Pooled RNAi screen identifies ubiquitin ligase Itch as crucial for influenza A virus release from the endosome during virus entry

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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.

Section: Identification Of Rrp1b As An Important Cofactor For Influenzamentioning

confidence: 99%

“…Unfortunately, relatively few overlaps among the candidates were found. We therefore carried out a modified pooled genome-wide RNAi library screen (12). In total, 38 candidate genes, hit by two unique short hairpin RNAs (shRNAs) showing at least 40% reduction of viral replication, were identified as hits.…”

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confidence: 99%

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

Hsu

Lee

et al. 2015

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“…EGFR is a receptor tyrosine kinase (RTK) that acts upstream of MAPK1 and has been implicated in IAV infection. During a recently published RNA interference screen for host factors of IAV, EGFR was one of the best-performing candidates (29). In addition, Eierhoff and colleagues have demonstrated that EGFR is activated following IAV attachment, which in turn promotes the uptake of virions into cells (39), thereby providing evidence for a role of EGFR during IAV entry.…”

Section: Discussionmentioning

confidence: 99%

“…Genome-wide small interfering RNA (siRNA) screens are a powerful tool to identify host factors associated with viral infections. Several such screens have been performed to determine cellular factors required by IAV during infection (23)(24)(25)(26)(27)(28)(29)(30). Surprisingly, there was hardly any overlap between the primary hits identified in these screens (31).…”

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confidence: 99%

Prolidase Is Required for Early Trafficking Events during Influenza A Virus Entry

Pohl

Edinger

Stertz

2014

Influenza A virus (IAV) entry is a multistep process that requires the interaction of the virus with numerous host factors. In this study, we demonstrate that prolidase (PEPD) is a cellular factor required by IAV for successful entry into target cells. PEPD was selected as a candidate during an entry screen performed on nonvalidated primary hits from previously published genome-wide small interfering RNA (siRNA) screens. siRNA-mediated depletion of PEPD resulted in the decreased growth of IAV during mono-and multicycle growth. This growth defect was independent of cell type or virus strain. Furthermore, IAV restriction was apparent as early as 3 h postinfection, and experiments in the absence of protein biosynthesis revealed that the nuclear import of viral ribonucleoprotein complexes (vRNPs) was already blocked in the absence of PEPD. These results led us to investigate which step during entry was affected. Receptor expression, IAV attachment, or IAV internalization was not dependent on the presence of PEPD. However, when looking at the distribution of incoming IAV particles in PEPD-knockdown cells, we found a localization pattern that differed from that in control cells: IAV mostly localized to the cell periphery, and consequently, viral particles displayed reduced colocalization with early and late endosome markers and fusion between viral and endosomal membranes was strongly reduced. Finally, experiments using a competitive inhibitor of PEPD catalytic activity suggested that the enzymatic function of the dipeptidase is required for its proviral effect on IAV entry. In sum, this study establishes PEPD as a novel entry factor required for early endosomal trafficking of IAV. IMPORTANCEInfluenza A virus (IAV) continues to be a constant threat to public health. As IAV relies on its host cell for replication, the identification of host factors required by the virus is of importance. First, such studies often reveal novel functions of cellular factors and can extend our knowledge of cellular processes. Second, we can further our understanding of processes that are required for the entry of IAV into target cells. Third, the identification of host factors that contribute to IAV entry will increase the number of potential targets for the development of novel antiviral drugs that are of urgent need. Our study identifies prolidase (PEPD) to be a novel entry factor required by IAV for correct routing within the endosomal compartment following virus internalization. Thereby, we link PEPD, which has been shown to play a role during collagen recycling and growth factor signaling, to early events of viral infection.

“…Phosphorylation of M1 at Y132 mediates the nuclear import of M1 (23), whereas SUMOylation of M1 regulates the nuclear export of vRNPs and promotes virion assembly and budding (21). Ubiquitination of M1 has been implicated in IAV release from the endosomes (24). However, the precise modification site and the biological functions of M1 ubiquitination are not well understood.…”

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confidence: 99%

Interaction of NS2 with AIMP2 Facilitates the Switch from Ubiquitination to SUMOylation of M1 in Influenza A Virus-Infected Cells

Gao

Liu

et al. 2015

Influenza A viruses (IAVs) rely on host factors to support their life cycle, as viral proteins hijack or interact with cellular proteins to execute their functions. Identification and understanding of these factors would increase our knowledge of the molecular mechanisms manipulated by the viruses. In this study, we searched for novel binding partners of the influenza A virus NS2 protein, the nuclear export protein responsible for overcoming host range restriction, by a yeast two-hybrid screening assay and glutathione S-transferase-pulldown and coimmunoprecipitation assays and identified AIMP2, a potent tumor suppressor that usually functions to regulate protein stability, as one of the major NS2-binding candidates. We found that the presence of NS2 protected AIMP2 from ubiquitin-mediated degradation in NS2-transfected cells and AIMP2 functioned as a positive regulator of IAV replication. Interestingly, AIMP2 had no significant effect on NS2 but enhanced the stability of the matrix protein M1. Further, we provide evidence that AIMP2 recruitment switches the modification of M1 from ubiquitination to SUMOylation, which occurs on the same attachment site (K242) on M1 and thereby promotes M1-mediated viral ribonucleoprotein complex nuclear export to increase viral replication. Collectively, our results reveal a new mechanism of AIMP2 mediation of influenza virus replication. IMPORTANCEAlthough the ubiquitination of M1 during IAV infection has been observed, the precise modification site and the molecular consequences of this modification remain obscure. Here, we demonstrate for the first time that ubiquitin and SUMO compete for the same lysine (K242) on M1 and the interaction of NS2 with AIMP2 facilitates the switch of the M1 modification from ubiquitination to SUMOylation, thus increasing viral replication. Influenza A virus (IAV) is a significant cause of morbidity and mortality in both humans and animal species owing to its ability to cause yearly epidemics and occasional pandemics (1-3). Like other viruses, IAV hijacks the host cellular machinery to support its life cycle. Thus, identification and characterization of the interactions between viral components and specific host factors would help provide an understanding of the mechanisms by which the viruses acquire human pandemic potential.IAV belongs to the Orthomyxoviridae family, and its genome consists of eight negative-sense RNA segments encoding up to 17 viral proteins (4-9). The viral RNA (vRNA) exists as a form of viral ribonucleoprotein complexes (vRNPs) containing vRNA, nucleoprotein (NP), and three viral polymerase proteins (PB1, PB2, and PA). Unlike most other RNA viruses, influenza virus transcribes and replicates its genome in the nucleus. Thus, after it enters a host cell, vRNPs enter the nucleus to complete transcription and replication (10). The newly synthesized vRNPs are exported from the nucleus for packaging into progeny virions (11). In this regard, efficient nuclear export of vRNPs is essential for productive infection.NS2, also kn...