scholarly journals Human Norovirus NS3 Has RNA Helicase and Chaperoning Activities

2017 ◽  
Vol 92 (5) ◽  
Author(s):  
Teng-Feng Li ◽  
Myra Hosmillo ◽  
Hella Schwanke ◽  
Ting Shu ◽  
Zhaowei Wang ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Rna Viruses ◽  
Rna Helicase ◽  
Diverse Group ◽  
Rna Helicases ◽  
Human Norovirus ◽  
Content Type ◽  
Rna Remodeling ◽  
Positive Strand Rna

ABSTRACTRNA-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, theirin vitroactivities and their roles in infected cells largely remain elusive. Noroviruses are a diverse group of positive-strand RNA viruses in the familyCaliciviridaeand 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 synthesisin vitroby 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 byCaliciviridaeand highlight the functional significance of NS3 in the noroviral life cycle.IMPORTANCENoroviruses 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 facilitatein vitroviral 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.

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

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Philipp Schult ◽  
Maren Nattermann ◽  
Chris Lauber ◽  
Stefan Seitz ◽  
Volker Lohmann
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Rna Replication ◽  
Rna Dependent Rna Polymerase ◽  
Internal Initiation ◽  
Positive Strand Rna

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 template-switching 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 RNA-dependent 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.

scholarly journals Reconstitution of an RNA Virus Replicase in Artificial Giant Unilamellar Vesicles Supports Full Replication and Provides Protection for the Double-Stranded RNA Replication Intermediate

2020 ◽  
Vol 94 (18) ◽  
Author(s):  
Nikolay Kovalev ◽  
Judit Pogany ◽  
Peter D. Nagy
Keyword(s):  
Virus Replication ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Host Factors ◽  
Replication Process ◽  
Double Stranded Rna ◽  
Replication Intermediate ◽  
Cellular Factors ◽  
Positive Strand Rna

ABSTRACT Positive-strand RNA [(+)RNA] viruses are important pathogens of humans, animals, and plants and replicate inside host cells by coopting numerous host factors and subcellular membranes. To gain insights into the assembly of viral replicase complexes (VRCs) and dissect the roles of various lipids and coopted host factors, we have reconstituted Tomato bushy stunt virus (TBSV) replicase using artificial giant unilamellar vesicles (GUVs). We demonstrate that reconstitution of VRCs on GUVs with endoplasmic reticulum (ER)-like phospholipid composition results in a complete cycle of replication and asymmetrical RNA synthesis, which is a hallmark of (+)RNA viruses. TBSV VRCs assembled on GUVs provide significant protection of the double-stranded RNA (dsRNA) replication intermediate against the dsRNA-specific RNase III. The lipid compositions of GUVs have pronounced effects on in vitro TBSV replication, including (−) and (+)RNA synthesis. The GUV-based assay has led to the discovery of the critical role of phosphatidylserine in TBSV replication and a novel role for phosphatidylethanolamine in asymmetrical (+)RNA synthesis. The GUV-based assay also showed stimulatory effects by phosphatidylinositol-3-phosphate [PI(3)P] and ergosterol on TBSV replication. We demonstrate that eEF1A and Hsp70 coopted replicase assembly factors, Vps34 phosphatidylinositol 3-kinase (PI3K) and the membrane-bending ESCRT factors, are required for reconstitution of the active TBSV VRCs in GUVs, further supporting that the novel GUV-based in vitro approach recapitulates critical steps and involves essential coopted cellular factors of the TBSV replication process. Taken together, this novel GUV assay will be highly suitable to dissect the functions of viral and cellular factors in TBSV replication. IMPORTANCE Understanding the mechanism of replication of positive-strand RNA viruses, which are major pathogens of plants, animals, and humans, can lead to new targets for antiviral interventions. These viruses subvert intracellular membranes for virus replication and coopt numerous host proteins, whose functions during virus replication are not yet completely defined. To dissect the roles of various host factors in Tomato bushy stunt virus (TBSV) replication, we have developed an artificial giant unilamellar vesicle (GUV)-based replication assay. The GUV-based in vitro approach recapitulates critical steps of the TBSV replication process. GUV-based reconstitution of the TBSV replicase revealed the need for a complex mixture of phospholipids, especially phosphatidylserine and phosphatidylethanolamine, in TBSV replication. The GUV-based approach will be useful to dissect the functions of essential coopted cellular factors.

scholarly journals Role of Viral RNA and Co-opted Cellular ESCRT-I and ESCRT-III Factors in Formation of Tombusvirus Spherules Harboring the Tombusvirus Replicase

2016 ◽  
Vol 90 (7) ◽  
pp. 3611-3626 ◽  
Author(s):  
Nikolay Kovalev ◽  
Isabel Fernández de Castro Martín ◽  
Judit Pogany ◽  
Daniel Barajas ◽  
Kunj Pathak ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Viral Rna ◽  
Electron Microscopic ◽  
Content Type ◽  
Endosomal Sorting ◽  
Infected Cells ◽  
Membrane Bound ◽  
Positive Strand Rna

ABSTRACTPlus-stranded RNA viruses induce membrane deformations in infected cells in order to build viral replication complexes (VRCs).Tomato bushy stunt virus(TBSV) co-opts cellular ESCRT (endosomal sorting complexes required for transport) proteins to induce the formation of vesicle (spherule)-like structures in the peroxisomal membrane with tight openings toward the cytosol. In this study, using a yeast (Saccharomyces cerevisiae)vps23Δbro1Δ double-deletion mutant, we showed that the Vps23p ESCRT-I protein (Tsg101 in mammals) and Bro1p (ALIX) ESCRT-associated protein, both of which bind to the viral p33 replication protein, play partially complementary roles in TBSV replication in cells and in cell extracts. Dual expression of dominant-negative versions ofArabidopsishomologs of Vps23p and Bro1p inhibited tombusvirus replication to greater extent than individual expression inNicotiana benthamianaleaves. We also demonstrated the critical role of Snf7p (CHMP4), Vps20p, and Vps24p ESCRT-III proteins in tombusvirus replication in yeast andin vitro. Electron microscopic imaging ofvps23Δ yeast revealed the lack of tombusvirus-induced spherule-like structures, while crescent-like structures are formed in ESCRT-III deletion yeasts replicating TBSV RNA. In addition, we also showed that the length of the viral RNA affects the sizes of spherules formed inN. benthamianacells. The 4.8-kb genomic RNA is needed for the formation of spherules 66 nm in diameter, while spherules formed during the replication of the ∼600-nucleotide (nt)-long defective interfering RNA in the presence of p33 and p92 replication proteins are 42 nm. We propose that the viral RNA serves as a “measuring string” during VRC assembly and spherule formation.IMPORTANCEPlant positive-strand RNA viruses, similarly to animal positive-strand RNA viruses, replicate in membrane-bound viral replicase complexes in the cytoplasm of infected cells. Identification of cellular and viral factors affecting the formation of the membrane-bound viral replication complex is a major frontier in current virology research. In this study, we dissected the functions of co-opted cellular ESCRT-I (endosomal sorting complexes required for transport I) and ESCRT-III proteins and the viral RNA in tombusvirus replicase complex formation usingin vitro, yeast-based, and plant-based approaches. Electron microscopic imaging revealed the lack of tombusvirus-induced spherule-like structures in ESCRT-I or ESCRT-III deletion yeasts replicating TBSV RNA, demonstrating the requirement for these co-opted cellular factors in tombusvirus replicase formation. The work could be of broad interest in virology and beyond.

scholarly journals RNA Helicase A Regulates the Replication of RNA Viruses

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 361
Author(s):  
Rui-Zhu Shi ◽  
Yuan-Qing Pan ◽  
Li Xing
Keyword(s):  
Virus Replication ◽  
Rna Binding ◽  
Rna Viruses ◽  
Rna Helicase ◽  
Rna Helicase A ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
N Terminus

The RNA helicase A (RHA) is a member of DExH-box helicases and characterized by two double-stranded RNA binding domains at the N-terminus. RHA unwinds double-stranded RNA in vitro and is involved in RNA metabolisms in the cell. RHA is also hijacked by a variety of RNA viruses to facilitate virus replication. Herein, this review will provide an overview of the role of RHA in the replication of RNA viruses.

scholarly journals Association of the Cold Shock DEAD-Box RNA Helicase RhlE to the RNA Degradosome in Caulobacter crescentus

2017 ◽  
Vol 199 (13) ◽  
Author(s):  
Angel A. Aguirre ◽  
Alexandre M. Vicente ◽  
Steven W. Hardwick ◽  
Daniela M. Alvelos ◽  
Ricardo R. Mazzon ◽  
...  
Keyword(s):  
Low Temperature ◽  
Caulobacter Crescentus ◽  
Immunoblot Analysis ◽  
Rna Helicase ◽  
Rnase E ◽  
Rna Helicases ◽  
Content Type ◽  
Dead Box ◽  
Rna Degradosome ◽  
The Dead

ABSTRACT In diverse bacterial lineages, multienzyme assemblies have evolved that are central elements of RNA metabolism and RNA-mediated regulation. The aquatic Gram-negative bacterium Caulobacter crescentus, which has been a model system for studying the bacterial cell cycle, has an RNA degradosome assembly that is formed by the endoribonuclease RNase E and includes the DEAD-box RNA helicase RhlB. Immunoprecipitations of extracts from cells expressing an epitope-tagged RNase E reveal that RhlE, another member of the DEAD-box helicase family, associates with the degradosome at temperatures below those optimum for growth. Phenotype analyses of rhlE, rhlB, and rhlE rhlB mutant strains show that RhlE is important for cell fitness at low temperature and its role may not be substituted by RhlB. Transcriptional and translational fusions of rhlE to the lacZ reporter gene and immunoblot analysis of an epitope-tagged RhlE indicate that its expression is induced upon temperature decrease, mainly through posttranscriptional regulation. RNase E pulldown assays show that other proteins, including the transcription termination factor Rho, a second DEAD-box RNA helicase, and ribosomal protein S1, also associate with the degradosome at low temperature. The results suggest that the RNA degradosome assembly can be remodeled with environmental change to alter its repertoire of helicases and other accessory proteins. IMPORTANCE DEAD-box RNA helicases are often present in the RNA degradosome complex, helping unwind secondary structures to facilitate degradation. Caulobacter crescentus is an interesting organism to investigate degradosome remodeling with change in temperature, because it thrives in freshwater bodies and withstands low temperature. In this study, we show that at low temperature, the cold-induced DEAD-box RNA helicase RhlE is recruited to the RNA degradosome, along with other helicases and the Rho protein. RhlE is essential for bacterial fitness at low temperature, and its function may not be complemented by RhlB, although RhlE is able to complement for rhlB loss. These results suggest that RhlE has a specific role in the degradosome at low temperature, potentially improving adaptation to this condition.

scholarly journals Factors Required for the Uridylylation of the Foot-and-Mouth Disease Virus 3B1, 3B2, and 3B3 Peptides by the RNA-Dependent RNA Polymerase (3Dpol) In Vitro

2005 ◽  
Vol 79 (12) ◽  
pp. 7698-7706 ◽  
Author(s):  
Arabinda Nayak ◽  
Ian G. Goodfellow ◽  
Graham J. Belsham
Keyword(s):  
Rna Polymerase ◽  
Disease Virus ◽  
Rna Synthesis ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Coding Region ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Positive Strand Rna

ABSTRACT The 5′ terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 3B (VPg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3Dpol. To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3Dpol in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV cre has been identified previously to be within the 5′ untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 3B peptides has now been determined, and the role of the FMDV cre (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.

Identification of a putative RNA helicase (HRH1), a human homolog of yeast Prp22

1994 ◽  
Vol 14 (11) ◽  
pp. 7611-7620
Author(s):  
Y Ono ◽  
M Ohno ◽  
Y Shimura
Keyword(s):  
Pcr Primers ◽  
Rna Helicase ◽  
Protein Family ◽  
Temperature Sensitive ◽  
Human Homolog ◽  
Rna Helicases ◽  
Sr Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rich Domain

In the budding yeast Saccharomyces cerevisiae, a number of PRP genes known to be involved in pre-mRNA processing have been genetically identified and cloned. Three PRP genes (PRP2, PRP16, and PRP22) were shown to encode putative RNA helicases of the family of proteins with DEAH boxes. However, any such splicing factor containing the helicase motifs in vertebrates has not been identified. To identify human homologs of this family, we designed PCR primers corresponding to the highly conserved region of the DEAH box protein family and successfully amplified five cDNA fragments, using HeLa poly(A)+ RNA as a substrate. One fragment, designated HRH1 (human RNA helicase 1), is highly homologous to Prp22, which was previously shown to be involved in the release of spliced mRNAs from the spliceosomes. Expression of HRH1 in a S. cerevisiae prp22 mutant can partially rescue its temperature-sensitive phenotype. These results strongly suggest that HRH1 is a functional human homolog of the yeast Prp22 protein. Interestingly, HRH1 but not Prp22 contains an arginine- and serine-rich domain (RS domain) which is characteristic of some splicing factors, such as members of the SR protein family. We could show that HRH1 can interact in vitro and in the yeast two-hybrid system with members of the SR protein family through its RS domain. We speculate that HRH1 might be targeted to the spliceosome through this interaction.

scholarly journals A Unique 5′ Translation Element Discovered in Triticum Mosaic Virus

2015 ◽  
Vol 89 (24) ◽  
pp. 12427-12440 ◽  
Author(s):  
Robyn Roberts ◽  
Jincan Zhang ◽  
Laura K. Mayberry ◽  
Satyanarayana Tatineni ◽  
Karen S. Browning ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Translation Initiation ◽  
Plant Virus ◽  
Plant Viruses ◽  
Leader Sequence ◽  
Content Type ◽  
Internal Translation ◽  
Positive Strand Rna ◽  
Independent Translation

ABSTRACTSeveral plant viruses encode elements at the 5′ end of their RNAs, which, unlike most cellular mRNAs, can initiate translation in the absence of a 5′ m7GpppG cap. Here, we describe an exceptionally long (739-nucleotide [nt]) leader sequence in triticum mosaic virus (TriMV), a recently emerged wheat pathogen that belongs to thePotyviridaefamily of positive-strand RNA viruses. We demonstrate that the TriMV 5′ leader drives strong cap-independent translation in both wheat germ extract and oat protoplasts through a novel, noncanonical translation mechanism. Translation preferentially initiates at the 13th start codon within the leader sequence independently of eIF4E but involves eIF4G. We truncated the 5′ leader to a 300-nucleotide sequence that drives cap-independent translation from the 5′ end. We show that within this sequence, translation activity relies on a stem-loop structure identified at nucleotide positions 469 to 490. The disruption of the stem significantly impairs the function of the 5′ untranslated region (UTR) in driving translation and competing against a capped RNA. Additionally, the TriMV 5′ UTR can direct translation from an internal position of a bicistronic mRNA, and unlike cap-driven translation, it is unimpaired when the 5′ end is blocked by a strong hairpin in a monocistronic reporter. However, the disruption of the identified stem structure eliminates such a translational advantage. Our results reveal a potent and uniquely controlled translation enhancer that may provide new insights into mechanisms of plant virus translational regulation.IMPORTANCEMany members of thePotyviridaefamily rely on their 5′ end for translation. Here, we show that the 739-nucleotide-long triticum mosaic virus 5′ leader bears a powerful translation element with features distinct from those described for other plant viruses. Despite the presence of 12 AUG start codons within the TriMV 5′ UTR, translation initiates primarily at the 13th AUG codon. The TriMV 5′ UTR is capable of driving cap-independent translationin vitroandin vivo, is independent of eIF4E, and can drive internal translation initiation. A hairpin structure at nucleotide positions 469 to 490 is required for the cap-independent translation and internal translation initiation abilities of the element and plays a role in the ability of the TriMV UTR to compete against a capped RNAin vitro. Our results reveal a novel translation enhancer that may provide new insights into the large diversity of plant virus translation mechanisms.

scholarly journals Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA

2017 ◽  
Vol 115 (2) ◽  
pp. E162-E171 ◽  
Author(s):  
François Ferron ◽  
Lorenzo Subissi ◽  
Ana Theresa Silveira De Morais ◽  
Nhung Thi Tuyet Le ◽  
Marion Sevajol ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Mutation Rates ◽  
Bifunctional Enzyme ◽  
Antiviral Compound ◽  
Mismatch Correction ◽  
Limited Efficacy

Coronaviruses (CoVs) stand out among RNA viruses because of their unusually large genomes (∼30 kb) associated with low mutation rates. CoVs code for nsp14, a bifunctional enzyme carrying RNA cap guanine N7-methyltransferase (MTase) and 3′-5′ exoribonuclease (ExoN) activities. ExoN excises nucleotide mismatches at the RNA 3′-end in vitro, and its inactivation in vivo jeopardizes viral genetic stability. Here, we demonstrate for severe acute respiratory syndrome (SARS)-CoV an RNA synthesis and proofreading pathway through association of nsp14 with the low-fidelity nsp12 viral RNA polymerase. Through this pathway, the antiviral compound ribavirin 5′-monophosphate is significantly incorporated but also readily excised from RNA, which may explain its limited efficacy in vivo. The crystal structure at 3.38 Å resolution of SARS-CoV nsp14 in complex with its cofactor nsp10 adds to the uniqueness of CoVs among RNA viruses: The MTase domain presents a new fold that differs sharply from the canonical Rossmann fold.

scholarly journals Identification of Determinants Involved in Initiation of Hepatitis C Virus RNA Synthesis by Using Intergenotypic Replicase Chimeras

2007 ◽  
Vol 81 (10) ◽  
pp. 5270-5283 ◽  
Author(s):  
Marco Binder ◽  
Doris Quinkert ◽  
Olga Bochkarova ◽  
Rahel Klein ◽  
Nikolina Kezmic ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Negative Strand ◽  
Virus Rna ◽  
Nontranslated Region ◽  
Genotype 2 ◽  
Positive Strand Rna

ABSTRACT The 5′ nontranslated region (NTR) and the X tail in the 3′ NTR are the least variable parts of the hepatitis C virus (HCV) genome and play an important role in the initiation of RNA synthesis. By using subgenomic replicons of the HCV isolates Con1 (genotype 1) and JFH1 (genotype 2), we characterized the genotype specificities of the replication signals contained in the NTRs. The replacement of the JFH1 5′ NTR and X tail with the corresponding Con1 sequence resulted in a significant decrease in replication efficiency. Exchange of the X tail specifically reduced negative-strand synthesis, whereas substitution of the 5′ NTR impaired the generation of progeny positive strands. In search for the proteins involved in the recognition of genotype-specific initiation signals, we analyzed recombinant nonstructural protein 5B (NS5B) RNA polymerases of both isolates and found some genotype-specific template preference for the 3′ end of positive-strand RNA in vitro. To further address genotype specificity, we constructed a series of intergenotypic replicon chimeras. When combining NS3 to NS5A of Con1 with NS5B of JFH1, we observed more-efficient replication with the genotype 2a X tail, indicating that NS5B recognizes genotype-specific signals in this region. In contrast, a combination of the NS3 helicase with NS5A and NS5B was required to confer genotype specificity to the 5′ NTR. These results present the first genetic evidence for an interaction between helicase, NS5A, and NS5B required for the initiation of RNA synthesis and provide a system for the specific analysis of HCV positive- and negative-strand syntheses.

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