A Nucleolar Protein, Ribosomal RNA Processing 1 Homolog B (RRP1B), Enhances the Recruitment of Cellular mRNA in Influenza Virus Transcription

Abstract

Influenza A virus (IAV) undergoes RNA transcription by a unique capped-mRNA-dependent transcription, which is carried out by the viral RNA-dependent RNA polymerase (RdRp), consisting of the viral PA, PB1, and PB2 proteins. However, how the viral RdRp utilizes cellular factors for virus transcription is not clear. Previously, we conducted a genome-wide pooled short hairpin RNA (shRNA) screen to identify host factors important for influenza A virus replication. Ribosomal RNA processing 1 homolog B (RRP1B) was identified as one of the candidates. RRP1B is a nucleolar protein involved in ribosomal biogenesis. Upon IAV infection, part of RRP1B was translocated from the nucleolus to the nucleoplasm, where viral RNA synthesis likely takes place. The depletion of RRP1B significantly reduced IAV mRNA transcription in a minireplicon assay and in virus-infected cells. Furthermore, we showed that RRP1B interacted with PB1 and PB2 of the RdRp and formed a coimmunoprecipitable complex with RdRp. The depletion of RRP1B reduced the amount of capped mRNA in the RdRp complex. Taken together, these findings indicate that RRP1B is a host factor essential for IAV transcription and provide a target for new antivirals. IMPORTANCEInfluenza virus is an important human pathogen that causes significant morbidity and mortality and threatens the human population with epidemics and pandemics every year. Due to the high mutation rate of the virus, antiviral drugs targeting viral proteins might ultimately lose their effectiveness. An alternative strategy that explores the genetic stability of host factors indispensable for influenza virus replication would thus be desirable. Here, we characterized the rRNA processing 1 homolog B (RRP1B) protein as an important cellular factor for influenza A virus transcription. We showed that silencing RRP1B hampered viral RNA-dependent RNA polymerase (RdRp) activity, which is responsible for virus transcription and replication. Furthermore, we reported that RRP1B is crucial for RdRp binding to cellular capped mRNA, which is a critical step of virus transcription. Our study not only provides a deeper understanding of influenza virus-host interplay, but also suggests a potential target for antiviral drug development.

Zinc Finger-Containing Cellular Transcription Corepressor ZBTB25 Promotes Influenza Virus RNA Transcription and Is a Target for Zinc Ejector Drugs

“…Identification of ZBTB25 as a positive regulator of IAV replication. To identify cellular regulators involved in IAV replication, our group previously utilized a highthroughput pooled RNA interference (RNAi) library screen to search for potential candidate genes (31)(32)(33). In this study, a secondary RNAi screening was performed to confirm these genes.…”

The Nucleolar Protein LYAR Facilitates Ribonucleoprotein Assembly of Influenza A Virus

“…Previous studies have shown that there is an intense functional interplay between IAV and nucleoli ( 25 , 58 ). IAV vRNP hijacks the nucleolar proteins, such as NCL, NPM1, RRP1B, and some ribosomal proteins, to facilitate virus transcription and replication, or vRNP nuclear export ( 25 – 28 ), of which rRNA processing 1 homolog B (RRP1B) interacts with PB1 and PB2, enhancing viral transcription ( 27 ). NPM1 interacts with reconstituted vRNP, promoting viral polymerase activity ( 28 ).…”

“…The nucleolus is known to be the site of rRNA gene transcription, rRNA processing, and assembly into preribosomal subunits ( 22 , 23 ), and its nonclassical functions include mRNA transport, sequestration of regulation complex, and regulation of cellular metabolism and cellular stress responses ( 24 ). IAV also interacts with the nucleoli to usurp host cell functions and recruits nucleolar proteins to aid its replication in the nucleus; in particular, the vRNP hijacks many nucleolar proteins for its function ( 25 – 27 ). Mayer et al identified 45 cellular proteins interacting with vRNP or polymerase ( 28 ); 11 of them are nucleolar proteins, including the two major constitutive proteins of the nucleolus, nucleolin (NCL or C23) and nucleophosmin (NPM1 or B23), and ribosomal proteins, some of which have also been identified by others using similar proteomic approaches or other experiments ( 29 , 30 ).…”

Selective regulation in ribosome biogenesis and protein production for efficient viral translation

“…Similarly, both nucleolar proteins of ribosomal RNA processing 1 homolog B (RRP1B) and nucleolin are involved in ribosomal biogenesis (Chamousset et al 2010 ). RRP1B associated with pre-60S ribosomal subunits was translocated to the nucleoplasm upon viral infection of influenza virus, where RRP1B improves viral RNA-dependent RNA polymerase (RdRp) activity, which is responsible for virus transcription and replication (Su et al 2015 ). Nucleolin, which is thought to control RNA metabolism and ribosome biogenesis (Cong, et al 2012 ; Allain 2000 ), is prevented from entering the nucleus in poliovirus-infected cells (Waggoner 1998 ); this allows stimulation of IRES-mediated translation of viral transcripts (Izumi 2001 ).…”