Abstract
The influenza A virus RNA polymerase is a heterotrimeric complex responsible for viral genome transcription and replication in the nucleus of infected cells. We recently carried out a proteomic analysis of purified polymerase expressed in human cells and identified a number of polymerase-associated cellular proteins. Here we characterise the role of one such host factors, SFPQ/PSF, during virus infection. Down-regulation of SFPQ/PSF by silencing with two independent siRNAs reduced the virus yield by 2–5 log in low-multiplicity infections, while the replication of unrelated viruses as VSV or Adenovirus was almost unaffected. As the SFPQ/PSF protein is frequently associated to NonO/p54, we tested the potential implication of the latter in influenza virus replication. However, down-regulation of NonO/p54 by silencing with two independent siRNAs did not affect virus yields. Down-regulation of SFPQ/PSF by siRNA silencing led to a reduction and delay of influenza virus gene expression. Immunofluorescence analyses showed a good correlation between SFPQ/PSF and NP levels in infected cells. Analysis of virus RNA accumulation in silenced cells showed that production of mRNA, cRNA and vRNA is reduced by more than 5-fold but splicing is not affected. Likewise, the accumulation of viral mRNA in cicloheximide-treated cells was reduced by 3-fold. In contrast, down-regulation of SFPQ/PSF in a recombinant virus replicon system indicated that, while the accumulation of viral mRNA is reduced by 5-fold, vRNA levels are slightly increased. In vitro transcription of recombinant RNPs generated in SFPQ/PSF-silenced cells indicated a 4–5-fold reduction in polyadenylation but no alteration in cap snatching. These results indicate that SFPQ/PSF is a host factor essential for influenza virus transcription that increases the efficiency of viral mRNA polyadenylation and open the possibility to develop new antivirals targeting the accumulation of primary transcripts, a very early step during infection.
Highlights
The influenza A viruses belong to the family Orthomyxoviridae and contain a segmented, single-stranded RNA genome of negative polarity
These results suggest that SFPQ/PSF might be required for virus primary transcription and the observed reduction in vRNA accumulation would be an indirect consequence, since viral protein expression is essential for viral RNA replication [58]
Concluding remarks The results presented here show that SFPQ/PSF is required for influenza virus multiplication and indicate that this cellular factor is essential for the transcription of viral RNPs during both primary and secondary mRNA synthesis
Summary
The influenza A viruses belong to the family Orthomyxoviridae and contain a segmented, single-stranded RNA genome of negative polarity A number of human cell factors have been described as interactors of influenza virus polymerase and in some specific cases their role in virus replication has been studied [23,24,25,26,27,28,29,30,31,32,33,34,35,36] In one such studies, we identified the human SFPQ/PSF factor as associated in vivo to influenza virus polymerase by proteomic analysis of purified complexes [34]. In agreement with the SFPQ/PSF association with PTB, its binding has been reported to elements in the splicing machinery, like the U4/U6U5 tri-snRNP and many other splicing factors [44,45,46] and the RNA pol II CTD [47], probably in a RRM-dependent manner
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