Analysis of the interaction of influenza virus polymerase complex with human cell factors

Abstract

The influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. Here we present the characterisation of the complexes formed intracellularly by the influenza polymerase in human cells. The virus polymerase was expressed by cotransfection of the polymerase subunits cDNAs, one of which fused to the tandem-affinity purification (TAP) tag. The intracellular complexes were purified by the TAP approach, which involves IgG-Sepharose and calmodulin-agarose chromatography, under very mild conditions. The purified complexes contained the heterotrimeric polymerase and a series of associated proteins that were not apparent in purifications of untagged polymerase used as a control. Several influenza polymerase-associated proteins were identified by MALDI-MS and their presence in purified polymerase-containing complexes were verified by Western blot. Their relevance for influenza infection was established by colocalisation with virus ribonucleoproteins in human infected cells. Most of the associated human factors were nuclear proteins involved in cellular RNA synthesis, modification and nucleo-cytoplasmic export, but some were cytosolic proteins involved in translation and transport. The interactions recognised in this proteomic approach suggest that the influenza polymerase might be involved in steps of the infection cycle other than RNA replication and transcription.

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).…”

Human G3BP1 interacts with β-F1-ATPase mRNA and inhibits its translation

“…Most of these proteins are known to be involved in virtually all stages of mRNA life cycle and have roles in more than one RNA-processing step, which suggests that they are multifunctional proteins (Huttelmaier et al, 2005;Martin and Ephrussi, 2009;Mongelard and Bouvet, 2007;Moore, 2005). In addition, they are present in other nuclear and cytoplasmic RNP complexes, such as the spliceosome (Chen et al, 2007), the Ago1 complex (Hock et al, 2007), IMP1 granules (Jonson et al, 2007) and FMRP granules (Khandjian et al, 2004), as well as in viral RNPs (Jorba et al, 2008;Mayer et al, 2007;Pacheco et al, 2008). The finding that these proteins built an RNP complex of oxidative phosphorylation highlights for the first time their relevance as post-transcriptional regulators in the biogenesis and function of mitochondria.…”

Heterogeneous Nuclear Ribonucleoprotein M Facilitates Enterovirus Infection

“…9). Previous studies have implicated a role for hnRNP M in replication of influenza A virus and Semliki Forest virus (SFV) (67,68). Moreover, depletion of hnRNP M enhances SFV gene expression and replication, suggesting that hnRNP M may be antiviral.…”