RNA Exosome Complex Regulates Stability of the Hepatitis B Virus X-mRNA Transcript in a Non-stop-mediated (NSD) RNA Quality Control Mechanism

Hussein H Aly,Junya Suzuki,Koichi Watashi,Kazuaki Chayama,Shin-Ichi Hoshino,Makoto Hijikata,Takanobu Kato,Takaji Wakita

doi:10.1074/jbc.m116.724641

Abstract

Hepatitis B virus (HBV) is a stealth virus, minimally inducing the interferon system required for efficient induction of both innate and adaptive immune responses. However, 90% of acutely infected adults can clear the virus, suggesting the presence of other, interferon-independent pathways leading to viral clearance. Given the known ability of helicases to bind viral nucleic acids, we performed a functional screening assay to identify helicases that regulate HBV replication. We identified the superkiller viralicidic activity 2-like (SKIV2L) RNA helicase (a homolog of the Saccharomyces cerevisiae Ski2 protein) on the basis of its direct and preferential interaction with HBV X-mRNA. This interaction was essential for HBV X-mRNA degradation at the RNA exosome. The degradation of HBV X-mRNA at the RNA exosome was also mediated by HBS1L (HBS1-like translational GTPase) protein, a known component of the host RNA quality control system. We found that the redundant HBV-precore translation initiation site present at the 3'-end of HBV X-mRNA (3' precore) is translationally active. The initiation of translation from this site without a proper stop codon was identified by the non-stop-mediated RNA decay mechanism leading to its degradation. Although 3' precore is present in the five main HBV-RNA transcripts, only X-mRNA lacks the presence of an upstream start codons for large, middle, and small (L, M, and S) HBV surface proteins. These upstream codons are in-frame with 3' precore translation initiation site, blocking its translation from the other HBV-mRNA transcripts. To our knowledge, this is the first demonstration of the anti-viral function of the non-stop-mediated RNA decay mechanism.

Highlights

Viral infections are initially detected and counteracted by the host through the innate immune response [1]
We identified the superkiller viralicidic activity 2-like (SKIV2L) RNA helicase on the basis of its direct and preferential interaction with Hepatitis B virus (HBV) X-mRNA
SiRNA silencing of SKIV2L yielded increased HBV X-mRNA titers (Fig. 4, C (p Ͻ 0.05) and D (p Ͻ 0.05)), the RIP assay showed that silencing of SKIV2L suppressed HBV X-mRNA interaction with RNA exosome components EXOSC4 (Fig. 4E) and EXOSC5 (Fig. 4F). These results show that SKIV2L is required for the HBV XmRNA/RNA exosome interaction

Summary

Introduction

Viral infections are initially detected and counteracted by the host through the innate immune response [1]. Three independent experiments were performed in triplicate, and data are presented as the mean Ϯ S.D. D, HepG2 cells were co-transfected with HBV-D60 plasmid or empty vector (pUC19; mock) together with pEF4-SKIV2L-MycHis or pEF4-MycHis. Three days later intracellular HBV-RNA levels were assessed by Northern blot.

Results

Conclusion