronaviruses [1,4,5]. This strategy bypasses the normally rate limiting steps of deadenylation and decapping to effect rapid mRNA degradation by host exonucleases [1]

Methods

Results

Conclusion