Characterization of a virtually full-length human immunodeficiency virus type 1 genome of a prevalent intersubtype (C/B') recombinant strain in China

Abstract

A molecular epidemiology study was conducted among more than 100 human immunodeficiency virus type 1 (HIV-1) subtype C seropositive intravenous drug users (IDUs) from China. Genotyping based on the envelope C2V3 coding region revealed the highest homology of the most prevalent virus strains circulating throughout China to subtype C sequences of Indian origin. Based on these results, a virtually full-length genome representing the most prevalent class of clade C strains circulating throughout China was directly amplified from peripheral blood mononuclear cells of a selected HIV-infected IDU and subcloned. Sequence analysis identified a mosaic structure, suggesting extensive intersubtype recombination events between genomes of the prevalent clade C and (B')-subtype Thai virus strains of that geographic region. Recombinant Identification Program analysis and phylogenetic bootstrapping suggested that there were 10 breakpoints (i) in the gag-pol coding region, (ii) in vpr and at the 3' end of the vpu gene, and (iii) in the nef open reading frame. (B')-sequences therefore include (i) several insertions in the gag-pol coding region; (ii) 3'-vpr, the complete vpu gene, and the first exons of tat and rev; and (iii) the 5' half of the nef gene. Breakpoints located in the vpr/vpu coding region as well as in the nef gene of 97cn54 were found at almost identical positions of all subtype C strains isolated from IDUs living in different areas of China, suggesting a common ancestor for the C/B' recombinant strains. More than 50% of well-defined subtype B-derived cytotoxic T-lymphocyte epitopes within Gag and Pol and 10% of the known epitopes in Env were found to exactly match sequences within in this clade C/B' chimeric reference strain. These results may substantially facilitate a biological comparison of clade C-derived reference strains as well as the generation of useful reagents supporting vaccine-related efforts in China.

FIG. 1

Phylogenetic relationship of the env gene C2V3 coding region from clone 97cn54 with the representatives of the major HIV-1 (group M) subtypes. cn-con-c represents the env consensus sequence of HIV-1 subtype C strains prevalent in China. The phylogenetic tree was constructed using the neighbor-joining method. Values at the nodes indicate the percent bootstraps in which the cluster to the right was supported. Bootstraps of 70% and higher only are shown. Brackets on the right represent the major subtype sequences of HIV-1 group M.

FIG. 2

RIP (version 1.3) analysis of the complete gag-pol coding region of 97cn54 (window size, 200; threshold for statistical significance, 90%; gap handling, STRIP). Positions of the gag and pol open reading frames are indicated by arrows above the diagram. RIP analysis was based on background alignments using reference sequences derived from selected virus strains representing the most relevant HIV-1 subtypes. The standard representatives are marked by different colors as indicated. The x axis indicates the nucleotide positions along the alignment. The y axis indicates the similarity of 97cn54 to the listed reference subtypes.

FIG. 3

Phylogenetic relationship of different regions within the 97cn54-derived gag-pol reading frames to standard representatives of the major HIV-1 (group M) subtypes. Phylogenetic trees were constructed using the neighbor-joining method based on the following sequence stretches: nucleotides 1 to 478 (A), 479 to 620 (B), 621 to 1290 (C), 1291 to 1830 (D), 1831 to 2220 (E), 2221 to 2520 (F), and 2521 to 2971 (G). The indicated positions refer to the first nucleotide of the gag open reading frame. Grey areas highlight clustering of the analyzed sequences either with clade C-derived (A, C, E, and G) or with clade B-derived (B, D, and F) reference strains. Values at the nodes indicate the percent bootstraps in which the cluster to the right was supported. Bootstraps of 70% and higher only are shown.

FIG. 4

RIP analysis (version 1.3) of different regions of 97cn54 (window size, 200; threshold for statistical significance, 90%; Gap handling, STRIP). (A and B) The analysis included a sequence stretch of 1,500 bp from the start codon of the vif gene to the 5′ end of env, including vif, vpr, exon 1 of tat and rev, vpu, and the first 200 bp of env (A) and a ca. 700-bp fragment overlapping 300 bp from the 3′ end of env encompassing the complete nef gene and parts of the 3′ LTR (B). Positions of the start codons of vpr, tat, vpu, env, and nef, as well as the 5′ end of the 3′ LTR, are indicated by arrows above the diagrams. RIP analysis was based on background alignments using sequences derived from selected virus strains representing the most relevant HIV-1 subtypes. The indicated standard representatives are marked by different colors. The x axis indicates the nucleotide positions along the alignment. The y axis indicates the similarity of the 97cn54 to the listed reference subtypes. (C and D) RIP analysis of sequences from two independent clade C isolates (xj24 [C] and xj158 [D]) from China overlapping the vpr and vpu genes including the first exon of tat.

FIG. 5

Phylogentic tree analysis. Phylogenetic trees were constructed using the neighbor-joining method based on a 380-bp fragment overlapping the 3′ 150 bp of the vpr gene to the end of the vpu reading frame (A), based on the first 290 bp of the nef coding region (B), and based on the 3′ 320 bp of the nef gene (C). Values at the nodes indicate the percent bootstraps in which the cluster to the right was supported. Bootstraps of 70% and higher only are shown. Brackets on the right represent the major subtype sequences of HIV-1 group M.

FIG. 6

Schematic representation of the mosaic genome organization of 97cn54.

FIG. 7

Comparison between known and experimentally proven prototype B (HIV-1_LAI)-derived CTL epitopes and the corresponding amino acid sequences in the gag-, pol-, and env-encoded polypeptides of the clade C strain 97cn54. Functional domains in Gag (p17 matrix, p24 capsid, p15 nucleocapsid, and linker protein), Pol (protease [PR], RT, and integrase [IN]), and Env (gp120 external glycoprotein and gp41 transmembrane protein) are indicated. Numbers underneath the open reading frames indicate the amino acid position relative to the amino termini of the polypeptides. Haplotype restrictions of the known HIV-1_LAI-derived CTL epitopes are indicated in the left and right margins. Green bars represent sequence identity between the known epitope and the corresponding clade C sequence; blue bars indicate two or fewer conservative missmatches; red bars represent clade C-derived sequence stretches with more than two conservative missmatches or any nonconservative substitution compared to the corresponding LAI-derived epitope.