Recently the HCV protein NS5A has been implicated in the regulation of HCV genome replication, IRES-mediated translation of the viral polypeptide, and viral assembly.24,32
We have identified a NS5A/HSP complex composed of NS5A, HSP40, and HSP70 that may impact one or more of these stages of the viral life cycle. A potential interaction of NS5A with HSP70 (same specific family member, HSPA1A) has previously been reported by the Rice laboratory, where HSP70 was identified through a yeast two-hybrid screen with NS5A bait.33
Although no additional assays were performed to confirm the interaction in that study, siRNA knockdown of HSP70 significantly impaired virus production as well as viral RNA levels. Here we have presented additional evidence of an NS5A/HSP40 and NS5A/HSP70 interaction through identification of the complex through an alternative proteomic-based screen, coimmunoprecipitation, and colocalization by immunofluorescence in the perinuclear area where the virus replicates. In our laboratory, exogenous expression of tagged NS5A is very low relative to other tagged proteins, lowering the possibility that misfolding is the reason for its interaction with HSPs. This most likely represents a complex composed of all three proteins together, because HSP40 is well known to interact with and augment HSP70 activity.34
Although we have shown there to be a NS5A/HSP complex, we certainly cannot exclude the possibility that these HSPs are also interacting with other viral products.
The major role of HSP40 and HSP70 may not be in genome replication but rather in subsequent stages in the viral life cycle, because Quercetin treatment of subgenomic replicon-harboring cells did not affect viral RNA replication. HSP70 is implicated in NS5A-mediated IRES translation, as demonstrated by decreased IRES activity with HSP70 knockdown in the IRES reporter system. A yeast HSP40 homolog, and indirectly HSP70 by virtue of their existence as a complex, have previously been shown to be important for cellular IRES specific translation in yeast.26
The reduction in the IRES activity with knockdown of HSP40 is minimal, and with HSP70 is modest compared with Quercetin treatment and may be related to several factors. These proteins are highly abundant, rendering significant knockdown difficult. Furthermore, there are eight family members of HSP7035
and 41 of HSP40,36
clearly raising the possibility that loss of one member could be functionally complemented by one of the many others. Compensatory increased expression of numerous HSPs also has been shown to occur during heat shock with knockout of HSP70 in Drosophila
In mice, up-regulation of one family member of HSP70 has been seen with knockout of another HSP70 family member.38
We have further found knockdown of HSP40 results in increased HSP70 mRNA, and conversely, knockdown of HSP70 increases HSP40 mRNA expression (data not shown). The stronger impact Quercetin has on the virus compared with HSP knockdown can be explained by the fact that most of the HSP40 and HSP70 family members are more globally reduced by Quercetin.
Quercetin treatment of the subgenomic replicon system also reduced NS5A protein levels, demonstrating its effect on IRES translation, albeit an exogenous one (encephalomyocarditis virus). Our data suggest that Quercetin can also reduce baseline HCV IRES translation in the absence of NS5A expression as well as NS5A-augmented translation, as seen by the marked reduction of the ratio of IRES/cap reporter gene activity in the bicistronic reporter assay. Taken together, the IRES assay and the replicon studies suggest that Quercetin can inhibit viral IRES translation and could potentially be used to treat HCV as well as other viral infections. Quercetin has been shown to inhibit several kinases,39,40
and we cannot exclude this activity as a factor in its ability to reduce virus production. However, similar, yet modest, effects imparted by HSP knockdown support the argument that its HSP synthesis inhibitory properties are responsible for these effects.
In addition to the potential role HSPs and possibly an HSP/NS5A complex may play in IRES translation, we show that HSPs are important for viral production using an HCV cell culture reporter system. Viral replication was modestly reduced by HSP70 knockdown and more strongly by Quercetin treatment. The lack of significant effect by HSP40 knockdown and the modest reduction by HSP70 knockdown may be explained by similar mechanisms given for the IRES assay results. The decrease in infectious particle production appeared more dramatic than with viral replication, because HSP40 knockdown also affected it, and Quercetin further reduced levels to the lower limits of assay detection. This stronger effect raises the possibility that the HSPs also may play a role in viral assembly or secretion. Given the known role of HSPs as protein chaperones, it is reasonable to suggest these HSPs are important for several stages of the viral life cycle, including IRES translation, viral assembly, and secretion. Further studies to determine the role HSPs alone and in complex with NS5A in assembly and secretion are beyond the scope of this study and are currently being investigated.
The most striking finding in this study is the marked reduction in viral production imparted by HSP synthesis inhibitor Quercetin. HSP synthesis inhibition may be an attractive therapeutic avenue, because it does not block a viral-encoded protein directly but rather a cellular cofactor. This may reduce the chance of treatment-related viral escape mutants. The low toxicity and pharmacokinetics of Quercetin are well known,41,42
and it has been approved for other uses in clinical trials in the United Kingdom and the United States,41,43,44
rendering it an immediately viable candidate for treatment of HCV infection.