In the current study, we found that core genes were relatively conserved among different patients with acute HCV infection, and that all clones analyzed from a single patient at a single timepoint were identical at the nucleotide and amino acid level. This homogeneity could reflect a genetic bottleneck [31
] or selection of the most-fit viruses during transmission. Analysis of a higher number of clones from infected individuals may reveal minor quasispecies for the core coding region that have not been identified in our study. Indeed, a previous study that analyzed a larger number of clones revealed genetic variability in the HCV genome during acute infection [32
Our results indicate that despite the presence of relatively rare mutations in the core protein during acute infection, essential functions of core, such as immune-perturbing activities and targeting to lipid droplets, were generally the same for all isolates. Whether immune perturbing functions of core are critical for establishing chronic infection is currently unknown as all but one of our patients progressed to chronic infection. Nonetheless, mutations at amino acid 91 of core have been associated with liver cancer and virological non-responsiveness to IFN therapy[33
We found that expression of isolates of core from acute infection inhibited TNF-α induced nuclear translocation of NF-κB and NF-κB dependent transcription. This finding is in agreement with a previous study[35
], but at odds with other studies which found core activates NF-κB [36
]. Similarly, core has been reported to have opposing effects on the TNF-α system[46
]. The differences may relate to different cell line and expression systems or the different core sequences used.
The extent of mobility of the lipid targeting domain 2 (D2) of the HCV core protein is likely governed by the strength of binding of D2 to the surface of lipid droplets [26
]. From our FRAP analysis, substitutions at positions 130 (leucine replacing phenylalanine) and 161 (serine replacing glycine), both of which represent relatively conservative changes, did not dramatically affect mobility. These residues in D2 are located in amphipathic helices (position 130 in Helix I and position 161 in Helix II). However, mutations that affect the amphipathic nature of the helices, and hence their interaction with lipid on the surface of lipid droplets, have a more pronounced influence on mobility, reduce virus production, and can considerably decrease the stability of core [47
] (McLauchlan et al. unpublished). Indeed, alteration of phenylalanine at position 130 to glutamic acid induces proteasomal degradation of core and abrogates production of infectious virus [9
]. Therefore, the nature of the interaction of D2 with lipid droplets plays an important role in the process of assembly and release of virions. The possible exception is the A147V mutation found in 4 patients, which has been shown to enhance virus release in the JFH-1 system [26
]. Moreover, we showed that all core proteins were efficiently secreted from Huh7 cells, but the kinetics were significantly delayed in comparison to low m.o.i. infection with JFH-1. It is possible that the kinetics of core secretion change in the presence of other HCV proteins or in the context of virus replication. Indeed, NS5A is known to target to lipid droplets, where it interacts with core to facilitate virus assembly [10
With regard to the effect of the amino acid changes in core on virus replication and assembly, as discussed above, the G161S change in Domain 2 does not affect virus production in JFH1. With regard to the F130L substitution, while changing this residue to a glutamate abolishes virus production, a phenylalanine to leucine change would be predicted to be conservative and therefore unlikely to significantly affect virus production. It certainly had very little impact in the FRAP analysis and the ability of the domain to target droplets. As far as the changes in the signal peptide are concerned (Domain 3), we predict that the V187I change would have little impact. Previous studies with JFH1 indicate that changing the adjacent residue from threonine to leucine (T186L refererred to as mut3 in [7
] had barely any effect on virus production. Moreover, Murray et al [48
]made 3 alanine scanning mutations in the signal peptide (mutants 181-184, 185-188 and 189-191). All 3 mutants were viable and had little impact on virus production using the J6/JFH1 chimera. Thus, a single valine to isoleucine mutation at position 187 is unlikely to have any effect. Indeed, the Murray paper illustrates how difficult it can be to introduce mutations into the signal peptide and influence assembly because of the reported degeneracy in the cleavage site for SPP [7
]. For the changes in Domain 1, it is also difficult to predict any outcome on virus production. While the Murray paper [48
] introduced a set of 4 consecutive alanines into sequences across core and found that Domain 1 was sensitive to such alterations, these engineered sequences are not like a natural sequences. Indeed, in the case of the core sequences isolated from acutely infected patients, the changes were maximally at 4 amino acids scattered throughout domain 1. Some of the changes are rather conservative (arginine to lysine at positions 9 and 43; glycine to serine at position 45; arginine to histidine at position 101; threonine to serine at position 110) and therefore unlikely to be deleterious to virus production. Furthermore, an aspartate at position 68 (an alanine in most sequences) and alanine at position 75 (a threonine in most sequences) are found in both JFH1 and HC-J6 sequences and the core proteins from these strains produce virus in tissue culture. Moreover, the variable residue at position 91 (cysteine, methionine and arginine) is a leucine in JFH1 and HC-J6. Thus, most of the changes are very unlikely to have any significant on virus production.
In summary, core quasispecies tended to be conserved during acute HCV infection, both within a patient and over time. Despite demonstrable amino acid changes, core proteins had similar immune perturbing and lipid targeting functions. Acute core isolates had unique amino acid changes compared to core isolates from chronic infection.