The idea of NS5A as a regulator of events in the HCV lifecycle via phosphorylation by cellular kinases is not a new concept. Indeed, this idea has been prevalent in the HCV community since the discovery that NS5A exists in multiple phosphorylation states 
. Considerable progress has been made in deciphering the role of CKIα phosphorylation of NS5A in RNA replication 
. The functional significance of CKII phosphorylation of NS5A, however, has long been an enigma. Our data provides the first evidence for a function of these events, not in RNA replication, but in the process of virion assembly.
We have shown that CKII likely phosphorylates serine 457 of genotype 2a NS5A, and this modification is required for infectious virus production. Generating an NS5A that is incapable of serving as a phosphoacceptor (alanine mutation at position 457) does not alter RNA replication, but blocks the production of infectious virus. Generating an aspartic acid mutation at position 457 lowers RNA replication slightly but does not affect infectivity release. This lack of direct coupling of replication levels and infectivity release is in agreement with that observed for the JC1 chimeric virus 
, in which infectivity is increased ten fold with no change in RNA replication levels. Our first interpretation of this data was that phosphorylation of serine 457, mimicked by the aspartic acid mutation, destabilized the replicase leading to release of NS5A and viral RNA from the replicase for assembly events. This idea is roughly equivalent to the models of phosphorylation as a regulator of the replicase first proposed by Evans, et al. 
The case is clearly not this simple for our mutants, as we see NS5A participating in later events in assembly, such as association with lipid droplets even when serine 457 (and surrounding residues) are deleted. In this regard, our data is quite different than the recently described NS5A mutants in domain I that block infectious virus production by hindering the association of NS5A with lipid droplets 
. These mutations are severely impaired in RNA replication, yet release what appear to be non-infectious virus particles containing both HCV core and viral RNA. In contrast, our deletions and single amino acid serine 457 to alanine mutant in domain III does not alter HCV RNA replication, but blocks the production of infectious virus, and does not release large amounts of viral RNA and HCV core. We see no defect in the association of NS5A with lipid droplets with our deletion B mutant, further highlighting the difference between domain I and domain III mutants in NS5A assembly activities. Collectively, these data suggest our mutants affect a different step in the HCV assembly pathway than the domain I mutations 
. Our lack of understanding of the mechanisms of HCV assembly make it difficult to speculate exactly what step in assembly our mutations are affecting. We are clearly manipulating an early step in the assembly process, as we do not see virus particles inside the cell secretory pathway, suggesting, in conjunction with the lack of RNA and core release, that particles are simply not generated. The data suggests that we are affecting a later stage in assembly than the domain I mutants, as we see clear evidence of lipid droplet association of NS5A in the context of our mutant. What this later step of assembly might be is unknown. Perhaps it involves NS5A making productive interactions with the structural proteins at the lipid droplet, NS5A moving and releasing viral RNA, NS5A functioning to package viral RNA, or NS5A directly being packaged into virions as a minor component of the infectious virion. Unfortunately, our understanding of the properties of NS5A is still in its infancy, making directly addressing these issues difficult at the present time.
Although our genetic manipulations of NS5A suggest serine 457 is phosphorylated by CKII, and we can show phosphorylation of fusion protein substrates corresponding to this region of NS5A, we have not shown direct modification of this residue by this kinase in the context of an authentic virus infection. Indeed, only two sites of NS5A phosphorylation have been mapped to date, and not in the context of authentic RNA replication, despite intense efforts in this regard over the past 15 years 
. This is, at least in part, due to the large number of potential acceptor sites in NS5A and the unusual repetitive amino acid usage of potential acceptor sites in several regions of NS5A. Our efforts in attempting to identify serine 457 as a phosphoacceptor from NS5A isolated from infected cells by immunoprecipitation and mass spectrometry have not been successful due to poor coverage of the domain III region of NS5A in our spectra (data not shown). Based on the lack of evidence in the literature of documented NS5A phosphorylation sites, and our lack of understanding of what the differences in phosphorylation that generate the different phosphoforms of NS5A, it will likely be some time before the complete complement of NS5A phosphorylation sites are known. Even understanding whether this site is a component of the hypo or hyperphosphorylated form of NS5A is not a simple task. Serine 457 lies in a region of NS5A domain III that is believed to primarily contain sites of hypophosphorylation based on alanine scanning mutagenesis of potential acceptor sites in domain III, although this particular residue position has never been evaluated by mutagenesis in the context of the 2a NS5A sequence 
. Our Western blot data suggests serine 457 may either be a hyperphosphorylation site, or a residue that when phosphorylated, promotes hyperphosphorylation of NS5A. The later seems more likely, as aspartic acid mutations at serine 457 have normal levels of hyperphosphorylation, despite being incompetent as an acceptor site. The work of Miyanari, et al. shows that the lipid droplet associated form of NS5A, a requirement for virus assembly, is enriched for hyperphosphorylated NS5A relative to what is seen in viruses lacking the core protein, suggesting hyperphosphorylation might be important in assembly. This is, at least in broad terms, supported by the observation that hyperphosphorylated NS5A destabilizes the replicase. Nonetheless, in vitro phosphorylation reactions of NS5A with recombinant CKII only generate the hypophosphorylated form of NS5A, even in conditions of excess CKII 
. At least for CKI phosphorylation of NS5A in vitro, several acceptor sites require activation by pre-phosphorylation of upstream sites before they are modified, suggesting that in vitro reactions probably do not fully represent what occurs in vivo 
. Although we have shown serine 457 is an important residue in infectious virus production, it may be that this residue merely ‘activates’ other phosphoacceptor sites, as has been observed for CKIα phopshorylation. This concept fits well with our observations of altered levels of hyperphosphorylation with our mutants.
Although the cell culture infectious genotype 2a strain clearly possesses an insertion relative to other HCV genotypes (), it is unlikely that this insertion is directly related to the ability to generate infectious virus. Clearly, isolates of genotype 1a are infectious in cell culture, but lack this insertion 
. Indeed, we can delete this region without disrupting infectious virus production. The conservation of a CKII site around residue 457 seems to be a more likely candidate for an important conserved feature, with other defects preventing the production of infectious virus in cell culture for other genotypes. In this regard, it is worth noting that the web based NetPhosK 1.0 server 
can predict CKII sites in all 35 HCV genotype reference sequences in the Los Alamos HCV database 
that involve direct serine 457 modification. A recent paper looking at the sequence of patient isolated HCV RNA for genotypes 1a, 1b and 3 identified a number of potential CKII sites in NS5A, including sites very close to the location of serine 457 in genotype 2a, however the majority of the sites predicted have residues corresponding to serine 457 as sites of modification by another enzyme that generate a CKII recognition site just upstream of this position 
. This analysis was based on the site motif described by Pinna 
, in which most CK2 sites typically have a serine or threonine, followed by an acidic residue at the n+3 position. This work did not take into account, as mentioned by Pinna, that whenever the negatively charged determinant is absent at position n
+3, it is invariably present at position n
+1 and vice-versa, leading to some variability in CK2 sites 
. The majority of sites we predicted as conserved across all HCV reference strain had met these rules of the more conventional n+3, but many were of the less common n+1 variety. We also were able to predict the sites using an upstream CK2 site with serine 457 serving as an activator when phosphorylated, as described by Dal Pero and colleagues 
. Unfortunately, it will likely be some time before the intricacies of NS5A phosphorylation, RNA replication, and now virion assembly, will be fully understood, thereby addressing the question of what type of CK2 sites are utilized in this region of NS5A. The lack of solid information on the regulation of CKII further complicates our understanding of when CKII might be active on NS5A and at what location in the infected cell (see 
The requirement for phosphorylation of a viral non-structural viral replication protein by a host cell kinase for the production of infection virus particles, although novel for HCV, has been described previously for other viruses. Bluetongue virus (BTV) is perhaps the closest example of this phenomenon to what we observe in the case of HCV, despite the great evolutionary distance between these viruses. The BTV NS2 protein is phosphorylated by CKII, and this phosphorylation is required for the assembly of the site of replication virion assembly, termed viral inclusion bodies 
. As is the case for HCV, the precise mechanisms of this phenomenon are not fully understood in BTV biology. Viruses more closely related to HCV, such as the flaviviruses Dengue (DENV) and West Nile virus (WNV), require a functional c-Src (or c-Src like) kinase activity for the assembly and maturation of infectious virus particles at the site of RNA replication 
. The mechanism of these events is also unknown, but the relatively close relationship of DENV and WNV to HCV suggest c-Src and c-Src like activities and CKII may perform similar regulatory functions in switching between replication and assembly. It seems that the regulation of switching between replication and assembly by phosphorylation, or controlling later stages of assembly and mturation may be a common, but understudied mechanism in virology.
In summary, we have identified serine 457 of the HCV NS5A protein as a key determinant for the production of infectious virions and presented data showing this site is likely a target for CKII phosphorylation. Phosphorylation of this residue appears to be an important regulatory step in infectious virus production. Via genetic and chemical manipulations we have shown that HCV RNA replication can be uncoupled from infectious virus production without altering HCV RNA replication efficiency. It remains to be demonstrated whether these observations are valid in the context of an infected liver, but at least in cell culture these events appear to be a critical step in the virus lifecycle. Nonetheless, these new genetic and chemical tools, when combined with recently identified mutants in NS5A that alter localization to the site of virion assembly, will be of significant value in further dissection of the mechanisms of HCV RNA replication and virion biogenesis.