In the present study, we demonstrated the involvement of NS5A in the production of HCV particles via the interaction of NS5A with the core protein and identified its C-terminal serine cluster 3-B (aa 2428, 2430, and 2433), which is implicated in basal phosphorylation, as a key element for the interaction of NS5A with the core protein and for infectious virus production. Serine-to-alanine substitutions at the cluster, which have no impact on viral RNA replication, inhibit the interaction between NS5A and the core protein, thereby indicating that there is a connection between NS5A-core protein association and virus production. Finally, CL3B mutation leads to impairment of the association of the core protein with HCV RNA and, therefore, possibly RNA encapsidation.
Several reports have indicated that viral NS proteins are involved in the virion assembly of Flaviviridae
). For instance, mutations in yellow fever virus NS2A block production of infectious virus, and this perturbation can be released by a suppressor mutation in NS3 (25
), while the hydrophobic residues of Kunjin virus NS2A required for virus assembly have been mapped (26
). Miyanari et al. have shown that HCV core protein recruits NS proteins to the LD-associated membranes and that the NS proteins around the LDs participate in the assembly of infectious viral particles (33
). Furthermore, during preparation of the current article, two studies regarding participation of NS5A in the assembly of HCV particles were published. Appel et al. have demonstrated the essential role of domain III of NS5A in the formation of infectious particles, and deletions in this domain that disrupt colocalization of NS5A and the core protein abrogate virion production (2
). Tellinghuisen et al. identified a serine residue in domain III as a key determinant for viral particle production (50
). However, the mechanism by which NS proteins participate in virus assembly or the role of the interaction between structural and NS proteins in virus life cycles has not been fully elucidated. Here, we have clearly demonstrated that HCV NS5A interacts with the core protein in coimmunoprecipitation experiments not only with coexpression of each epitope-tagged protein but also with cells expressing the viral genome; and by using immunofluorescence and subcellular fractionation analysis, we have confirmed that mutations in CL3B abolish colocalization of NS5A and the core protein, presumably around LDs. In addition, the intracellular infectivity assay and IP-RT-PCR strongly suggest that impairment of the NS5A-core protein interaction results in disruption of virus production at an early stage of virion assembly. On the basis of the present results and findings in accompanying articles, one may infer the following events: newly synthesized HCV RNAs bound to NS5A are released from the replication complex-containing membrane compartment and can be captured by the core protein via interaction with domain III of NS5A at the surface of LDs or LD-associated membranes. Consequently, the viral RNAs are encapsidated, and virion assembly proceeds in the local environment. Recruitment of newly synthesized viral RNAs to the core protein could be important for efficient nucleocapsid formation in cells, where concentrations of the viral genome and the structural proteins are typically low, and may contribute to the selection of the viral genome to be packaged. Interaction between NS5A and the core protein has been previously reported, and the NS5A region containing an interferon sensitivity determining region and the PKR-binding sequence (aa 2212 to 2330) has been mapped to that required for binding with core protein by yeast two-hybrid and in vitro pull-down assays (13
). However, involvement of domain III in the NS5A-core protein interaction was not analyzed in detail, and a role for the NS5A-core protein interaction in the HCV life cycle was not examined in that study.
A growing body of evidence points to phosphorylation of NS5A as being important in controlling HCV RNA replication. Although the degree and the requirement for its hyperphosphorylation diverge between different HCV isolates, mutations that are associated with increased replicative fitness of HCV replicons frequently lead to a reduced level of NS5A hyperphosphorylation (1
). Inhibitors of serine/threonine protein kinases that block NS5A hyperphosphorylation facilitate replication of a non-culture-adapted replicon (3
). One model that has been proposed suggests that NS5A hyperphosphorylation negatively regulates HCV RNA replication by disrupting the interaction between NS5A and the vesicle-associated membrane protein-associated protein subtype A, a cellular factor considered necessary for efficient RNA replication (5
). However, the regulatory role of the basal phosphorylation of NS5A in the viral life cycle is poorly understood. It has been reported that the C-terminal region of NS5A (aa 2350 to 2419) is involved in basal phosphorylation (23
). There are highly conserved serine residues in this region, and alanine substitutions or in-frame deletion of the serine residues has been shown to impair basal phosphorylation but not to affect RNA replication in the genotype 1b isolate (1
). Consistently, a metabolic 32
P labeling experiment in the present study demonstrated that NS5A mutants of the JFH-1 isolate in the region impair the basal phosphorylation. Nevertheless, Tellinghuisen et al. noted that the serine at aa 2433 of JFH-1 is involved in generating hyperphosphorylated NS5A, as shown by Western blotting (50
). The basis for this difference is uncertain. To date, there is no clear evidence to determine which serine residues located in domain III are phosphoacceptor sites or whether these residues influence NS5A phosphorylation in an indirect fashion. Future study to map phosphoacceptor sites in the NS5A domain III by biochemical approaches is needed.
We found that two of the three serine residues at CL3B are responsible for regulating the interaction of NS5A with the core protein as well as for infectious virus production. To further evaluate the effect of constitutive serine phosphorylation at the cluster, we replaced the serine residues with glutamic acid, which mimics the presence of phosphoserines. The S2428/2430E mutant led to restoration of the interaction of NS5A with the core protein and virus production up to levels similar to the wild type. Somewhat unexpectedly, the triple glutamic acid substitution (CL3B/SE) exhibited only a slight restoration effect or none at all. It is considered that the degree of negative charge on the glutamic acid residue is not completely equivalent to that of phosphoserine. It is likely that the range of acidity at the local environment of the NS5A domain III that will allow interaction with the core protein is rather narrow. Induction of a conformational change in NS5A by the incorporation of phosphate may also be important for its interaction with the core protein. Tellinghuisen et al. reported that a single serine-to-alanine substitution at aa 2433 blocks the production of infectious virus and that casein kinase II likely phosphorylates the residue (50
). Although this seems inconsistent with our results, these investigators also showed that deletions producing a lack of all three serine residues in the cluster inhibited virus production more severely than a single mutation. We observed that a single substitution of S2428A, S2430A, or S2433A resulted in a moderate decrease in the virus released from the transfected cells; however, more evident perturbation was obtained from double or triple substitutions (Fig. ). Tellinghuisen et al. determined the HCV production at 48 h after RNA transfection and found a marked inhibition by the single substitution S2433A. In our study, as indicated in Fig. , the reduction caused by the S2433A mutant was approximately 90% at 48 h after transfection; however, the virus production from the mutant reached a similar level to that of the wild type at 96 h posttransfection.
Several previous studies have found that apolipoproteins B (apoB) and E (apoE), microsomal triglyceride transfer protein, and HCV p7 protein are key factors for production of the infectious HCV particles (4
). Assembly and maturation of the viral particles appear to depend on the formation of very-low-density lipoprotein, a large particle containing apoB, apoE, and large amounts of neutral lipids in hepatic cells. p7 protein is primarily involved in a late step of virus particle production, and the findings support the idea that p7 acts as viroporin, which has the capacity to compromise cell membrane integrity and thus favors the release of viral progeny. How the early step in virion production regulated by the NS5A-core protein interaction links with the later step(s) involved in the very-low-density lipoprotein assembly or p7 function remains an interesting question to be addressed.
In summary, we demonstrated that the C-terminal serine cluster of NS5A (aa 2428, 2430, and 2433), which is involved in generating the basal phosphorylated form, is a determinant of NS5A interaction with the core protein and the subcellular localization of NS5A. Mutation of this cluster blocks the NS5A-core protein interaction, resulting in perturbation of association between the core protein and HCV RNA. It is thus tempting to consider that NS5A plays a key role in transporting the viral genome RNA synthesized by the replication complex to the surface of LDs or LD-associated membranes, where the core protein localizes, leading to facilitation of nucleocapsid formation. Structural analysis of the NS5A domain III-core protein complex should provide greater insight into the mode of interaction between these viral proteins. Identification of residues at the interface that are involved in important interactions will be of significant value in designing novel structure-based inhibitors to block the early step of HCV particle formation.