Acoplamiento funcional entre el virus de la gripe y la maquinaria de transcripción celular relación con la patogenicidad viral

Resumen

Influenza virus polymerase is a heterotrimeric complex consisting of three subunits PB1, PB2 and PA. The polymerase, together with the nucleoprotein and the viral RNA, forms viral ribonucleoproteins (RNPs) that carry out viral transcription and replication. In a previous search for cellular associated factors using PA as bait in a yeast two-hybrid system, two proteins were identified: CHD6 and hCLE. hCLE co-immunoprecipitates with different phosphorylated forms of the RNAP polymerase II (RNAP II) and is a positive modulator of its activity. Since viral and cellular transcriptions are functionally coupled, we studied the possible role of hCLE protein during influenza virus infection. We have observed that the activity of reconstituted viral RNPs in hCLEsilenced cells is decreased. On the other hand, hCLE silencing has little effect on viral particles production and viral protein synthesis. However, the accumulation levels of hCLE protein increase along the infection and by immune electron microscopy we observe the presence of hCLE in viral particles released from infected cells. Recently, It has been described that influenza virus polymerase associates with RNAP II. To gain information about the role of this interaction, we explored if changes in RNAP II occur during infection. We observe that influenza virus infection causes the specific degradation of the hypophosphorylated form of the largest subunit of RNAP II without affecting the accumulation of its hyperphosphorylated forms. The analysis of synthesized mRNAs in isolated nuclei of infected cells indicates that cellular transcription decreases concomitantly with RNAP II degradation. Furthermore, the expression of viral polymerase from its cloned cDNAs is sufficient to cause the degradation and a recombinant virus that encodes a PA point mutant with decreased proteolytic activity delays the effect, suggesting a contribution of PA subunit to the RNAP II degradation process. To evaluate if this process is a general feature of virulent viruses, we studied the behavior of the influenza virus strains A/PR8/8/34 (PR8) and the cold-adapted A/Ann Arbor/6/60, which are currently used as the donor strains for vaccine seeds due to their attenuated phenotype. We have observed that upon infection with these strains, degradation of the RNAP II does not occur. Moreover, by run-off experiments we observe that PR8 has a reduced ability to inhibit cellular mRNA transcription. In addition, a hypervirulent PR8 variant that multiplies much faster than standard PR8 in infected cells and is more virulent in mice than the parental PR8 virus efficiently induces RNAP II degradation. When infection with natural human and avian isolates was examined, we observed that all these viruses efficiently induce the proteolytic process. Finally, we have observed that reassortant viruses with exchanged PA between strains that induce degradation and PR8 have a concomitant increased or decreased ability to degrade the RNAP II. Together, the data indicate that PA protein is involved in the RNAP II degradation process and that the capacity of the influenza virus to degrade RNAP II and inhibit the host-cell transcription machinery is a feature of influenza A viruses that might contribute to its virulence.