Host factor Ebp1: Selective inhibitor of influenza virus transcriptase

The RNA-dependent RNA polymerase (RdRp) of influenza A virus is a heterotrimeric complex composed of the PB1, PB2, and PA subunits. The interplay between host factors and the three subunits of the RdRp is critical to enable viral RNA synthesis to occur in the nuclei of infected cells. In this study, we newly identified host factor DnaJA1, a member of the type I DnaJ/Hsp40 family, acting as a positive regulator for influenza virus replication. We found that DnaJA1 associates with the bPB2 and PA subunits and enhances viral RNA synthesis both in vivo and in vitro. Moreover, DnaJA1 could be translocated from cytoplasm into the nucleus upon influenza virus infection. The translocation of DnaJA1 is specifically accompanied by PB1-PA nuclear import. Interestingly, we observed that the effect of DnaJA1 on viral RNA synthesis is mainly dependent on its C-terminal substratebinding domain and not on its typical J domain, while the J domain normally mediates the Hsp70-DnaJ interaction required for regulating Hsp70 ATPase activity. Therefore, we propose that DnaJA1 is co-opted by the influenza A virus to enter the nucleus and to enhance its RNA polymerase activity in an Hsp70 cochaperone-independent manner. IMPORTANCEThe interplay between host factors and influenza virus RNA polymerase plays a critical role in determining virus pathogenicity and host adaptation. In this study, we newly identified a host protein, DnaJA1/Hsp40, that is co-opted by influenza A virus RNA polymerase to enhance its viral RNA synthesis in the nuclei of infected cells. We found that DnaJA1 associates with both PB2 and PA subunits and translocates into the nucleus along with the nuclear import of the PB1-PA dimer during influenza virus replication. Interestingly, the effect of DnaJA1 is mainly dependent on its C-terminal substrate-binding domain and not on its typical J domain, which is required for its Hsp70 cochaperone function. To our knowledge, this is the first report on a member of the Hsp40s that is specifically involved in regulating influenza virus RNA polymerase. Targeting the interactions between polymerase subunits and DnaJA1 may provide a novel strategy to develop antiviral drugs.

Section: Discussionmentioning

confidence: 99%

DnaJA1/Hsp40 Is Co-Opted by Influenza A Virus To Enhance Its Viral RNA Polymerase Activity

Cao

Wei

Zhao

et al. 2014

“…In fact, from a recent genomic screening, only two factors that negatively modulate viral polymerase activity were found (75), and, furthermore, the physiological role of only a very few cases of influenza virus repressors has been characterized. Standing out among these are the characterization of Ebp-1, a suppressor of the androgenmediated transcription that inhibits RNA synthesis of the viral polymerase in vitro (76); cyclophilin E, which negatively regulates virus life cycle in infected cells (77); annexin 6, which impairs virus budding (78); and the HAX1 protein, which impedes the nuclear translocation of the PA polymerase subunit (79).…”

Section: Discussionmentioning

confidence: 99%

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

Alfonso

Rodríguez

Rodriguez

et al. 2013

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...

“…Hsp90 was shown to interact with PB2 and enhance influenza viral RNA synthesis (35). Ebp1 (ErbB3-binding protein) was identified as a PB1 interactor and found to selectively interfere with in vitro RNA synthesis by influenza virus RNA polymerase (17). For these given examples, there has been no evidence that the interactions vary between species or could account for host range restrictions.…”

Section: Discussionmentioning

confidence: 99%

Evidence for Avian and Human Host Cell Factors That Affect the Activity of Influenza Virus Polymerase

Moncorgé

Mura

Barclay

2010

Typical avian influenza A viruses do not replicate efficiently in humans. The molecular basis of host range restriction and adaptation of avian influenza A viruses to a new host species is still not completely understood. Genetic determinants of host range adaptation have been found on the polymerase complex (PB1, PB2, and PA) as well as on the nucleoprotein (NP). These four viral proteins constitute the minimal set for transcription and replication of influenza viral RNA. It is widely documented that in human cells, avian-derived influenza A viral polymerase is poorly active, but despite extensive study, the reason for this blockade is not known. We monitored the activity of influenza A viral polymerases in heterokaryons formed between avian (DF1) and human (293T) cells. We have discovered that a positive factor present in avian cells enhances the activity of the avian influenza virus polymerase. We found no evidence for the existence of an inhibitory factor for avian virus polymerase in human cells, and we suggest, instead, that the restriction of avian influenza virus polymerases in human cells is the consequence of the absence or the low expression of a compatible positive cofactor. Finally, our results strongly suggest that the well-known adaptative mutation E627K on viral protein PB2 facilitates the ability of a human positive factor to enhance replication of influenza virus in human cells.