Evidence for Internal Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B In Cellulo

Abstract

Initiation of RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) NS5B has been extensively studied in vitro and in cellulo. Intracellular replication is thought to rely exclusively on terminal de novo initiation, as it conserves all genetic information of the genome. In vitro, however, additional modes of initiation have been observed. In this study, we aimed to clarify whether the intracellular environment allows for internal initiation of RNA replication by the HCV replicase. We used a dual luciferase replicon harboring a terminal and an internal copy of the viral genomic 5= untranslated region, which was anticipated to support noncanonical initiation. Indeed, a shorter RNA species was detected by Northern blotting with low frequency, depending on the length and sequence composition upstream of the internal initiation site. By introducing mutations at either site, we furthermore established that internal and terminal initiation shared identical sequence requirements. Importantly, lethal point mutations at the terminal site resulted exclusively in truncated replicons. In contrast, the same mutations at the internal site abrogated internal initiation, suggesting a competitive selection of initiation sites, rather than recombination or templateswitching events. In conclusion, our data indicate that the HCV replicase is capable of internal initiation in its natural environment, although functional replication likely requires only terminal initiation. Since many other positive-strand RNA viruses generate subgenomic messenger RNAs during their replication cycle, we surmise that their capability for internal initiation is a common and conserved feature of viral RdRps. IMPORTANCE Many aspects of viral RNA replication of hepatitis C virus (HCV) are still poorly understood. The process of RNA synthesis is driven by the RNAdependent RNA polymerase (RdRp) NS5B. Most mechanistic studies on NS5B so far were performed with in vitro systems using isolated recombinant polymerase. In this study, we present a replicon model, which allows the intracellular assessment of noncanonical modes of initiation by the full HCV replicase. Our results add to the understanding of the biochemical processes underlying initiation of RNA synthesis by NS5B by the discovery of internal initiation in cellulo. Moreover, they validate observations made in vitro, showing that the viral polymerase acts very similarly in isolation and in complex with other viral and host proteins. Finally, these observations provide clues about the evolution of RdRps of positive-strand RNA viruses, which might contain the intrinsic ability to initiate internally.

Gene Expression Profiling of Different Huh7 Variants Reveals Novel Hepatitis C Virus Host Factors

“…Preparation of sub-genomic HCV reporter RNAs and electro-transfection into Huh7 cells was done as described [57]. Sub-genomic reporter replicons based on HCV genotypes 1b (Con1-ET) and 2a (JFH-1) have been described before (Con1-ET [27], JFH-1 [58]).…”

Positive strand RNA viruses differ in the constraints they place on the folding of their negative strand

“…To assess the potential for Rbzs to fold and cleave the [-] RNA of a positive strand RNA virus in cis , we initially chose to focus on HCV. The basis for this decision was because of evidence that HCV allows folding of relatively complex native structures in its [-] RNA (26,29). Additionally we opted to use subgenomic replicon-based constructs because they enable the relevant stages of virus genome replication to be studied in isolation from other stages of the virus replicative cycle - such as entry, packaging and egress.…”

“…This is because the complementary nature of the [-] and [+] RNAs promotes dsRNA formation. Despite this several viruses harbour structured CREs in their [-] RNA; CREs that act as promoters for genomic and subgenomic RNA production (25)(26)(27)(28)(29). Thus, at some point the duplex base pairing masking these CREs has to be separated in such a way that intramolecular base pairing is promoted while intermolecular base pairing is prevented.…”

“…Thus, at some point the duplex base pairing masking these CREs has to be separated in such a way that intramolecular base pairing is promoted while intermolecular base pairing is prevented. To date, the only way used to monitor RNA folding in the [-] RNA has been to employ CRE-dependent replication as a read-out (25,26,29). As CRE function is specific to the virus, this does not offer a uniform way to examine RNA folding across the [-] RNA of different viruses.…”

Hepatitis C Viral Replication Complex

“…Structural evidence indicates that NS5B uses de novo initiation to replicate the HCV RNA genome in cells [ 69 , 94 , 98 ]. Moreover, NS5B is capable of internal initiation via functional replication and likely only requires terminal initiation in its natural environment [ 99 ]. It is believed that initiation begins at the 3’ end of the HCV genomic RNA and requires high levels of GTP that bind to an allosteric site in the NS5B (β-flap domain) to act as structural support to prime the initiation step [ 100 , 101 ].…”