Heat shock proteins are stress response factors which also regulate several cellular processes 
. The Hsp40 family chaperones are known to play important roles in protein folding, translocation, cell signaling and apoptosis 
. Very often they are targeted by viral components for successful virus replication. For example, Hsp40 is known to interact with HIV type 2 Vpx protein and facilitate nuclear import of the pre-integration complex 
. HIV type 1 Nef protein interacts with Hsp40 to enhance viral gene expression 
. Hsp40 is also known to interact with the HBV core protein and affect viral turnover 
. Heat shock proteins are known to affect the viral replication of influenza viruses also. For example Hsp90 is known to interact with influenza virus polymerase components and aid in viral RNA synthesis 
. Hsp70 is also known to be involved in the nuclear export of the RNP complex and play a role in temperature dependence of IAV replication 
. Likewise, Hsp40 is also known to regulate PKR signaling in influenza virus infected cells 
. Similarly, the IAV NP is also a multifunctional protein that interacts with a wide variety of viral and cellular macromolecules, including RNA, PB1 and PB2 subunits of the viral RNA-dependent RNA polymerase and the viral matrix protein 
. It also binds to several host factors which include CRM1, UAP56, Alpha-importin 1 and NF90 
. Through these interactions, IAV-NP is known to encapsidate the viral genome, regulate virus transcription and replication, contribute towards pathogenicity of virus, and help in interspecies transmission of the virus 
. However, so far IAV NP is not reported to play any role in modulating the host antiviral response.
A key component of mammalian antiviral response mechanism is dsRNA dependent protein kinase PKR, which is activated by viral dsRNA 
. Upon activation, PKR gets dimerized and autophosphorylated at multiple serine and threonine residues. Activated PKR phosphorylates eukaryotic translation initiation factor eIF2α, which in phosphorylated state cannot participate in mRNA translation 
. This is an important strategy of the host to arrest translation of viral mRNAs thereby limiting viral replication 
. Another crucial host pathway which is activated by PKR is IRF3-mediated IFNβ production. Activation of PKR is known to enhance IRF3 phosphorylation and nuclear movement where it drives expression of Interferon β production and built up of antiviral host response 
. Similarly, PKR also has other substrates such as MAPK and iKKß which upon phosphorylation trigger various signaling pathways leading to apoptosis or interferon response 
. Being such a crucial molecule, PKR is very often the target of viral factors 
. In case of influenza virus infection, viral NS1 protein is known to bind directly to PKR and inhibit its activation 
. NS1 also inhibits the function of retinoic acid inducible gene-I (RIG-I), a cytosolic pathogen sensor involved in the antiviral response 
. Apart from that, PKR activity is controlled by another mechanism where P58IPK
, the cellular inhibitor of PKR is activated in influenza virus infected cells 
. Further, P58IPK
itself is inhibited by Hsp40 and is present as P58IPK
-Hsp40 complex under normal conditions. However upon influenza virus infection, it is released from the Hsp40 complex and inhibits PKR activation 
. In a recent report, it was shown that M2 protein of influenza A virus stabilizes the P58IPK
-Hsp40 complex and activates PKR phosphorylation, probably during later stage of infection 
. However the mechanism of dissociation of Hsp40-P58IPK
complex and concomitant PKR inhibition during influenza virus infection remain unknown.
Here, we report that IAV NP interacts with the human chaperone Hsp40 and employs this interaction to mitigate PKR-mediated antiviral response of the host. NP-Hsp40 interaction was identified through a yeast two-hybrid screen and confirmed in a cell-free translation system, in transfected cells and in influenza virus infected cells. The interaction was found to be conserved across different influenza A viruses, ranging from seasonal, avian H5N1 virus and the 2009 H1N1 pandemic virus despite substantial amino acid differences that range from 0–5% within a subtype/group and 6–10% between the subgroups in NP amino-acid sequence. Our findings demonstrate that IAV NP is the viral component that dissociates P58IPK
from the P58IPK
-Hsp40 complex during influenza virus infection in mammalian cells. It was observed that during the course of influenza virus infection in lung epithelial cells, a gradual increase in the association of NP with Hsp40 coincided with a concomitant decrease in P58IPK
association with Hsp40. Increased activity of P58IPK
, promoted by NP, should lead to the inhibition of PKR activation and subsequent downstream effects (). In accordance with the above hypothesis, we observed that ectopic expression of IAV NP in mammalian cells substantially reduced the phosphorylation levels of PKR and eIF2α. Furthermore, siRNA-mediated inhibition of NP expression during influenza virus infection led to increased phosphorylation of PKR and eIF2α, confirming the role of NP in the negative regulation of PKR. Although eIF2α is phosphorylated by other kinases also, namely, HRI, GCN2 and PERK which are activated during stress condition, only PKR is known to be targeted by viral inhibitors 
. In line with this, NP and NS1 had similar effects on PKR mediated eIF2α phosphorylation; however their synergistic effect was higher than their individual effects ().
Proposed model for inhibition of PKR mediated host response by influenza nucleoprotein.
Activation of PKR signaling during virus infections is known to result in IRF3 phosphorylation and concomitant IFNβ production. However IRF3 is not a direct substrate of PKR and it can get activated by the RIG I pathway, NFκB pathway and other unknown mechanisms 
. Influenza NS1 protein is known to inhibit PKR, RIG I and NFκB pathways, thus it is expected to have greater impact on IRF3 phosphorylation as compared to NP, which may inhibit only PKR mediated IRF3 phosphorylation 
. In line with this, we observed that NP inhibition during IAV infection led to enhanced IRF3 phosphorylation, IFNβ production and reduced viral replication; however inhibition of NS1 had greater impact on these events. As expected the synergistic effect of NP and NS1 inhibition on IRF3 activity was higher than their individual effects. The effect of NP on IFNβ production is also reflected on virus replication as siRNA-mediated inhibition of NP led to reduced vRNA production. This effect may also be attributed to the essential requirement of NP for proper functioning of influenza virus polymerase. However the inhibitory action of NP on PKR-mediated host response may also contribute to the reduced virus replication in case of siRNA-mediated inhibition of NP.
Based on our findings, we proposed a model for PKR inhibition by influenza virus nucleoprotein as shown in . According to this model, IAV NP interacts with Hsp40 and facilitates the release of P58IPK
from it, which in turn inhibits PKR activation (). Reduced PKR activity, on one hand leads to reduced eIF2α phosphorylation and ensures continued translation from viral mRNAs and on the other hand leads to reduced IRF3 mediated IFNβ production. Therefore, apart from the NS1 protein which is already known to inhibit PKR activation and IRF3 phosphorylation 
, NP also participates in this process, but through a different mechanism involving Hsp40. With structure information of both NP and Hsp40 being available 
, it would be interesting to see which domains and key amino-acid residues are involved in this interaction. Since the NP-Hsp40 interaction is conserved across influenza viruses of various subtypes including the 2009 pandemic H1N1 virus, it serves as an important target for developing anti-viral strategies.