HCV quasispecies evolution: association with progression to end-stage liver disease in hemophiliacs infected with HCV or HCV/HIV

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.…”

“…18,19 Early evolution of the viral quasispecies was shown to predict the clinical outcome of acute hepatitis C. 20,21 Likewise, the long-term viral evolution was shown to correlate with the severity of liver disease. 22,23 The HCV quasispecies has been well characterized for different viral genomic regions. The highest degrees of amino acid diversity within an infected individual (10%) 20 has been observed in variable regions of viral envelope genes (E1 and E2).…”

Hepatitis C virus quasispecies in HIV‐infected women

“…28,42,43 Similarly, narrowing of HVR1 complexity and diversity was associated with progressive liver disease in HCV/HIV coinfected hemophiliacs. 44 Despite the fact that HIV/HCV coinfection is common and may have grave clinical consequences, few studies have analyzed factors affecting HCV quasispecies in this setting. Two different studies found correlation between CD4ϩ cell count and HCV diversity, assessed as genetic distance, 29,30 but in a third study correlation was statistically not significant.…”