A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase

Levin, Mikhail K.; Gurjar, Madhura M.; Patel, Smita S.

doi:10.1038/nsmb920

Cited by 128 publications

(173 citation statements)

References 44 publications

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“…The molecular mechanism underlying RNA unwinding is not yet clear. However, recent kinetic analyses have shown that the enzyme undergoes highly coordinated cycles of fast dsRNA unwinding with high processivity for about 20 nt (23)(24)(25). Thereafter, the enzyme "pauses" and may fall off the template or start a new cycle of duplex unwinding.…”

Section: Components Of the Hcv Replication Complexmentioning

confidence: 99%

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

Appel

Schaller

Pénin

et al. 2006

Journal of Biological Chemistry

177

120

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With an estimated number of about 180 million affected people worldwide and a limited therapy option, infection with the hepatitis C virus (HCV) 2 is an important medical problem. Initial limitations in propagating HCV in cell culture have been overcome step-by-step in the past few years and culminated in the recent development of a system allowing efficient propagation of infectious HCV in tissue culture. Nevertheless, structural and biochemical studies of viral proteins as well as molecular analysis of viral RNA replication are still major challenges. The recent resolution of the three-dimensional structures of some HCV proteins or domains along with elegant reverse genetic and cell biological studies provided first insights into how HCV amplifies its RNA, exploits the cellular machinery, and eventually overcomes innate immune responses. Organization of the Viral GenomeHCV is a positive strand RNA virus that has been classified as the genus Hepacivirus in the Flaviviridae family. The HCV genome is an uncapped, linear molecule with a length of ϳ9600 nucleotides (nt; Fig. 1A). It carries a long open reading frame that is flanked at the 5Ј-and 3Ј-ends by short highly structured non-translated regions (NTRs). The 5Ј-NTR has a length of about 340 nt and contains an internal ribosome entry site (IRES) required for translation of the HCV genome (1). This RNA element binds the 40 S ribosomal subunit in the absence of other translation initiation factors in a way that the initiation codon is placed in the immediate vicinity of the P site (2). Part of the IRES (domain II) overlaps with RNA signals essential for viral replication (Fig. 1A) arguing for a possible role of domain II in regulating a translation-RNA replication switch. The 3Ј-NTR has a tripartite structure composed of an ϳ40-nt-long variable region downstream of the HCV coding sequence, a poly(U/UC) tract of heterogeneous length, and a highly conserved 98-nt-long sequence designated X-tail (Fig. 1A). Genetic studies have shown that a poly(U/UC) tract of at least ϳ25 nt as well as the complete X-tail are required for RNA replication in cell culture and for infectivity of the viral genome in vivo. An additional cis-acting RNA element (CRE) has been identified in the 3Ј-terminal coding region of NS5B (Fig. 1A). This CRE (designated 5BSL3.2) (3) forms a long distance RNA-RNA interaction with SL2 in the X-tail (4), which is indispensable for RNA replication.Expression of the viral proteins from the monocistronic genome is primarily achieved by production of a polyprotein that is proteolytically cleaved into the structural proteins (core, envelope proteins E1 and E2), the hydrophobic peptide p7, and the non-structural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (Fig. 1A) (1, 5). Processing of the core to p7 region is mediated by host cell signalases, and in the case of the core protein, in addition by signal peptide peptidase. All remaining cleavages are carried out by two viral proteases: the NS2/3 protease mediating cleavage between NS2 and NS3 and the NS3...

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Section: Components Of the Hcv Replication Complexmentioning

confidence: 99%

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

Appel

Schaller

Pénin

et al. 2006

Journal of Biological Chemistry

177

120

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“…These enzymes use the energy of NTP hydrolysis to unwind double-stranded RNA, and NS3 unwinds RNA and DNA homoduplexes and heteroduplexes in a 3፱ to 5፱ direction 25 . The helicase mechanism is not yet fully understood, but recent kinetic analyses show that the NS3 helicase behaves like a ratcheting two-stroke motor 26 and seems to function as a dimer that incrementally rips apart 18-base-pair stretches of substrate RNA 27 . In addition, NS3 helicase activity can be regulated by interactions between the serine protease and helicase domains of NS3 (refs 28, 29), indicating that these two enzyme activities may be somehow coordinated during replication.…”

Section: Dissecting the Structure And Function Of Hcv Ns Proteinsmentioning

confidence: 99%

Unravelling hepatitis C virus replication from genome to function

Lindenbach

Rice

2005

Nature

751

561

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Since the discovery of the hepatitis C virus over 15 years ago, scientists have raced to develop diagnostics, study the virus and find new therapies. Yet virtually every attempt to dissect this pathogen has met with roadblocks that impeded progress. Its replication was restricted to humans or experimentally infected chimpanzees, and efficient growth of the virus in cell culture failed until very recently. Nevertheless hard-fought progress has been made and the first wave of antiviral drugs is entering clinical trials.virus life cycle: first, as a messenger RNA (mRNA) for translation of the viral proteins; second as a template for RNA replication; and third, as a nascent genome packaged within new virus particles. Virions presumably form by budding into the endoplasmic reticulum (ER) and leave the cell through the secretory pathway.Researchers have followed each aspect of the virus life cycle in turn. Just as infection starts from an HCV genome entering the cytoplasm and progressing through translation, replication and particle production, our understanding has progressed from having a genome sequence to understanding translation and the viral gene products, characterizing RNA replication, and establishing systems to characterize virus particles and infectivity. In this review, we summarize the current understanding of HCV replication with special emphasis on recent developments. As space is limited, we cannot be comprehensive; readers may wish to consult other reviews for detail and breadth 5,13,14. Initial studies: HCV translation and polyprotein processingThe identification of HCV yielded a partial viral genome sequence 5 . Research in the early 1990s focused on dissecting HCV gene expression and characterizing the gene products. Much has been learned about the biochemistry of three key enzymes, and structural information is now available at the atomic level for roughly half of the proteincoding region.Translation of the HCV genome, which lacks a 5፱ cap, depends on an internal ribosome entry site (IRES) within the 5፱-noncoding region (NCR). The HCV IRES binds 40S ribosomal subunits directly and avidly, bypassing the need for pre-initiation factors, and inducing an mRNA-bound conformation in the 40S subunit 15 . The IRES-40S complex then recruits eukaryotic initiation factor (eIF) 3 and the ternary complex of Met-tRNA-eIF2-GTP to form a non-canonical 48S intermediate, before a kinetically slow transition to the translationally active 80S complex 16,17 .Once initiated, translation of the HCV genome produces a large polyprotein that is proteolytically cleaved to produce 10 viral proteins (Fig. 2a). The amino-terminal one-third of the polyprotein encodes the virion structural proteins: the highly basic core (C) protein, and glycoproteins E1 and E2. After the structural region comes a small integral membrane protein, p7, which seems to function as an ion channel 18,19 . The remainder of the genome encodes the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B, which coordinate the intracellular process...

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“…Despite recent progress in the study of these motor proteins (2-4), the physical mechanisms by which they move and catalyze the strand separation are not well understood. Helicase models ranging from a pure Brownian ratchet to a pure power stroke action have been discussed (5)(6)(7)(8), but experimental data to support them for RNA helicases have been lacking. The hepatitis C virus NS3 protein is a superfamily 2, 3Ј to 5Ј RNA helicase (9) known to be essential for virus replication and, thus, an antiviral drug target (10).…”

mentioning

confidence: 99%

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

Cheng¹,

Dumont

Tinoco³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

149

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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.hairpin ͉ molecular motors ͉ optical tweezers ͉ processivity A wide range of RNA metabolic activities (1) require the breaking of base pairs in double-stranded RNA (dsRNA) by RNA helicases. Despite recent progress in the study of these motor proteins (2-4), the physical mechanisms by which they move and catalyze the strand separation are not well understood. Helicase models ranging from a pure Brownian ratchet to a pure power stroke action have been discussed (5-8), but experimental data to support them for RNA helicases have been lacking. The hepatitis C virus NS3 protein is a superfamily 2, 3Ј to 5Ј RNA helicase (9) known to be essential for virus replication and, thus, an antiviral drug target (10). Recently, NS3 has been the subject of single-molecule manipulation studies (4), which revealed that NS3 makes steps of 11 bp each made up of three substeps (4). RNA molecules contain various kinetic barriers to mechanical unfolding (11). How these barriers influence the activity of motor proteins that work on RNA will depend on the mechanism of the motor. To probe the molecular mechanism of NS3 unwinding activity, we have designed and characterized RNA molecules with various mechanical unfolding barriers and used single-molecule methods to investigate how these barriers affect the velocity, pausing, and processivity of the helicase in real time. ResultsDesign and Characterization of RNA Hairpin Substrates. We first designed two RNA hairpin substrates that terminate both on a tetraloop (Fig. 1A) to monitor the response of a single NS3 helicase to weak and strong sequence barriers. Substrate RNA-AG has 30 A⅐U pairs followed by 30 G⅐C pairs; RNA-GA has the A⅐U and G⅐C sequences interchanged. The sequences within A⅐U and G⅐C regions were chosen to minimize the formation of alternative secondary structures other than the desired 60-bp hairpin. In our experiment, a single RNA hairpin was attached between a microsphere in an optical trap and a microsphere placed on the end of a micropipette through hybrid RNA-DNA handles to se...

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A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase

Cited by 128 publications

References 44 publications

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

Unravelling hepatitis C virus replication from genome to function

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

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