Systematic Analysis of Enhancer and Critical
            <i>cis</i>
            -Acting RNA Elements in the Protein-Encoding Region of the Hepatitis C Virus Genome

Chu, Derrick M.; Ren, Shuo; Hu, Stacy; Wang, Wei Gang; Subramanian, Aparna; Contreras, Deisy; Kanagavel, Vidhya; Chung, Eunna; Ko, Justine S.; Appadorai, Ranjit Singh Amirtham Jacob; Sinha, Sanjeev; Jalali, Ziba; Hardy, David; French, Samuel W.; Arumugaswami, Vaithilingaraja

doi:10.1128/jvi.00840-12

Cited by 32 publications

(43 citation statements)

References 72 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3, purple boxes). Five elements correspond to regulatory motifs that have been well characterized: the IRES domains II-IV, SL9098, and CRE (13)(14)(15)(16)(17). We focused on the four uncharacterized RNA elements (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Functionally conserved architecture of hepatitis C virus RNA genomes

Mauger¹,

Golden

Yamane

et al. 2015

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

122

190

View full text Add to dashboard Cite

Hepatitis C virus (HCV) infects over 170 million people worldwide and is a leading cause of liver disease and cancer. The virus has a 9,650-nt, single-stranded, messenger-sense RNA genome that is infectious as an independent entity. The RNA genome has evolved in response to complex selection pressures, including the need to maintain structures that facilitate replication and to avoid clearance by cell-intrinsic immune processes. Here we used highthroughput, single-nucleotide resolution information to generate and functionally test data-driven structural models for three diverse HCV RNA genomes. We identified, de novo, multiple regions of conserved RNA structure, including all previously characterized cisacting regulatory elements and also multiple novel structures required for optimal viral fitness. Well-defined RNA structures in the central regions of HCV genomes appear to facilitate persistent infection by masking the genome from RNase L and double-stranded RNA-induced innate immune sensors. This work shows how structure-first comparative analysis of entire genomes of a pathogenic RNA virus enables comprehensive and concise identification of regulatory elements and emphasizes the extensive interrelationships among RNA genome structure, viral biology, and innate immune responses.RNA structure | evolution | motif discovery | functional validation H epatitis C virus (HCV) currently infects over 170 million people. There is no vaccine, and therapy, generally involving treatment with IFN and ribavirin, is often ineffective (1). Efficacious anti-HCV therapeutics are becoming available (2), but the extent to which they will mitigate the hepatitis C disease burden remains to be seen. Roughly 70% of acutely infected individuals fail to clear the virus and become lifelong HCV carriers, at risk for progressive hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (3).HCV genomes are single-stranded, ∼9,650-nt, messengersense RNA molecules (4). The naked RNA initiates autonomous replication when transfected into cells and establishes chronic HCV infection in chimpanzees (5, 6). The HCV genomic RNA carries genetic information at two levels: a single large ORF encodes viral proteins and complex RNA structural elements regulate the viral replication cycle (4). Viral replication begins when conserved RNA elements in the 5′ UTR bind the 40S ribosome subunit and recruit essential translation factors (7). Translation produces a viral polyprotein that is cleaved by cellular and viral proteases to generate 10 viral proteins (4). The HCV genome is replicated through a negative-strand RNA intermediate by a viral RNA-dependent RNA polymerase (NS5B) in a process controlled by conserved RNA elements (8-17).The HCV genomic RNA is physically compact (18) and highly structured (19). These features likely facilitate persistent HCV infections in humans by protecting the genome from degradation by innate antiviral defenses (20,21). Two elements of this defense are RNase L, which cleaves in single-stranded regions (22), and diverse double...

show abstract

Section: Resultsmentioning

confidence: 99%

“…Translation produces a viral polyprotein that is cleaved by cellular and viral proteases to generate 10 viral proteins (4). The HCV genome is replicated through a negative-strand RNA intermediate by a viral RNA-dependent RNA polymerase (NS5B) in a process controlled by conserved RNA elements (8)(9)(10)(11)(12)(13)(14)(15)(16)(17).…”

mentioning

confidence: 99%

Functionally conserved architecture of hepatitis C virus RNA genomes

Mauger¹,

Golden

Yamane

et al. 2015

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

122

190

View full text Add to dashboard Cite

show abstract

“…Many of these structures have since been validated by mutational analysis in cell culture models of HCV replication and infectivity, and they have helped assign functional roles for specific RNA structural elements (Diviney et al, 2008; Friebe and Bartenschlager, 2002; Friebe et al, 2005; Friebe et al, 2001; Kolykhalov et al, 2000; Lee et al, 2004; McMullan et al, 2007; Oakland et al, 2013; Vassilaki et al, 2008; You and Rice, 2008; You et al, 2004). However, these methodologies have been less successful in identifying and characterizing RNA structures that occur elsewhere in the HCV genome (Chu et al, 2013). …”

Section: Introductionmentioning

confidence: 99%

The Coding Region of the HCV Genome Contains a Network of Regulatory RNA Structures

et al. 2016

View full text Add to dashboard Cite

RNA is a versatile macromolecule that accommodates functional information in primary sequence, secondary and tertiary structure. We use a combination of chemical probing, RNA structure modeling, comparative sequence analysis and functional assays to examine the role of RNA structure in the hepatitis C virus (HCV) genome. We describe a set of conserved but functionally diverse structural RNA motifs that occur in multiple coding regions of the HCV genome, and we demonstrate that conformational changes in these motifs influence specific stages in the virus’s life cycle. Our study shows that these types of structures can pervade a genome, where they play specific mechanistic and regulatory roles, constituting a “code within the code” for controlling biological processes.

show abstract

“…Human hepatoma cell line (Huh-7) derivatives Huh-7.5, Huh-7.5.1 and Huh7-Lunet cell lines are commonly used for evaluating viral growth [11][12][13]15 . The peak viral production of JFH-1 strain in Huh7-Lunet cells is at 3-4 days post-transfection, whereas in Huh-7.5.1 cells it is at 21 days post-transfection 19,20 . For viral production from in vitro transcribed RNA, early passage human hepatoma cell line is preferred.…”

Section: Discussionmentioning

confidence: 94%

A Protocol for Analyzing Hepatitis C Virus Replication

Ren

Contreras

Arumugaswami

2014

JoVE

Self Cite

View full text Add to dashboard Cite

Hepatitis C Virus (HCV) affects 3% of the world's population and causes serious liver ailments including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points posttransfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle. Video LinkThe video component of this article can be found at

show abstract

Systematic Analysis of Enhancer and Critical cis -Acting RNA Elements in the Protein-Encoding Region of the Hepatitis C Virus Genome

Cited by 32 publications

References 72 publications

Functionally conserved architecture of hepatitis C virus RNA genomes

Functionally conserved architecture of hepatitis C virus RNA genomes

The Coding Region of the HCV Genome Contains a Network of Regulatory RNA Structures

A Protocol for Analyzing Hepatitis C Virus Replication

Contact Info

Product

Resources

About