Polymorphism of the Protease and Reverse Transcriptase and Drug Resistance Mutation Patterns of HIV-1 Subtype B Prevailing in China

To the Editor: HIV replication can be markedly inhibited by highly active antiretroviral therapy, whereas some mutations in the genes of reverse transcriptase (RT) and protease, which endow the virus with drug resistance ability, are considered a major cause of treatment failure. Although many studies have focused on characterization of the polymorphisms or drug resistance mutations of protease and RT gene, HIV-1 subtype B isolates of North America and Europe were studied in most cases. In this study, we analyzed the polymorphism of protease and RT gene in HIV-1 subtype B prevailing in China. The treatment-associated resistance mutation spectrum of HIV-1 RT gene during RT inhibitors (RTIs) therapy was also studied. The subjects of this survey were from 4 midland provinces of China (Anhui, Henan, Shanxi, and Sichuan) where the HIV-1 subtype B is mostly prevalent; all of the samples were collected in 2004. RNA was extracted from plasma samples, and the sequences of protease and RT were obtained by reverse transcription nested polymerase chain reaction and sequencing. All sequences belonging to subtype B were compared with the subtype B consensus sequence in Stanford HIV Drug Resistance Database (http://hivdb.stanford.edu) using the HIVdb software (version, 4.1.2). The χ2 tests or the Fisher exact tests were used to compare the differences of the proportion of the amino acid substitution at each position between different groups or to determine the correlations between different mutation positions. One hundred seventy-four sequences of protease and 197 sequences of RT from 197 subjects infected with HIV-1 subtype B were obtained. All of the 197 participants were of Han nationality, predominantly aged from 30 to 50 years (81.7%). The percentage of men was 50.8%. The large majority of patients indicated that they were most likely infected with HIV through blood donation (89.8%). All of the patients were treatment-naive of protease inhibitors (PIs). Seventy-one patients (36.0%) never received antiretroviral therapy; 126 other patients (64.0%) experienced RTIs therapy; 55 patients (27.9%) used a regimen of azidothymidine (AZT), didanosine (ddI), and nevirapine (NVP); 66 patients (33.5%) used a regimen of stavudine (d4T), ddI, and NVP; 4 patients (2.0%) used a regimen of AZT, lamivudine (3TC), and NVP; and 1 patient (0.5%) changed the regimen from d4T, ddI, and NVP to AZT, 3TC, and efavirenz (EFV). In this study, polymorphism is the variation from the consensus sequence within the subtype B, arbitrarily defined as position-specific amino acid differences occurring in more than 1% of protease or RT sequences isolated from those patients who are treatment-naive of PIs or RTIs. If the proportion of an amino acid is more than 50%, then this amino acid is considered to be the inherent amino acid at this position. We found that polymorphisms occurred at 27 codons (27.3%) of the total 99 amino acids of protease (Table 1); the proportions of amino acid substitution at positions 63 (100.0%), 77 (96.6%), 72 (94.3%), 93 (92.0%), and 35 (81.0%) were all more than 50%; the L63P (90.2%), V77I (96.6%), I72V (77.0%), I93L (91.4%), and E35D (79.9%) were the inherent amino acid of the protease of the HIV-1 subtype B strains prevailing in China compared with the strains prevailing in America and Europe. In addition, the proportions of amino acid substitution were between 20% and 50% at the positions 71 (32.2%) and 62 (20.7%).TABLE 1: The Incidence of Amino Acid Substitution in Protease of HIV-1 Subtype B Isolated from 174 HIV/AIDS Patients Never Treated with PIsPositions 63, 71, 77, and 93 are also the polymorphic positions in HIV-1 subtype B prevailing in America and Europe, and these positions are considered as the secondary drug resistance mutation positions. Previous studies indicated that, in America and Europe, about 55% of isolates have amino acid substitution (amino acid other than L) at positions 63, about 25% at positions 77 and 93, and about 5% at position 71 in untreated persons; in heavily treated patients, those proportions reached 90% at position 63, 40% at positions 77 and 93, and 70% at position 71.1,2 In this study, the proportions of L63P, V77I, and I93L in untreated patients were even higher than those in heavily treated patients described previously. In addition, the proportion of amino acid substitutions at protease position 71 was higher than that of subtype B prevailing in America and Europe. Although secondary mutations do not significantly alter the susceptibility of the PIs, these mutations, which exist before the emergence of a primary mutation, may alter the rate of development of drug resistance or the subsequent evolution of mutations.3 It is reported that a relatively poorer response to therapy was associated with high-baseline protease polymorphism and with the presence of I93L and A71V/T at baseline 4. Therefore, the high frequencies of baseline amino acid substitutions at positions 63, 71, 77, and 93 of protease in HIV-1 subtype B prevailing in China may have potential influence on the curative effects of PIs that will be introduced in antiretroviral therapy of China hereafter. In this study, 71 RT sequences were obtained from untreated patients. Seventy-four (24.7%) of the first 300 amino acid positions of RT were found to be polymorphic. The proportions of amino acid substitution at positions 278 (100.0%), 162 (95.8%), 200 (90.1%), 135 (83.1%), and 277 (74.2%) were more than 50%; the Q278E (100%), S162C (93.0%), T200A (81.7%), I135V (69.0%), and K277R (74.2%) were the inherent amino acid of RT. In addition, relatively high proportions of amino acid substitution were found at positions 211 (31.0%) and 293 (24.2%). All amino acid substitutions previously mentioned have not been reported to be associated with the RTIs resistance, except for position 135. Previous study indicated that those strains containing the I135M/L/T substitution in combination with the L283I substitution showed decreases in susceptibility to all 3 of the nonnucleoside reverse transcriptase inhibitors (NNRTIs).5 In our study, although the total proportion of I135L/T is 9.8%, the polymorphism at position 135 would not influence the susceptibility of HIV-1 to drugs because there was no L238I existing together with I135L/T. In this study, only 3 isolates from 71 treatment-naive patients harbored 3 nucleoside reverse transcriptase inhibitors (NRTIs) resistance-associated mutations (A62V, Y115F, and T215A); no NNRTIs resistance mutation was found. These results demonstrated that the frequency of baseline RTIs resistance mutations was very low among these treatment-naive patients. In the RT sequences from 126 patients undergoing RTIs therapy, we found 6 kinds of NRTIs resistance mutations at 4 amino acid positions: K65R (3.2%), D67G (1.6%), D67N (1.6%), L74V (4.8%), T215F (0.8%), and T215Y (2.4%). In contrast, we found 10 kinds of NNRTIs resistance mutations at 7 amino acid positions: K101E (2.4%), K103N (34.1%), K103S (1.6%), V106A (4.0%), Y181C (23.8%), Y188C (0.8%), Y188L (0.8%), G190A (15.1%), G190S (1.6%), and K238T (1.6%). Notably, the proportions of K103N, Y181C, and G190A mutations were significantly higher in treated patients than in untreated patients (P = 0.000, 0.000, and 0.001, respectively). These results indicated that the emergence of NNRTIs resistance mutations is also an important problem during antiretroviral therapy in China. To find whether there were novel mutations in RT associated with anti-HIV therapy, the proportions of amino acid substitution at each mutation position were compared between RTI-treated and untreated groups. We found that position 221 was highly conserved in HIV-1 isolates from untreated group (proportion, 0/71), whereas amino acid substitution at this position occurred in treated patients with higher proportion (14/126). Moreover, as the most frequent amino acid substitution of this position, the proportion of H221Y was significantly higher in RTI-treated patients (13/126 [10.3%]) than that in untreated patients (P = 0.012), which indicated that H221Y mutation was associated with RTIs treatment. Furthermore, we compared the proportions of H221Y between 2 groups with and without K103N or Y181C mutations-the 2 most frequent drug resistance mutations in RT after RTIs treatment in our study. The proportion of H221Y in the group with Y181C was significantly higher than that in the group without Y181C (40.0% vs 1.0%, P = 0.000). However, there was no significant difference in proportion of H221Y between 2 groups with and without K103N (14.0% vs 8.4%, P = 0.511). H221Y is a novel therapy-related mutation first reported by Gonzales6 in 2003. Gonzales et al proposed that H221Y was associated with NRTI therapy. But this proposition is discrepant with the results of a recent study in which the H221Y was indicated to be associated with NNRTI resistance.7 In our study, the H221Y mutation was found to be correlated with Y181C mutation (ø = 0.545), whereas Y181C has been reported to be NNRTIs resistance mutation; thus, we speculate that the H221Y mutation is more likely associated with the NNRTIs resistance. In summary, there are inherent amino acids at some positions of protease and RT of HIV-1 subtype B strains prevailing in China compared with the strains in America and Europe. As the secondary PIs resistance mutations, the high frequencies of L63P, V77I, and I93L may have potential influence on the curative effect of PIs that will be used in China. The mutations K103N, Y181C, and G190A were the 3 most prevalent drug resistance mutations after antiretroviral therapy in China, and H221Y was a novel mutation more likely associated with the NNRTI therapy. Shulin Jiang, PhD Hui Xing, MSc Xuefeng Si, MSc Yu Wang, MSc Yiming Shao, PhD Division of Research on Virology and Immunology, State Key Laboratory for Infectious Disease Prevention and Control (Chinese Center for Disease Control and Prevention), National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing People's Republic of China