Dataset Information

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork.

ABSTRACT: RNA helicases regulate virtually all RNA-dependent cellular processes. Although much is known about helicase structures, very little is known about how they deal with barriers in RNA and the factors that affect their processivity. The hepatitis C virus encodes NS3, an RNA helicase that is essential for viral RNA replication. We have used optical tweezers to determine at the single-molecule level how the local stability of the RNA substrate affects the enzyme rate of strand separation, whether separation occurs by an active or a passive mechanism, and whether processivity is affected. We show that sequence barriers in RNA modulate NS3 activity. NS3 processivity depends on barriers ahead of the opening fork. Our results rule out a model where NS3 passively waits for the thermal fraying of double-stranded RNA. Instead, we find that NS3 destabilizes the duplex before separating the strands. Failure to do so before a strong barrier leads to helicase dissociation and limits the processivity of the enzyme.

SUBMITTER: Cheng W

PROVIDER: S-EPMC1955789 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Publications

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork.

Cheng Wei W Dumont Sophie S Tinoco Ignacio I Bustamante Carlos C

Proceedings of the National Academy of Sciences of the United States of America 20070820 35

RNA helicases regulate virtually all RNA-dependent cellular processes. Although much is known about helicase structures, very little is known about how they deal with barriers in RNA and the factors that affect their processivity. The hepatitis C virus encodes NS3, an RNA helicase that is essential for viral RNA replication. We have used optical tweezers to determine at the single-molecule level how the local stability of the RNA substrate affects the enzyme rate of strand separation, whether se ...[more]

PMID: 17709749

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Project description:RNA-remodeling proteins, including RNA helicases and chaperones, act to remodel RNA structures and/or protein-RNA interactions and are required for all processes involving RNAs. Although many viruses encode RNA helicases and chaperones, their in vitro activities and their roles in infected cells largely remain elusive. Noroviruses are a diverse group of positive-strand RNA viruses in the family Caliciviridae and constitute a significant and potentially fatal threat to human health. Here, we report that the protein NS3 encoded by human norovirus has both ATP-dependent RNA helicase activity that unwinds RNA helices and ATP-independent RNA-chaperoning activity that can remodel structured RNAs and facilitate strand annealing. Moreover, NS3 can facilitate viral RNA synthesis in vitro by norovirus polymerase. NS3 may therefore play an important role in norovirus RNA replication. Lastly, we demonstrate that the RNA-remodeling activity of NS3 is inhibited by guanidine hydrochloride, an FDA-approved compound, and, more importantly, that it reduces the replication of the norovirus replicon in cultured human cells. Altogether, these findings are the first to demonstrate the presence of RNA-remodeling activities encoded by Caliciviridae and highlight the functional significance of NS3 in the noroviral life cycle.IMPORTANCE Noroviruses are a diverse group of positive-strand RNA viruses, which annually cause hundreds of millions of human infections and over 200,000 deaths worldwide. For RNA viruses, cellular or virus-encoded RNA helicases and/or chaperones have long been considered to play pivotal roles in viral life cycles. However, neither RNA helicase nor chaperoning activity has been demonstrated to be associated with any norovirus-encoded proteins, and it is also unknown whether norovirus replication requires the participation of any viral or cellular RNA helicases/chaperones. We found that a norovirus protein, NS3, not only has ATP-dependent helicase activity, but also acts as an ATP-independent RNA chaperone. Also, NS3 can facilitate in vitro viral RNA synthesis, suggesting the important role of NS3 in norovirus replication. Moreover, NS3 activities can be inhibited by an FDA-approved compound, which also suppresses norovirus replicon replication in human cells, raising the possibility that NS3 could be a target for antinoroviral drug development.

Dataset Information

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork.

Publications

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork.

Similar Datasets

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