CHD6, a Cellular Repressor of Influenza Virus Replication, Is Degraded in Human Alveolar Epithelial Cells and Mice Lungs during Infection

Abstract

The influenza virus polymerase associates to an important number of transcription-related proteins, including the largest subunit of the RNA polymerase II complex (RNAP II). Despite this association, degradation of the RNAP II takes place in the infected cells once viral transcription is completed. We have previously shown that the chromatin remodeler CHD6 protein interacts with the influenza virus polymerase complex, represses viral replication, and relocalizes to inactive chromatin during influenza virus infection. In this paper, we report that CHD6 acts as a negative modulator of the influenza virus polymerase activity and is also subjected to degradation through a process that includes the following characteristics: (i) the cellular proteasome is not implicated, (ii) the sole expression of the three viral polymerase subunits from its cloned cDNAs is sufficient to induce proteolysis, and (iii) degradation is also observed in vivo in lungs of infected mice and correlates with the increase of viral titers in the lungs. Collectively, the data indicate that CHD6 degradation is a general effect exerted by influenza A viruses and suggest that this viral repressor may play an important inhibitory role since degradation and accumulation into inactive chromatin occur during the infection. The influenza virus contains a segmented genome of eight negative-sense and single-stranded RNA molecules, whose expression takes place in the nucleus of the infected cell. Genomic RNAs (vRNAs) form ribonucleoprotein complexes (vRNPs) that are constituted by the three subunits of the polymerase (PB1, PB2, and PA) and the nucleoprotein (NP), which are responsible for genome expression (1-5). For viral replication, the vRNAs are copied to form full-length positive-stranded RNAs (cRNAs), which serve as templates for vRNA synthesis. During transcription, capped and polyadenylated viral mRNAs are synthesized by the viral polymerase. The mRNA synthesis is primed by shortcapped oligonucleotides of around 10 to 12 nucleotides scavenged from de novo synthesized host cell pre-mRNAs by a viral endonuclease activity (6). This transcription strategy involves a functional coupling between viral and cellular transcription for the capsnatching process, but this functional association is broader and applies to different steps of viral mRNA metabolism. Indeed, two of the viral transcripts are spliced (7,8), but the influenza virus does not possess a viral splicing system, since it is dependent on the host splicing machinery (9), an activity related to the RNA polymerase II (RNAP II) transcription. Furthermore, the influenza virus uses the mRNA export machinery of the infected cell at least for some of its mRNAs, and an active RNA polymerase II is required to facilitate nuclear export of selected viral mRNAs (10). In agreement with this transcriptional association, interaction of the viral polymerase with host cell transcription-related factors has been reported, among which the interaction with the largest subunit of the RNAP II (11) should be emphasi...

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

“…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).…”

CHD6 (chromodomain helicase DNA binding protein 6)

“…Additionally, CHD6 is a DNA dependent ATPase. CHD6 protein is expected to function as a transcriptional repressor and it has been shown to be interact with RNA Pol II proteins (Lutz et al, 2006) and to be involved in the cellular repression of viral replication (Fertey et al, 2010;Alfonso et al, 2011;Alfonso et al, 2013 …”

“…It associates with viral ribonuclear proteins in infected cells (Alfonso et al, 2011). Loss of CHD6 by siRNA silencing results in an increased viral replication, while in lungs of infected mice, CHD6 is degraded upon infection and exposure to the three subunits of the viral polymerase (Alfonso et al, 2013). Recent reports also indicate that CHD6 interacts with human papillomavirus proteins, binding and repressing the expression of oncogenes (Fertey et al, 2010).…”

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.…”