Mutations in Hepatitis C Virus RNAs Conferring Cell Culture Adaptation

Abstract

As an initial approach to studying the molecular replication mechanisms of hepatitis C virus (HCV), a major causative agent of acute and chronic liver disease, we have recently developed selectable self-replicating RNAs. These replicons lacked the region encoding the structural proteins and instead carried the gene encoding the neomycin phosphotransferase. Although the replication levels of these RNAs within selected cells were high, the number of G418-resistant colonies was reproducibly low. In a search for the reason, we performed a detailed analysis of replicating HCV RNAs and identified several adaptive mutations enhancing the efficiency of colony formation by several orders of magnitude. Adaptive mutations were found in nearly every nonstructural protein but not in the 5 or 3 nontranslated regions. The most drastic effect was found with a single-amino-acid substitution in NS5B, increasing the number of colonies ϳ500-fold. This mutation was conserved with RNAs isolated from one cell line, in contrast to other amino acid substitutions enhancing the efficiency of colony formation to a much lesser extent. Interestingly, some combinations of these nonconserved mutations with the highly adaptive one reduced the efficiency of colony formation drastically, suggesting that some adaptive mutations are not compatible.The Hepatitis C virus (HCV) is a distinct member of the family Flaviviridae, comprising a group of enveloped viruses to which the flaviviruses, with the prototype Yellow fever virus, and the animal-pathogenic pestiviruses like Classical swine fever virus and Bovine viral diarrhea virus (BVDV) belong (41). These viruses have in common a single-stranded RNA genome of positive polarity carrying one long open reading frame (ORF) that is flanked at the 5Ј and 3Ј ends by nontranslated regions (NTRs). In HCV, the genome has a length of ϳ9,600 nucleotides and encodes a ϳ3,000-amino-acid-long polyprotein carrying the structural proteins in the amino-terminal quarter and the nonstructural (NS) proteins in the remainder (for reviews, see references 4 and 43).During and after translation, the polyprotein is cleaved in the structural region by host cell enzymes, and the NS proteins are cleaved by two viral proteinases, giving rise to at least 10 different products. These are arranged from the amino to the carboxy terminus as follows: core (C)-envelope protein 1 (E1)-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B (22, 23, 54). The amino-terminal products C, E1, and E2 are the major constituents of the virus particle, and they are released from the polyprotein precursor by host cell signal peptidases (23). The function of the small hydrophobic polypeptide p7 is unknown. NS2 and the amino-terminal domain of NS3 constitute the NS2-3 proteinase, responsible for polyprotein cleavage at the NS2/3 junction (20, 24). NS3 carries two enzymatic activities residing in two well-defined globular domains (62): a chymotrypsin-like serine proteinase spanning the ϳ180 aminoterminal NS3 residues, and nucleoside triphosphatase (NTPase)/helicase activ...

Genetic and catalytic efficiency structure of an HCV protease quasispecies

“…Nested PCR was then performed with a 5Ј oligonucleotide (HCVproL2) encoding an EcoRI site, residues 21 to 34 of NS4, and a dipeptide linker, Gly-Gly, along with residues 2 to 8 of NS3 (residues 3411 to 3431 of the HCV-J strain) (5Ј-GGGTTGAATTCTATG-GCTCCTATTGGATCTGTTGTTATTGTTGG AA-GAATTATTTTGTCTGGAAGAGGAGGACCTAT-CACGGCCTACTCCCAA-3Ј) and a 3Ј oligonucleotide (HCV proR) that was complementary to residues 3930 to 3950 of the HCV-J strain (amino acid residues 175 to 181 of NS3) and encoded an in-frame stop codon flanked by an XhoI site (5Ј-GGGAGGGGCTCGAGTCAA-GACCGCATAGTAGTTTCCAT-3Ј). The PCR products were digested with EcoRI and XhoI and ligated to pBSK-(Stratagene) to generate a ␤gal-HCV NS3 2-181 / 4 [21][22][23][24][25][26][27][28][29][30][31][32][33][34] protease fusion protein (pHCVNS3 2-181 /4 [21][22][23][24][25][26][27][28][29][30][31][32][33][34] protease). To ensure that multiple HCV NS3/4 protease templates were present in each analyzed quasispecies, for each sample, 40 different PCR amplifications were performed and pooled before cloning.…”

“…The catalytic efficiency of the different HCV NS3/4 proteases was determined using a bacteriophage lambda ( )-based genetic screen as previously described. 26,27 Escherichia coli JM109 cells containing plasmid pcI.HCVcro 27 that included the NS4B/NS5A (NEDCSTPCSGSWLRDVW) or the NS5A/NS5B (ASEDVVCCSMSYTWTGA) cleavage site were then transformed with plasmid pH-CVNS3 2-181 /4 [21][22][23][24][25][26][27][28][29][30][31][32][33][34] protease. The resulting cells were grown overnight at 30°C in the presence of 0.2% maltose, harvested by centrifugation, and resuspended to an optical density at 600 nm of 2.0 per ml in 10 mM MgSO 4 .…”

“…The resulting cells were grown overnight at 30°C in the presence of 0.2% maltose, harvested by centrifugation, and resuspended to an optical density at 600 nm of 2.0 per ml in 10 mM MgSO 4 . To induce the expression of HCV NS3 2-181 /4 [21][22][23][24][25][26][27][28][29][30][31][32][33][34] protease, cells (200 l) were incubated in 1 ml of Luria-Bertani medium containing 12.5 g tetracycline, 20 g ampicillin, 0.2% maltose, 10 mM MgSO 4 , and 1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) for 1 hour. Thereafter, cell cultures were infected with 10 7 PFU of phage.…”

Experimental models for hepatitis C viral infection

“…Introduction of these specific mutations in the wild-type consensus sequence significantly enhanced viral replication in vitro. [16][17][18][19][20] Mutational hot spots were found clustered primarily in the NS3, NS4B, and NS5A regions. The mechanisms behind the enhanced replication caused by these tissue-culture-adaptive mutations are still largely unknown, and the interesting fact that these mutations are not commonly found in patients suggests that these may have a toll on the viral fitness.…”

Hepatitis C virus comes full circle: Production of recombinant infectious virus in tissue culture

“…Interestingly, certain amino acid substitutions (i.e., cell culture-adaptive mutations) were found to increase the efficiency of replication by several orders of magnitude. 41,42 Such adaptive mutations cluster in certain regions, such as the central region of nonstructural protein 5A (NS5A), the C-terminal portion of the NS3 serine protease and the N-terminal portion of the NS3 RNA helicase domains as well as two positions in NS4B. 43 In addition, the efficiency of replicon RNA amplification was found to be determined by selection for particularly permissive cells within a given population of Huh-7 cells.…”