Genotyping and phenotyping of Human Immunodeficiency Virus Type 1 by Protease Inhibitor Resistance Testing

• Main Authors: Wang Sheng Fan, 王聖帆
• Other Authors: Wu-Tse Liu
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/72689479563360945637

碩士 === 國立陽明大學 === 醫學生物技術研究所 === 89 === Human immunodeficiency Virus type1 (HIV-1) is the major pathogen known for causing acquired immunodeficiency syndrome (AIDS). Development of antiretroviral therapy has been focus on inhibitors of viral enzyme including the reverse transcriptase and viral protease. Recently a great advance has been made against HIV-1 by combination antiretroviral therapy, however , there appears failure of the treatment with certain reasons. One of the important reasons is the occurrence of drug-resistant quasispecies during the therapy, especially that resistant to protease inhibitors (PI) is a major obstacle. Information on the genetic subtypes of HIV-1 is also important for understanding global evolution and this has an impact on drug susceptibility, on the determination of drug resistance mutations and measurement of viral load as well . In this study, we determined the HIV-1 subtypes in Taiwan by vpu gene analysis, the vpu genes were amplified by nested PCR of proviral DNA extracted from 119 HIV-1- infected individuals and the product were directly sequenced. The result showed that most strains were clustered in subtype B [ 89/119 (74.4%) ] ,subtype E [ 26/119 (21.8%)] and a few strains were clustered in subtype C [4/119 (3.36%)]. In this study, the RT-PCR method was used to analyze the prevalence of mutations occurred in HIV-1 protease genes in 83 HIV-1 infected patients which were treated with at least one regimen containing a protease inhibitor ( Saqiunavir, Ritonavir, Indinavir ). The result showed that most frequent PR mutations were at L63P(95%), M36I(44%), K20R(18%), A71V(12%), G73S(10%), L90M(7%), L10I(5%), I54V(4%), V82A(3%), I84V(3%), A77V(1%). There were two special cases with 7 or 10 PI-resistant associated mutations which were all stepwisely accumulated in 1~4 years. A region in HIV protease substrate (p17/p24) with mutations associated with PI-resistance was cloned into an E.coli expression vector , the pET43b .The full length of the expressed substrate precursor is predicted at the size of 81.8KD, which would be subjected to cis-cleavage, resulting in a 12.2KD band as analyzed by a western blot. In the presence of the PI, the drug susceptibility conferred by each mutation can be monitored by the intensity of the band representing substrate precursor. The mutant M36I, A71V, L10V+L63P+A71V+V77I were showed more active proteolytic function and higher resistance than wild type in 62.5uM PI. The mutant V82A, L90M were showed lower cleavage function and resistance than wild type. In summary, this system PRS-PR can express functionally in E.coli and it can be used to study drug resistant associated mutations in viral protease.