Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

An efficient model is currently used to study hepatitis C virus (HCV) replication in cell culture. It involves transfection in Huh7, a hepatoma-derived cell line, of an antibiotic (neomycin) selectable HCV subgenomic replicon encoding the non-structural (NS) proteins from NS3 to NS5B. However, strong and sustained replication is achieved only on the appearance of adaptive mutations in viral proteins. The most effective of these adaptive mutations are concentrated mainly in NS5A, not only into the original Con1 but also in the recently established HCV-BK and HCV-H77 isolate-derived replicons. This suggests that the expression of wild-type (wt) NS5A may not allow efficient HCV RNA replication in cell culture. With the use of a b-lactamase reporter gene as a marker for HCV replication and TaqMan RNA analysis, the replication of different HCV replicons in cotransfection experiments was investigated. Comparing wt with NS5A-adapted replicons, the strong evidence accumulated showed that the expression of wt NS5A was actually able to inhibit the replication of NS5A-adapted replicons. This feature was characterized as a dominant negative effect. Interestingly, an NS5B (R2884G)-adapted replicon, containing a wt NS5A, was dominant negative on an NS5A-adapted replicon but was not inhibited by the original Con1 replicon. In conclusion, these studies revealed that the original wt Con1 replicon is not only incompetent for replication in cell culture, but is also able to interfere with NS5A-adapted replicons.

Section: Methodsmentioning

confidence: 99%

Dominant negative effect of wild-type NS5A on NS5A-adapted subgenomic hepatitis C virus RNA replicon

Graziani¹,

Paonessa²

2004

“…NC_009823) contains one stemloop structure (stem-loop I, SLI) (Honda et al, 1999), and two microRNA 122 (miR-122) binding sites (S1 and S2) (Henke et al, 2008;Jopling, 2008;Jopling et al, 2005;Machlin et al, 2011). It has been shown that the SLI is important for viral RNA replication (Friebe et al, 2001;Luo et al, 2003), and we demonstrated that it is essential for the viability of infectious HCV genotype 1-6 recombinants (Li et al, 2011a). miR-122 is a liver-abundant miRNA (Chang et al, 2004), and it stimulates viral RNA replication and translation by directly binding to S1 and S2, as well as to upstream nucleotides, in the HCV 5¢UTR (Fig.…”

Section: Introductionmentioning

confidence: 99%

Functional analysis of microRNA-122 binding sequences of hepatitis C virus and identification of variants with high resistance against a specific antagomir

Pham

Uzcátegui

et al. 2016

MicroRNA 122 (miR-122) stimulates the replication and translation of hepatitis C virus (HCV) RNA by binding to two adjacent sites, S1 and S2, within the HCV 5¢UTR. We demonstrated previously that the miR-122 antagomir miravirsen (SPC3649) suppresses the infection of HCV strain JFH1-based recombinants with HCV genotypes 1-6 5¢UTR-NS2 in human hepatoma Huh7.5 cells. However, specific S1 mutations were permitted and conferred virus resistance to miravirsen treatment. Here, using the J6 (genotype 2a) 5¢UTR-NS2 JFH1-based recombinant, we performed reverse-genetics analysis of S1 (ACACUCCG, corresponding to miR-122 seed nucleotide positions 8-1), S2 (CACUCC, positions 7-2), and ACCC (positions 1-4) at the 5¢ end of the HCV genome (5¢E); the CC at positions 2-3 of 5¢E is involved in miR-122 binding. We demonstrated that the 5¢E required four nucleotides for optimal function, and that G or A at position 3 or combined GA at positions 2-3 of 5¢E was permitted. In S1 and S2, several single mutations were allowed at specific positions. A UCC fi CGA change at positions 4-3-2 of S1, S2, or both S1 and S2 (S1/S2), as well as a C fi G change at position 2 of S1/S2 were permitted. We found that 5¢E mutations did not confer virus resistance to miravirsen treatment. However, mutations in S1 and S2 induced virus resistance, and combined S1 and/or S2 mutations conferred higher resistance than single mutations. Identification of miR-122 antagomir resistance-associated mutations will facilitate the study of additional functions of miR-122 in the HCV life cycle and the mechanism of virus escape to host-targeting antiviral approaches.

“…1b). The extreme 59 end of the genome including domain I is not part of the IRES but is essential for replication (Rijnbrand et al, 1995;Friebe et al, 2001). Nine cloned variants corresponded to the consensus sequence and make up the prevalent quasispecies.…”

Section: Quasispecies Distribution In the 5 § Ntrmentioning

confidence: 99%

“…Since the HCV IRES contains cis-acting elements for translation as well as replication (Friebe et al, 2001;Reusken et al, 2003), we tested the stem-loop IIIa variant in selectable HCV replicons, which enable the study of viral RNA replication in cell culture (Lohmann et al, 1999). In these replicons the region encoding the structural proteins is replaced by a neomycin phosphotransferase gene, which upon expression confers resistance to G418.…”

Section: Effect Of Stem-loop Iiia Mutations On Replicationmentioning

confidence: 99%

“…This polyprotein is co-translationally and post-translationally cleaved into ten structural and non-structural proteins (Bartenschlager & Lohmann, 2000). Flanking the open reading frame at each end are a 59 and a 39 non-translated region (NTR), approximately 342 and 225 nt in length, which contain signals required for replication (Friebe et al, 2001;Friebe & Bartenschlager, 2002). Translation of the HCV open reading frame is mediated via the 59 NTR that includes a structural RNA element identified as an internal ribosomal entry site (IRES) (Tsukiyama-Kohara et al, 1992;Wang et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Evolution of naturally occurring 5′ non-translated region variants of hepatitis C virus genotype 1b in selectable replicons

Leeuwen

Reusken

Roeten

et al. 2004

Quasispecies shifts are essential for the development of persistent hepatitis C virus (HCV) infection. Naturally occurring sequence variations in the 59 non-translated region (NTR) of the virus could lead to changes in protein expression levels, reflecting selective forces on the virus. The extreme 59 end of the virus' genome, containing signals essential for replication, is followed by an internal ribosomal entry site (IRES) essential for protein translation as well as replication. The 59 NTR is highly conserved and has a complex RNA secondary structure consisting of several stem-loops. This report analyses the quasispecies distribution of the 59 NTR of an HCV genotype 1b clinical isolate and found a number of sequences differing from the consensus sequence. The consensus sequence, as well as a major variant located in stem-loop IIIa of the IRES, was investigated using self-replicating HCV RNA molecules in human hepatoma cells. The stem-loop IIIa mutation, which is predicted to disrupt the stem structure, showed slightly lower translation efficiency but was severely impaired in the colony formation of selectable HCV replicons. Interestingly, during selection of colonies supporting autonomous replication, mutations emerged that restored the base pairing in the stem-loop. Recloning of these altered IRESs confirmed that these second site revertants were more efficient in colony formation. In conclusion, naturally occurring variants in the HCV 59 NTR can lead to changes in their replication ability. Furthermore, IRES quasispecies evolution was observed in vitro under the selective pressure of the replicon system. INTRODUCTIONA major complication of hepatitis C virus (HCV) infection is its asymptomatic progression into chronic hepatitis, which can lead to cirrhosis of the liver and ultimately hepatocellular carcinoma and liver failure (Farci & Purcell, 2000). Therapy with interferon alpha and ribavirin, a guanoside analogue, provides the highest sustained virological response rates pivotal to the treatment for chronic HCV liver disease (Pawlotsky, 2003). A contributory factor for development of persistent infection of HCV is that its genome shows considerable genetic heterogeneity as a result of accumulation of mutations during viral RNA replication. This variability, which is characteristic of RNA viruses, results from the fact that the viral RNA polymerase lacks a proofreading activity needed for repair of misincorporated nucleotides during RNA synthesis. Mutations that occur during virus replication might confer selective advantages or disadvantages to the progeny virus, depending on varying local environment conditions including the presence of innate and adaptive immune response, antivirus treatment and availability of permissive cells. The existence of such a population of variants within a single individual is called a quasispecies (Farci & Purcell, 2000).HCV, which is a distinct member of the family Flaviviridae, has a positive-stranded RNA genome of~9600 nt that encodes a single long open reading frame of~3...