Impact of HIV-1 reverse transcriptase H221Y mutation on replication capacity of Y181C mutant viral strain
Objective To explore the development of Y181C and H221Y mutations among HIV-1 infected patients upon antiretroviral therapy in China and to investigate the impact of H221Y mutation on replication capacity of Y181C mutant.Methods Three HIV-1 subtype B' infected patients with Y181C and H221Y mutations upon highly active antiretroviral therapy from Henan province were enrolled in this study.Follow-up blood samples collected every six months from the patients after therapy between 2004 to 2006 were detected for viral load evaluation.Fragments of pol gene were amplified by nested PCR.The purified PCR products were ligated into pMD18-T vector to construct recombinant plasmids,which were further sequenced and compared with the HIV reference strain from Stanford HIVDB for analyzing the development of Y181C and H221Y mutations.Plasmids containing Y181C mutation alone and plasmids containing Y181C and H221Y double mutations were constructed and cotransfected respectively with envelope-expression plasmid pVSV-G into HEK 293T cells to obtain HIV-1 pseudotyped viruses.Replication capacity of each pseudotyped virus was analyzed after a single-cycle infection in TZM-bl cells.Results A total of 57 TA cloned sequences from six follow-up blood samples of three patients were obtained.Y181C mutation alone was found in 46 clones and Y181C and H221Y mutations were found in 35 clones.Moreover,H221Y mutation was only found in clones in which Y181C mutation had already developed.Between two pairs of pseudotyped virus from two patients,replication capacity of those viruses containing Y181C and H221Y mutations were 1.42 and 1.62 fold higher than the viruses containing Y181C mutation alone,respectively.Conclusion H221Y mutation might be a compensatory mutation of Y181C mutation occurred to improve the replication capacity of viral strains with Y181C mutant in HIV-1 subtype B' infected patients upon antiretroviral therapy.The finding suggests that it's significantly important to further perform the genotypic test for HIV-1 subtype B' drug resistance in order to guide antiretroviral treatment in clinical in China. 
 
Key words: 
HIV-1;  Y181C;  H221Y;  Replication capacity