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

Abstract

Influenza virus transcription requires functional coupling with cellular transcription for the cap-snatching process. Despite this fact, RNA polymerase II (RNAP II) is degraded during infection in a process triggered by the viral polymerase. Reassortant viruses from the A/PR/8/34 (PR8) strain that induce (hvPR8) or do not induce (lvPR8) RNAP II degradation led to the identification of PA and PB2 subunits as responsible for the degradation process. Three changes in the PB2 sequence (I105M, N456D, and I504V) and two in PA (Q193H and I550L) differentiate PA and PB2 of lvPR8 from those of hvPR8. Using recombinant viruses, we observed that changes at position 504 of PB2, together with 550 of PA, confer the ability on lvPR8 for RNAP II degradation and, conversely, abolish hvPR8 degradation capacity. Since hvPR8 is more pathogenic than lvPR8 in mice, we tested the potential contribution of RNAP II degradation in a distant viral strain, the 2009 pandemic A/California/04/09 (CAL) virus, whose PA and PB2 subunits are of avian origin. As in the hvPR8 virus, mutations at positions 504 of PB2 and 550 of PA in CAL virus abolished its RNAP II degradation capacity. Moreover, in an in vivo model, the CAL-infected mice lost more body weight, and 75% lethality was observed in this situation compared with 100% survival in mutant-CAL-or mock-infected animals. These results confirm the involvement of specific PB2 and PA residues in RNAP II degradation, which correlates with pathogenicity in mice of viruses containing human or avian polymerase PB2 and PA subunits. IMPORTANCEThe influenza virus polymerase induces the degradation of RNAP II, which probably cooperates to avoid the antiviral response. Here, we have characterized two specific residues located in the PA and PB2 polymerase subunits that mediate this degradation in different influenza viruses. Moreover, a clear correlation between RNAP II degradation and in vivo pathogenicity in mice was observed, indicating that the degradative process constitutes a viral pathogenicity factor.

The Cellular Factor NXP2/MORC3 Is a Positive Regulator of Influenza Virus Multiplication

“…Plasmids pCMVPB1, pCMVPB2, pCMVPA, and pCMVNP, expressing the influenza virus polymerase subunits and the NP, were described previously (28). Plasmid pHHCAT, which transcribes a virus-like cat gene in negative polarity (29), was provided by A. Rodriguez-Frandsen.…”

Influenza Virus and Chromatin: Role of the CHD1 Chromatin Remodeler in the Virus Life Cycle

“…This degradation is reflected as a shorter protein half-life and is proteasome independent (24). In vivo experiments indicate that RNAP II degradation correlates with pathogenicity in mice (27).…”

“…The hvPR8 strain (high virulence, RNAP II degradation inducer) bears PA 550L and PB2 504V, whereas the lvPR8 strain (low virulence, noninducer of RNAP II degradation) has PA 550I and PB2 504I. A defined combination of PA 550L and PB2 504V confers on the PR8 background the ability to degrade RNAP II, as it also does for a natural isolate, the pandemic CAL 2009 strain (27). Individual changes of PA 550L or PB2 504V to isoleucine attenuate the ability of these recombinant viruses to degrade RNAP II, and the double mutation (DM) eliminates this ability.…”

“…Individual changes of PA 550L or PB2 504V to isoleucine attenuate the ability of these recombinant viruses to degrade RNAP II, and the double mutation (DM) eliminates this ability. The specific PA 550L-PB2 504V combination restores the ability of lvPR8DM to degrade RNAP II, and conversely, the double mutation of these amino acids to isoleucine inhibits the degradation abilities of CAL and hvPR8 (27).…”

“…Despite the coupling between viral and cellular transcription, reports showed that in a process triggered by the viral polymerase, influenza virus infection induces RNAP II degradation once viral transcription is complete and synthesis of cellular mRNA is no longer needed (24)(25)(26). We recently characterized the involvement pf the PB2 and PA subunits in RNAP II degradation, as well as the contribution of specific subunit residues to this process, which correlates with pathogenicity in mice (27). We also showed that the CHD6 chromatin remodeler is specifically degraded after influenza virus infection in cultured cells and in a mouse model (28).…”