Molecular phylogenetic analysis of human immunodeficiency virus type 1 strains obtained from Korean patients: env gene sequences.

Abstract

303 HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 subtypes have mainly been analyzed by nucleotide sequencing, by the DNA heteroduplex mobility assay, and by serotyping using synthetic peptides.5 Myers et al.6 have divided the subtypes into two groups, major (subtype A±J) and outlier (Group O), on the basis of the nucleotide sequences of the complete gag and env genes. These HIV-1 subtypes are generally confined to specific geographic locations. Because HIV-1 subtypes can be grouped on the basis of the nucleotide sequences of the V3 loop and its flanking regions C2±C3, analysis of the nucleotide sequences of these regions contributes to our understanding of the evolution and spread of HIV-1, in addition to providing data on prevention strategies for that population. Since the first case of HIV infection was reported in Korea in December 1985, the number of HIV-infected patients has increased annually and 647 cases of HIV infection have been reported as of March 31, 1997.7 Of the known cases of HIV infection, 37.7% are known to be infected through heterosexual contact abroad and the remainder are infected through in-country contact. To investigate the genetic diversity and epidemiological distribution of HIV-1 in Korea, we collected blood samples from 58 HIV-1-seropositive patients enrolled at the Center for AIDS Research (National Institute of Health [NIH], Seoul, Korea). The donors were from geographically different regions of South Korea. Of the subjects (whose sex ratio was 50:8, male:female), 23 patients were infected through heterosexual contact and 25 through homosexual contact. The remaining 10 patients were infected by blood transfusion or by using blood products. In the clinical aspects, the study subjects consisted of 21 patients with AIDS or AIDS related-complex and 37 with asymptomatic infection. Seven AIDS patients died during the course of the study. The clinical profile of each subject is presented in Table 1. HIV-1 DNA sequences encoding approximately 242 bp (nucleotides 6990±7234, HIVNL43 sequence) and 375 bp (nucleotides 6990±7367, HIVNL43 sequence) of the gp120 C2-toV3 region were amplified by nested PCR from uncultured peripheral blood mononuclear cells. The cDNAs were cloned into the pGEMT vector (Promega, Madison, WI) and an average of three clones from each sample were sequenced, except for samples KR4, KR10, KR11, KR15, KR16, and KR37. For the comparison of sequences of different size, the C2±V3 region (220 bp) in the 242-bp products was used for analysis, excluding the primer regions. The deduced amino acid sequences of the C2±V3 region of the env gene of HIV-1 from 58 Korean isolates and a consensus sequence derived from them are shown in Fig. 1. All of the isolates have cysteines at both ends of the V3 termini. At the crown of the V3 loop, eight different tetrameric sequences were found. GPGS and GPGR motifs were found in 45 (83%) isolates. Forty-four isolates among these, i.e., all except KR48, were clustered as subtype B. One remarkable finding is that a novel motif, RPRS, was found in three clones of KR45. This has never been reported before, and it belongs to subtype B. Other motifsÐ GPGQ (six cases), APGS (three cases), APGR, GPGG, and GPGKÐ were also found. All six cases with the GPGQ motif belonged to a non-B subtype. According to the Los Alamos Database, GPGQ is mainly found in subtype A, and also is distributed in subtypes E, C, F, B, and G.6 Phylogenetic analysis was carried out with nucleotide sequences from each representative clone of isolates by using parsimony and neighbor-joining methods included with the PHYLIP package.8 Phylogenetic analysis indicated that different clones in one patient never clustered as different subtypes (data not shown), and 51 of 58 isolates (88%) were clustered as subtype B (Fig. 2). Seven patients belonging to a non-B sub-