Abstract
The hepatitis C virus genotype 2a isolate, JFH-1, exhibits much more efficient genome replication than other isolates. Although basic replication mechanisms must be conserved, this raises the question of whether the regulation of replication might exhibit isolate- and/or genotype-specific characteristics. Exemplifying this, the phenotype of NS5A hyperphosphorylation is genotype-dependent; in genotype 1b a loss of hyperphosphorylation correlates with an enhancement of replication. In contrast, the replication of JFH-1 is not regulated by hyperphosphorylation. We previously identified a novel phosphorylation site in JFH-1 NS5A: S146. A phosphomimetic substitution (S146D) had no effect on replication but correlated with a loss of hyperphosphorylation. In genotype 1b, residue 146 is alanine and we therefore investigated whether the substitution of A146 with a phosphorylatable (S), or phosphomimetic, residue would recapitulate the JFH-1 phenotype, decoupling hyperphosphorylation from replication. This was not the case, as A146D exhibited both a loss of hyperphosphorylation and a reduction in replication, accompanied by a perinuclear restriction of replication complexes, reductions in lipid droplet and PI4P lipid accumulation, and a disruption of NS5A dimerization. In contrast, the S232I culture-adaptive mutation in the low-complexity sequence I (LCSI) also exhibited a loss of hyperphosphorylation, but was associated with an increase in replication. Taken together, these data imply that hyperphosphorylation does not directly regulate replication. In contrast, the loss of hyperphosphorylation is a consequence of perturbing genome replication and NS5A function. Furthermore, we show that mutations in either domain I or LCSI of NS5A can disrupt hyperphosphorylation, demonstrating that multiple parameters influence the phosphorylation status of NS5A.
Highlights
Hepatitis C virus (HCV) is a leading cause of liver disease worldwide, with an estimated 71 million chronic infections that can lead to liver fibrosis, cirrhosis or hepatocellular carcinoma [1]
This observation in genotype 1b was exemplified by a culture-adaptive mutation, S232I, within low-complexity sequence I (LCSI), which presumably acted by directly disrupting the proposed hierarchical phosphorylation within this serine-rich sequence that has been associated with the hyperphosphorylated species
The results of this study demonstrate that in the context of the genotype 1b Con1 isolate, residue 146 of NS5A plays a role in the regulation of the genome replication, sub-c ellular localization and dimerization of NS5A, with corresponding effects on NS5A hyperphosphorylation
Summary
Hepatitis C virus (HCV) is a leading cause of liver disease worldwide, with an estimated 71 million chronic infections that can lead to liver fibrosis, cirrhosis or hepatocellular carcinoma [1]. Virus genome replication has become the target for a number of potent direct-a cting antivirals (DAAs) that have revolutionized therapy for HCV infection. These small molecule inhibitors are targeted directly to the NS3 protease, NS5B polymerase and NS5A. NS5A is a phosphoprotein that has multiple functions in virus genome replication, assembly and modulation of host cell biology [3]. NS5A is highly phosphorylated [8], and two species with differing migration on SDS-PAGE are designated p56 (basal) and p58 (hyper). Phosphorylation of NS5A appears to be a highly regulated process and
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