An influenza virus replicon system in yeast identified Tat-SF1 as a stimulatory host factor for viral RNA synthesis

Abstract

Influenza viruses infect vertebrates, including mammals and birds.Influenza virus reverse-genetics systems facilitate the study of the structure and function of viral factors. In contrast, less is known about host factors involved in the replication process. Here, we developed a replication and transcription system of the negativestrand RNA genome of the influenza virus in Saccharomyces cerevisiae, which depends on viral RNAs, viral RNA polymerases, and nucleoprotein (NP). Disruption of SUB2 encoding an orthologue of human RAF-2p48/UAP56, a previously identified viral RNA synthesis stimulatory host factor, resulted in reduction of the viral RNA synthesis rate. Using a genome-wide set of yeast single-gene deletion strains, we found several host factor candidates affecting viral RNA synthesis. We found that among them, Tat-SF1, a mammalian homologue of yeast CUS2, was a stimulatory host factor in influenza virus RNA synthesis. Tat-SF1 interacted with free NP, but not with NP associated with RNA, and facilitated formation of RNA-NP complexes. These results suggest that Tat-SF1 may function as a molecular chaperone for NP, as does RAF-2p48/UAP56. This system has proven useful for further studies on the mechanism of influenza virus genome replication and transcription. molecular chaperone ͉ replication ͉ RNA-dependent RNA polymerase ͉ nucleoprotein

Interaction of the Influenza A Virus Nucleocapsid Protein with the Viral RNA Polymerase Potentiates Unprimed Viral RNA Replication

“…These cRNA and vRNA products presumably initiate with a ribonucleoside triphosphate, as shown by other studies of cRNA and vRNA synthesis (6). Because the purified polymerase and NP protein preparations used in our assays were obtained from insect cells and contain no major contaminating proteins, our results suggest that the initiation of unprimed RNA synthesis does not require previously reported mammalian host factors (12,13,21,23,24,26,39). Nonetheless, our results do not rule out the possibility that such mammalian host factors might regulate or further enhance viral RNA replication in infected cells.…”

“…In addition, amino acids in PB1 and PA have been implicated in virulence in ferrets and/or ducks (11,38). It has been postulated that influenza virus RNA synthesis requires host factors that differ between species (5,17,25,40,42), and evidence for the participation of host factors in influenza virus RNA synthesis has been reported (12,13,21,23,24,26,39).…”

The influenza virus RNA synthesis machine

“…61,117 The incorporation of NP into new vRNPs is facilitated by the activity of cellular factors UAP56 and Tat-SF1, that may act as chaperones. 118,119 Viral polymerase should also be required stoichiometrically, as the end product is not vRNA but a vRNP. The question remains whether the polymerase complex associated to the progeny vRNP is identical or distinct to the polymerase that actually performs the replication reaction.…”

pp32, an INHAT component, is a transcription machinery recruiter for maximal induction of IFN-stimulated genes

“…Furthermore, we used pU6-puro-sipp32 and pU6-purosiEGFP plasmids, which express 21-nucleotide hairpin-type siRNAs against pp32 and EGFP, respectively, under the control of the U6 promoter (Haruki et al, 2006;Naito et al, 2007). The target sequence of pU6-puro-sipp32, 5Ј-AAGA -ACTTGTCCTGGACAACAGT-3Ј, differs from that of pp32 Stealth siRNA, 5Ј-AATTCCTATTGTGATTTGACTGTTT-3Ј.…”