Adaptive Evolution of Human Immunodeficiency Virus-Type 1 During the Natural Course of Infection

Abstract

The rate of progression to disease varies considerably among individuals infected with human immunodeficiency virus-type 1 (HIV-1). Analyses of semiannual blood samples obtained from six infected men showed that a rapid rate of CD4 T cell loss was associated with relative evolutionary stasis of the HIV-1 quasispecies virus population. More moderate rates of CD4 T cell loss correlated with genetic evolution within three of four subjects. Consistent with selection by the immune constraints of these subjects, amino acid changes were apparent within the appropriate epitopes of human leukocyte antigen class I-restricted cytotoxic T lymphocytes. Thus, the evolutionary dynamics exhibited by the HIV-1 quasispecies virus populations under natural selection are compatible with adaptive evolution.

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.…”

“…S varies from 0 (no complexity) to 1 (maximum complexity). 31 To determine possible selective pressures, the proportion of synonymous substitutions per potential synonymous sites and the proportion of nonsynonymous substitutions per potential nonsynonymous sites was calculated with the WET software program (Windows Easy Tree software package, http://www.cnb.uam.es/ϳdopazo/software/ wet.html). To estimate codon-specific selection pressures, we used a maximum likelihood method implemented in the CODEML software program from the PAML version 3.14 software package.…”

“…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.…”

“…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.…”