Effect of Bottlenecking on Evolution of the Nonstructural Protein 3 Gene of Hepatitis C Virus during Sexually Transmitted Acute Resolving Infection

Abstract

Sexual partners of patients infected with the hepatitis C virus (HCV) often have detectable HCV-specific T-cell responses in the absence of seroconversion, suggesting unapparent, spontaneously resolving infection. To determine whether differences in the evolutionary potential of bottlenecked inoculum may explain the low rate of HCV persistence after sexual exposure, we have investigated changes in the entire HCV nonstructural 3 (NS3) gene over time in a chronic carrier and compared his viral quasispecies with that of the acute-phase isolate of his sexual partner, who developed acute resolving hepatitis C. The overall rate of accumulation of mutations, estimated by regression analysis of six consecutive consensus NS3 sequences over 8 years, was 1.5 ؋ 10 ؊3 mutations per site per year, with small intersample fluctuations related to changes in environmental conditions. Comparison of quasispecies parameters in one isolate of the chronic carrier with those of the acute-phase isolate of the infected partner revealed a higher heterogeneity and lower proportion of nonsynonymous mutations in the former. All NS3 sequences from the acute-phase isolate clustered with a single sequence from the chronic isolate, despite complete HLA mismatch between the patients, suggesting bottlenecking during transmission. The low risk of viral persistence after sexual exposure to HCV may be related to the selection of a limited number of viral particles carrying a particular combination of mutations which may further limit the potential of a relatively homogeneous quasispecies to rapidly diversify and overcome the immune response of the exposed host.

Genetic and catalytic efficiency structure of an HCV protease quasispecies

“…After incubation at 37°C for 6 hours, the resulting phage plaques were counted to score growth. In the experiments in which E. coli cells express the wild-type HCV I389/NS3-3 NS3/4 protease obtained from the subgenomic replicon, 33,34 phage replicated up to 6000-fold more efficiently than in cells that did not express the HCV NS3 2-181 /4 [21][22][23][24][25][26][27][28][29][30][31][32][33][34] construct.…”

“…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 .…”

“…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 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.…”

“…A smaller level of intraindividual amino acid diversity (less than 1%) has been reported for the NS3/4 protease coding region. 24 In the current work, we analyzed, at a high resolution (1%), the genotype and enzymatic activity of 3 HCV NS3/4 protease quasispecies. We determined the catalytic efficiency of each variant present in the quasispecies to establish the relationships between genotype, phenotype, and fitness.…”