We and others have previously reported the NS5A/HSP70 interaction (18
). However, direct interaction between NS5A and HSPs was not demonstrated due to the use of cellular systems. Here we report a direct interaction between NS5A and HSP70 using purified recombinant proteins and in vitro
pull-down assays. This suggests that no other viral or cellular protein is necessary for the interaction between NS5A and HSP70.
Deletion studies of NS5A domain I narrowed down the HSP70-binding site to the C terminus of NS5A domain I (C34). Recently it was shown that NS5A amino acids 221–302 may be responsible for NS5A/HSP70 interaction (26
). This region includes most of the N terminus of NS5A domain II and some of the linker peptide between domains I and II. While this region does not directly bind HSP70, as shown by our in vitro
interaction studies using purified proteins, it may represent an indirect interaction. In another report, a region of NS5A including the entire domain II and the first few amino acids of domain III was shown to inhibit NS5A-augmented IRES-mediated translation (17
). Both of these reports point to a common region in NS5A (i.e. domain II), and thus, it may be possible that NS5A domain II have a negative effect on IRES-mediated translation through (an) adaptor protein(s). However, further biochemical studies are needed to verify (an) indirect interaction(s).
Our data shows that the C34 hairpin is the site of NS5A/HSP70 interaction. This is supported by the fact that the HCV4 peptide is able to block pull-down of HSP70 with NS5A in vitro
as well as bind HSP70 in vivo
(). Furthermore, the C34 hairpin is the only region of C34 with a secondary structure, based on the crystal structure (24
). It has been reported that HSP27 also binds NS5A at residues 1–181 (27
). It is important to note that the C34 hairpin structure lies exactly at the C terminus of this region. Thus, it may be possible that, in addition to HSP70, other HSPs either interact with the C34 hairpin or are also in complex with HSP70. Recently an alternate crystal structure of dimeric NS5A domain I was published which displays the two domain I units to be closer together closing the proposed RNA-binding cleft (28
). Analysis of this structure reveals the two C34 hairpins to be also exposed.
HSP70 binds non-specifically to a large number of hydrophobic peptide sequences (nascent or denatured) through its C-terminal substrate-binding domain (SBD) allowing the client peptide to attain its native conformation (29
). The N-terminal nucleotide binding domain (NBD) of HSP70 does not interact with peptides directly. Rather, it binds ATP and hydrolyzes it to ADP to induce conformational changes in SBD to facilitate its function (29
). NS5A hydrophobicity plot reveals that C34 is not significantly hydrophobic, and the C34 hairpin is, in fact, hydrophilic (data not shown). Our findings reveal a specific and sequence-dependent interaction between HSP70 and NS5A as there are many regions in NS5A domain I as well as in other domains that are significantly more hydrophobic than C34. This specific interaction is further verified by our discovery that only HSP70-NBD binds NS5A directly. HSP70-NBD is known to interact with proteins that regulate the HSP70 chaperone activity. These proteins include BAG5, co-chaperones of J-domain proteins (such as HSP40), and HSP110 all of which bind HSP70-NBD (29
). Thus, interactions with NBD of HSP70 are an important regulatory mechanism, and it may be possible that NS5A modulates HSP70 activity as well resulting in increased IRES activity.
NS5A domains II and III have been implicated in viral genome replication and virion assembly (10
), and domains I, II, and III have been shown to bind viral RNA (33
). None of the NS5A domains, however, have been previously ascribed to regulation of viral IRES-mediated translation. In this study, we have shown that NS5A domain I plays an important role in regulation of IRES-mediated translation through the C34 beta-sheet hairpin structure. We have shown that C34 expression and treatment with the C34 hairpin peptide derivative (HCV4) block NS5A-augmented IRES-mediated translation. This effect is sequence specific as peptides of equal length from NS5A domains II and III fail to produce similar results. We have previously shown that Quercetin treatment and HSP70 knock-down block NS5A-augmented IRES-mediated translation (18
). Taken together, our data implicates the C34 hairpin of NS5A domain I in the regulation of viral IRES-mediated translation.
Our current model of NS5A-augmented IRES-mediated translation consists of a complex of NS5A, HSP70, HSP40, and probably additional, yet unidentified, factors such as the ribosome which may stabilize the translation complex (). An RNA-binding cleft between the two claw-like domain I units has been predicted in the crystal structure (24
) (Fig. S1
). It has also been reported that HSP70 (specifically the HSPA1A isoform used in this study) is able to bind to the 40S ribosomal subunit (34
). Our finding that NS5A directly binds HSP70, therefore, provides a possible link between the NS5A dimers and the ribosome both of which interact with the RNA template. This NS5A/HSP70 interaction may be responsible for stabilizing the components of translation machinery on the RNA template during translation initiation and/or elongation.
The significance of C34 region and the hairpin within is further underlined by the mutation analysis of HCV genome (20
). As shown in Fig. S3
, the majority of insertion mutants in C34 and all mutants within the hairpin are detrimental for the virus. Quite interestingly, mutation of the region immediately upstream of the hairpin and the region a few amino acids downstream are well tolerated (Fig. S3
). This data supports our hypothesis that the C34 hairpin is critical for viral proliferation.
It is known that in the context of chronic HCV infection, a number of viral proteins including NS5A are able to dramatically alter host gene expression to evade the innate immune response to viral infection resulting in the low efficacy of the currently available pegylated interferon-α (PEG-IFN) and ribavirin treatment. Our findings demonstrate the significance of the NS5A/HSP70 interaction and the role of HSPs in HCV life cycle, in particular, IRES-mediated viral protein translation. Therefore, the C34 modified hairpin peptide may be a candidate for HCV therapy. Furthermore, considering the potency of the C34 hairpin peptide in suppressing viral translation levels, treatment with this peptide may significantly improve the efficacy of PEG-IFN and ribavirin treatment in patients resistant to these compounds or allow for IFN-free therapy in combination with other antiviral agents, which may be beneficial for patients unable to receive IFN therapy.