Hepatitis C Virus RNA Replication Depends on Specific Cis- and Trans-Acting Activities of Viral Nonstructural Proteins

Abstract

Many positive-strand RNA viruses encode genes that can function in trans, whereas other genes are required in cis for genome replication. The mechanisms underlying trans- and cis-preferences are not fully understood. Here, we evaluate this concept for hepatitis C virus (HCV), an important cause of chronic liver disease and member of the Flaviviridae family. HCV encodes five nonstructural (NS) genes that are required for RNA replication. To date, only two of these genes, NS4B and NS5A, have been trans-complemented, leading to suggestions that other replicase genes work only in cis. We describe a new quantitative system to measure the cis- and trans-requirements for HCV NS gene function in RNA replication and identify several lethal mutations in the NS3, NS4A, NS4B, NS5A, and NS5B genes that can be complemented in trans, alone or in combination, by expressing the NS3–5B polyprotein from a synthetic mRNA. Although NS5B RNA binding and polymerase activities can be supplied in trans, NS5B protein expression was required in cis, indicating that NS5B has a cis-acting role in replicase assembly distinct from its known enzymatic activity. Furthermore, the RNA binding and NTPase activities of the NS3 helicase domain were required in cis, suggesting that these activities play an essential role in RNA template selection. A comprehensive complementation group analysis revealed functional linkages between NS3-4A and NS4B and between NS5B and the upstream NS3–5A genes. Finally, NS5B polymerase activity segregated with a daclatasvir-sensitive NS5A activity, which could explain the synergy of this antiviral compound with nucleoside analogs in patients. Together, these studies define several new aspects of HCV replicase structure-function, help to explain the potency of HCV-specific combination therapies, and provide an experimental framework for the study of cis- and trans-acting activities in positive-strand RNA virus replication more generally.

Evolution and persistence of resistance‐associated substitutions of hepatitis C virus after direct‐acting antiviral treatment failures

“…Lethal mutations in viral genes can sometimes be rescued by trans ‐complementation. Among the five nonstructural proteins of HCV (ie, NS3, NS4A, NS4B, NS5A, NS5B), NS5A protein is well known for its trans ‐complementation effect, which allows for efficient rescue of defective viral RNA replication due to NS5A mutations . The presence of RASs in NS5A protein generally reduces viral RNA replication capacity (Figures B and B) and this defective genome replication could also be trans ‐complemented by the coexisting wild‐type NS5A protein which is present among viral quasispecies within the infected host.…”

Functional RNA structures throughout the Hepatitis C Virus genome

“…At the 3′-end of the ORF, within the region encoding NS5B, there is a set of stem-loops that interact with RNA motifs in the 3′ UTR, resulting in network of RNA elements that are essential for HCV replication [21]. Interaction between NS5B and these same structures may explain why NS5B expression is required in cis [22]. …”

Affinity Purification of the Hepatitis C Virus Replicase Identifies Valosin-Containing Protein, a Member of the ATPases Associated with Diverse Cellular Activities Family, as an Active Virus Replication Modulator

“…In addition to NS5A, we aimed to tag a replicase component other than NS4B without obviously attenuating viral replication. We selected NS5B because of its constitutive role in replicase assembly rather than for its enzymatic activity (26,27). It is tolerant to replacement with exogenous sequences and to insertion mutations (28,29).…”