Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity

Hiroko Miyamoto,Yoshimi Tsuda,Kazumichi Kuroda,Ayato Takada,Hiroshi Kida,Daisuke Fujikura,Reiko Yoshida,Rashid Manzoor,Masahiro Kajihara

doi:10.1074/jbc.m113.507798

Hiroko Miyamoto, Yoshimi Tsuda

Open Access

https://doi.org/10.1074/jbc.m113.507798

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Abstract

The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments.

Highlights

It has been shown that heat shock protein 70 (Hsp70) plays a role in influenza A virus replication
We investigated the role of Hsp70 in influenza virus transcription and replication in cells constitutively expressing Hsp70
We found that Hsp70 had modulatory effect on influenza virus replication, presumably by facilitating the viral polymerase activity when present at normal levels and by suppressing the polymerase activity when its levels were increased above normal levels

Summary

Background

It has been shown that heat shock protein 70 (Hsp70) plays a role in influenza A virus replication. We determined the role of Hsp in influenza virus replication in HeLa and HEK293T cells, which express Hsp constitutively. Each viral genomic RNA (vRNA) segment exists in association with multiple monomers of nucleoprotein (NP) and a single copy of a polymerase heterotrimer consisting of a polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA), thereby constituting a viral ribonucleoprotein complex (vRNP) These vRNPs are independent functional units capable of transcription and replication [1, 2]. Studies conducted to elucidate the role of Hsp in influenza virus replication have suggested that Hsp inhibits influenza virus replication either by preventing the nuclear export of the RNP complex [34] or by disrupting the binding of viral polymerase with viral RNA [35]. An increase in viral polymerase activity was observed during the heat shock phase compared with the recovery phase, coinciding with the subcellular movement of Hsp, suggesting that Hsp acted as chaperone for the viral polymerase complex

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