Influenza Virus Infection Causes Specific Degradation of the Largest Subunit of Cellular RNA Polymerase II

Abstract

It has been described that influenza virus polymerase associates with RNA polymerase II (RNAP II). To gain information about the role of this interaction, we explored if changes in RNAP II occur during infection. Here we show that influenza virus causes the specific degradation of the hypophosphorylated form of the largest subunit of RNAP II without affecting the accumulation of its hyperphosphorylated forms. This effect is independent of the viral strain and the origin of the cells used. Analysis of synthesized mRNAs in isolated nuclei of infected cells indicated that transcription decreases concomitantly with RNAP II degradation. Moreover, this degradation correlated with the onset of viral transcription and replication. The ubiquitinmediated proteasome pathway is not involved in virally induced RNAP II proteolysis. The expression of viral polymerase from its cloned cDNAs was sufficient to cause the degradation. Since the PA polymerase subunit has proteolytic activity, we tested its participation in the process. A recombinant virus that encodes a PA point mutant with decreased proteolytic activity and that has defects in replication delayed the effect, suggesting that PA's contribution to RNAP II degradation occurs during infection.The genome of influenza virus consists of eight singlestranded RNA molecules of negative polarity. The viral RNA polymerase is composed of three subunits, PB1, PB2, and PA (16,26,27), which together with the nucleoprotein perform all the activities required for viral RNA expression (15,18,28,33). The PB2 subunit is able to bind cap 1 structures of host cell hnRNAs (8, 57). The PB1 subunit contains both sequence motifs typical of the viral RNA-dependent RNA polymerases (46), which are essential for RNA synthesis (7), and the endonuclease activity responsible for the cleavage of host mRNA precursors (35). The PA subunit is a phosphoprotein with proteolytic activity (25,40,50,51). The phenotype of viral temperature-sensitive and protease mutants suggests that the PA subunit may be involved in the transition from mRNA transcription to replication (29, 37). The transcription process involves a cap-stealing mechanism by which 5Ј-capped oligonucleotides derived from newly synthesized RNA polymerase II (RNAP II) transcripts are used as primers and elongated by the viral polymerase (9, 45). In line with this transcription strategy, parental virion RNPs colocalize with active RNAP II in the infected-cell nucleus (I. Salanueva, personal communication). Due to the requirements for cellular capped mRNAs, virus transcription is inhibited by actinomycin D or ␣-amanitin (38). Viral RNA replication involves the synthesis of cap-independent, full-length positive-stranded RNAs complementary to the genomic viral RNAs (vRNAs), which serve as templates for amplification of the vRNAs and are not sensitive to actinomycin D or ␣-amanitin (53).Many viruses induce alterations in host cell gene expression. Among these, changes in the transcriptional machinery of the infected cells are broadly documented. RNAP ...

Interferon Antagonist NSs of La Crosse Virus Triggers a DNA Damage Response-like Degradation of Transcribing RNA Polymerase II

“…Two other viruses, herpes simplex virus and influenza A virus, were previously shown to trigger degradation of RNAP II (61)(62)(63)(64). However, for both these viruses, levels of total RNAP II or of IIa are affected rather than preferentially IIo as in the case of LACV.…”

“…However, for both these viruses, levels of total RNAP II or of IIa are affected rather than preferentially IIo as in the case of LACV. Herpes simplex virus achieves RNAP II degradation through the proteasomal system (61,62), whereas for influenza A virus the proteolytic viral protein PA is responsible (63,64). It should be noted that, in contrast to LACV, these viruses are directly dependent on RNAP II activity.…”

Mammalian Adaptive Mutations of the PA Protein of Highly Pathogenic Avian H5N1 Influenza Virus

“…Most importantly, it catalyzes host mRNA cleavage via its endonuclease activity (31)(32)(33). Other functions of the PA protein include degradation of viral and host protein via its protease activity (34)(35)(36) and supporting nuclear import via nuclear localization signals (NLSs) (37). NLSs are recognized by host proteins and allow complexes of virus and host proteins to jointly pass through nuclear pore complexes.…”

Specific Residues of PB2 and PA Influenza Virus Polymerase Subunits Confer the Ability for RNA Polymerase II Degradation and Virus Pathogenicity in Mice

“…The RNAP II and CHD6 degradation observed seems to be a general feature triggered by influenza A viruses (10,12), but we have previously reported that degradation of RNAP II does not occur upon infection with the attenuated A/PR/8/34 (PR8) strain (9), which would contribute to the attenuated phenotype of the strain. However, a hypervirulent PR8 (hvPR8) variant, which multiplies much faster than standard PR8 (lvPR8) in infected cells and is more virulent in mice than the parental PR8 virus, efficiently induces RNAP II degradation (9).…”

“…More recently, a degradative process affecting RNA polymerase II (RNAP II) (9)(10)(11), as well as CHD6 chromatin remodeler (12), has been reported. Degradation and disabling of host cell factors involved in genome expression may contribute to hijacking the metabolism of the infected cell and to suppressing the establishment of the host antiviral defense against viral pathogens, contributing to viral pathogenicity.…”