scholarly journals Role of the Hepatitis C Virus Core+1 Open Reading Frame and Core cis-Acting RNA Elements in Viral RNA Translation and Replication

2008 ◽  
Vol 82 (23) ◽  
pp. 11503-11515 ◽  
Author(s):  
Niki Vassilaki ◽  
Peter Friebe ◽  
Philipe Meuleman ◽  
Stephanie Kallis ◽  
Artur Kaul ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Gene ◽  
Stem Loop ◽  
Reading Frame ◽  
Rna Translation ◽  
The Core ◽  
Rna Elements

ABSTRACT Four conserved RNA stem-loop structures designated SL47, SL87, SL248, and SL443 have been predicted in the hepatitis C virus (HCV) core encoding region. Moreover, alternative translation products have been detected from a reading frame overlapping the core gene (core+1/ARFP/F). To study the importance of the core+1 frame and core-RNA structures for HCV replication in cell culture and in vivo, a panel of core gene silent mutations predicted to abolish core+1 translation and affecting core-RNA stem-loops were introduced into infectious-HCV genomes of the isolate JFH1. A mutation disrupting translation of all known forms of core+1 and affecting SL248 did not alter virus production in Huh7 cells and in mice xenografted with human liver tissue. However, a combination of mutations affecting core+1 at multiple codons and at the same time, SL47, SL87, and SL248, delayed RNA replication kinetics and substantially reduced virus titers. The in vivo infectivity of this mutant was impaired, and in virus genomes recovered from inoculated mice, SL87 was restored by reversion and pseudoreversion. Mutations disrupting the integrity of this stem-loop, as well as that of SL47, were detrimental for virus viability, whereas mutations disrupting SL248 and SL443 had no effect. This phenotype was not due to impaired RNA stability but to reduced RNA translation. Thus, SL47 and SL87 are important RNA elements contributing to HCV genome translation and robust replication in cell culture and in vivo.

scholarly journals Expression of the Novel Hepatitis C Virus Core+1/ARF Protein in the Context of JFH1-Based Replicons

2015 ◽  
Vol 89 (9) ◽  
pp. 5164-5170 ◽  
Author(s):  
Ioly Kotta-Loizou ◽  
Ioannis Karakasiliotis ◽  
Niki Vassilaki ◽  
Panagiotis Sakellariou ◽  
Ralf Bartenschlager ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Gene ◽  
Open Reading Frame ◽  
The Novel ◽  
Reading Frame ◽  
The Core ◽  
Virus Core ◽  
First Time ◽  
Arf Protein

Hepatitis C virus contains a second open reading frame within the core gene, designated core+1/ARF. Here we demonstrate for the first time expression of core+1/ARF protein in the context of a bicistronic JFH1-based replicon and report the production of two isoforms, core+1/L (long) and core+1/S (short), with different kinetics.

scholarly journals 3′ RNA Elements in Hepatitis C Virus Replication: Kissing Partners and Long Poly(U)

2007 ◽  
Vol 82 (1) ◽  
pp. 184-195 ◽  
Author(s):  
Shihyun You ◽  
Charles M. Rice
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Tertiary Structure ◽  
Coding Region ◽  
Pyrimidine Nucleotides ◽  
Stem Loop ◽  
Nontranslated Region ◽  
Rna Elements ◽  
Kissing Loop

ABSTRACT The hepatitis C virus (HCV) genomic RNA possesses conserved structural elements that are essential for its replication. The 3′ nontranslated region (NTR) contains several of these elements: a variable region, the poly(U/UC) tract, and a highly conserved 3′ X tail, consisting of stem-loop 1 (SL1), SL2, and SL3. Studies of drug-selected, cell culture-adapted subgenomic replicons have indicated that an RNA element within the NS5B coding region, 5BSL3.2, forms a functional kissing-loop tertiary structure with part of the 3′ NTR, 3′ SL2. Recent advances now allow the efficient propagation of unadapted HCV genomes in the context of a complete infectious life cycle (HCV cell culture [HCVcc]). Using this system, we determine that the kissing-loop interaction between 5BSL3.2 and 3′ SL2 is required for replication in the genotype 2a HCVcc context. Remarkably, the overall integrity of the 5BSL3 cruciform is not an absolute requirement for the kissing-loop interaction, suggesting a model in which trans-acting factor(s) that stabilize this interaction may interact initially with the 3′ X tail rather than 5BSL3. The length and composition of the poly(U/UC) tract were also critical determinants of HCVcc replication, with a length of 33 consecutive U residues required for maximal RNA amplification. Interrupting the U homopolymer with C residues was deleterious, implicating a trans-acting factor with a preference for U over mixed pyrimidine nucleotides. Finally, we show that both the poly(U) and kissing-loop RNA elements can function outside of their normal genome contexts. This suggests that the poly(U/UC) tract does not function simply as an unstructured spacer to position the kissing-loop elements.

scholarly journals Synonymous Mutations in the Core Gene Are Linked to Unusual Serological Profile in Hepatitis C Virus Infection

PLoS ONE ◽  
2011 ◽  
Vol 6 (1) ◽  
pp. e15871 ◽  
Author(s):  
Agata Budkowska ◽  
Athanassios Kakkanas ◽  
Eric Nerrienet ◽  
Olga Kalinina ◽  
Patrick Maillard ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Infection ◽  
Core Gene ◽  
Hepatitis C Virus Infection ◽  
Synonymous Mutations ◽  
The Core ◽  
Serological Profile

scholarly journals Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core–NS2 and NS5A of genotypes 1–7

2013 ◽  
Vol 94 (10) ◽  
pp. 2221-2235 ◽  
Author(s):  
Andrea Galli ◽  
Troels K. H. Scheel ◽  
Jannick C. Prentoe ◽  
Lotte S. Mikkelsen ◽  
Judith M. Gottwein ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Current Knowledge ◽  
Particle Assembly ◽  
Adaptive Mutations ◽  
Culture Systems ◽  
The Core ◽  
Cell Culture Systems ◽  
Significant Difference

Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets (cLDs) or on the endoplasmic reticulum (ER) at different stages of particle assembly. Current knowledge on assembly and release is primarily based on studies in genotype 2a cell culture systems; however, given the high genetic heterogeneity of HCV, variations might exist among genotypes. Here, we developed novel HCV strain JFH1-based recombinants expressing core–NS2 and NS5A from genotypes 1–7, and analysed core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of core–NS2/NS5A recombinants and putative adaptive mutations were analysed by reverse genetics. Adapted core–NS2/NS5A recombinants produced infectivity titres of 102.5–104.5 f.f.u. ml−1. Co-localization analysis demonstrated that the core and NS5A proteins from all genotypes co-localized extensively, and there was no significant difference in protein co-localization among genotypes. In addition, we found that the core and NS5A proteins were highly associated with cLDs at 12 h post-infection but became mostly ER associated at later stages. Finally, we found that different genotypes showed varying levels of core/cLD co-localization, with a possible effect on viral assembly/release. In summary, we developed a panel of HCV genotype 1–7 core–NS2/NS5A recombinants producing infectious virus, and an immunostaining protocol detecting the core and NS5A proteins from seven different genotypes. These systems will allow, for the first time, investigation of core/NS5A interactions during assembly and release of HCV particles of all major genotypes.

scholarly journals Efficient Infectious Cell Culture Systems of the Hepatitis C Virus (HCV) Prototype Strains HCV-1 and H77

2014 ◽  
Vol 89 (1) ◽  
pp. 811-823 ◽  
Author(s):  
Yi-Ping Li ◽  
Santseharay Ramirez ◽  
Lotte Mikkelsen ◽  
Jens Bukh
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Adaptive Mutations ◽  
Culture Systems ◽  
Infectious Clones ◽  
Cell Culture Systems ◽  
A Genome

ABSTRACTThe first discovered and sequenced hepatitis C virus (HCV) genome and the firstin vivoinfectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77Cin vivoinfectious clones. We initially adapted a genome with the HCV-1 5′UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3′UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 104.0focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 103.5and 104.4FFU/ml, respectively.IMPORTANCEHepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but notin vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developedin vitroinfectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, thein vitroinfectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.

scholarly journals Cell Culture Adaptation of Hepatitis C Virus and In Vivo Viability of an Adapted Variant

2007 ◽  
Vol 81 (23) ◽  
pp. 13168-13179 ◽  
Author(s):  
Artur Kaul ◽  
Ilka Woerz ◽  
Philip Meuleman ◽  
Geert Leroux-Roels ◽  
Ralf Bartenschlager
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cultured Cells ◽  
Infectious Hepatitis ◽  
Adaptive Mutation ◽  
Viral Genomes ◽  
Adaptive Mutations ◽  
Spread Of Infection

ABSTRACT Production of infectious hepatitis C virus in cell culture has become possible because of the unique properties of the JFH1 isolate. However, virus titers are rather low, limiting the utility of this system. Here we describe the generation of cell culture-adapted JFH1 variants yielding higher titers of infectious particles and enhanced spread of infection in cultured cells. Sequence analysis of adapted genomes revealed a complex pattern of mutations that differed in two independent experiments. Adaptive mutations were observed both in the structural and in the nonstructural regions, with the latter having the highest impact on enhancement of virus titers. The major adaptive mutation was identified in NS5A, and it enhanced titers of three intergenotypic chimeras consisting of the structural region of a genotype 1a, 1b, or 3a isolate and the remainder of the JFH1 isolate. The mutation resides at the P3 position of the NS5A-B cleavage site and slows down processing, implying that subtle differences in replication complex formation appear to determine the efficiency of virus formation. Highly adapted JFH1 viruses carrying six mutations established a robust infection in uPA-transgenic SCID mice xenografted with human hepatocytes. However, the mutation in NS5A which enhanced virus titers in cell culture the most had reverted to wild type in nearly half of the viral genomes isolated from these animals at 15 weeks postinoculation. These results argue for some level of impaired fitness of this mutant in vivo.

A second-generation method of genotyping hepatitis C virus by the polymerase chain reaction with sense and antisense primers deduced from the core gene

1996 ◽  
Vol 57 (1) ◽  
pp. 31-45 ◽  
Author(s):  
Hiroaki Okamoto ◽  
Satoyuki Kobata ◽  
Hajime Tokita ◽  
Taisuke Inoue ◽  
Graeme D. Woodfield ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Second Generation ◽  
Core Gene ◽  
Chain Reaction ◽  
The Core ◽  
Polymerase Chain

scholarly journals cis-Acting RNA Signals in the NS5B C-Terminal Coding Sequence of the Hepatitis C Virus Genome

2004 ◽  
Vol 78 (20) ◽  
pp. 10865-10877 ◽  
Author(s):  
Haekyung Lee ◽  
Hyukwoo Shin ◽  
Eckard Wimmer ◽  
Aniko V. Paul
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Genome ◽  
Coding Region ◽  
Subgenomic Replicon ◽  
Stem Loop ◽  
Coding Sequence ◽  
Cis Acting ◽  
Rna Elements

ABSTRACT The cis-replicating RNA elements in the 5′ and 3′ nontranslated regions (NTRs) of the hepatitis C virus (HCV) genome have been thoroughly studied before. However, no cis-replicating elements have been identified in the coding sequences of the HCV polyprotein until very recently. The existence of highly conserved and stable stem-loop structures in the RNA polymerase NS5B coding sequence, however, has been previously predicted (A. Tuplin, J. Wood, D. J. Evans, A. H. Patel, and P. Simmonds, RNA 8:824-841, 2002). We have selected for our studies a 249-nt-long RNA segment in the C-terminal NS5B coding region (NS5BCR), which is predicted to form four stable stem-loop structures (SL-IV to SL-VII). By deletion and mutational analyses of the RNA structures, we have determined that two of the stem-loops (SL-V and SL-VI) are essential for replication of the HCV subgenomic replicon in Huh-7 cells. Mutations in the loop and the top of the stem of these RNA elements abolished replicon RNA synthesis but had no effect on translation. In vitro gel shift and filter-binding assays revealed that purified NS5B specifically binds to SL-V. The NS5B-RNA complexes were specifically competed away by unlabeled homologous RNA, to a small extent by 3′ NTR RNA, and only poorly by 5′ NTR RNA. The other two stem-loops (SL-IV and SL-VII) of the NS5BCR domain were found to be important but not essential for colony formation by the subgenomic replicon. The precise function(s) of these cis-acting RNA elements is not known.

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