Phenotypic Analysis of HIV-1 Genotypic Drug-Resistant Isolates from China, Using a Single-Cycle System

AbstractBackground and Objectives: Drug resistance in HIV-1 is one of the main causes of failure of antiretroviral therapy. Phenotypic detection of drug-resistant HIV-1 can provide guidance in selecting the optimal treatment regimen. Traditional phenotype assays are labor intensive and time consuming. Thus, a rapid and convenient phenotype assay with a single cycle of replication was developed and used in this study. Methods: Two restriction endonuclease sites, ApaI and AgeI, were inserted into the plasmid pSG3,DΔenv using site-directed mutagenesis. The reverse transcriptase and protease genes of HIV-1 were amplified from patients and cloned into the modified pSG3Δenv. Sixteen original recombinant pseudoviruses were generated. The phenotypic susceptibility of these 16 recombinant pseudoviruses to 12 antiretro viral drugs was determined using a luciferase reporter system, and the phenotype and genotype results were compared. Results: A modified phenotype assay with a single-cycle system was established, and its reproducibility and feasibility were validated. Approximately 89% of the phenotype results were in agreement with the genotype results; this slight disagreement may have been due to complex and multiple resistance mutations. The phenotype results showed that individual pseudoviruses with four thymidine analog mutations (TAMs) [M41L, T67N, L210W, and T215Y] in combination with various other mutations had different levels of resistance to nucleoside reverse transcriptase inhibitors (NRTIs). Mutations E44A, T69D, and V118I influenced the pattern of resistance of TAMs. The level of resistance to non-NRTIs (NNRTIs) was also variable when different NNRTI-resistance mutations were combined. Conclusion: The single-cycle pseudovirus phenotypic susceptibility detection system reflects HIV-1 drug resistance, especially for complex resistance mutants, and could be used to screen new antiretroviral candidates.

[1]  D. Pillay,et al.  What is the risk of mortality following diagnosis of multidrug-resistant HIV-1? , 2008, The Journal of antimicrobial chemotherapy.

[2]  D. Ho,et al.  Isolation and Quantitation of HIV in Peripheral Blood , 1993, Current protocols in immunology.

[3]  L. Galli,et al.  The 118I Reverse Transcriptase Mutation Is the Only Independent Genotypic Predictor of Virologic Failure to a Stavudine-Containing Salvage Therapy in HIV-1-Infected Patients , 2006, Journal of acquired immune deficiency syndromes.

[4]  B. Marchand,et al.  Mutations E44D and V118I in the Reverse Transcriptase of HIV-1 Play Distinct Mechanistic Roles in Dual Resistance to AZT and 3TC* , 2003, Journal of Biological Chemistry.

[5]  Lynn Morris,et al.  Impact of HIV-1 Subtype and Antiretroviral Therapy on Protease and Reverse Transcriptase Genotype: Results of a Global Collaboration , 2005, PLoS medicine.

[6]  Robert W. Shafer,et al.  Algorithm Specification Interface for Human Immunodefiency Virus Type 1 Genotypic Interpretation , 2003, Journal of Clinical Microbiology.

[7]  Christos J. Petropoulos,et al.  A Novel Phenotypic Drug Susceptibility Assay for Human Immunodeficiency Virus Type 1 , 2000, Antimicrobial Agents and Chemotherapy.

[8]  R. Kutner,et al.  Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors , 2009, Nature Protocols.

[9]  K. Hong,et al.  Genetic and Neutralization Properties of HIV-1 env Clones From Subtype B/BC/AE Infections in China , 2008, Journal of acquired immune deficiency syndromes.

[10]  J. Schapiro,et al.  Genetic variation at NNRTI resistance-associated positions in patients infected with HIV-1 subtype C , 2004, AIDS.

[11]  J. Gallant.  Antiretroviral drug resistance and resistance testing. , 2005, Topics in HIV medicine : a publication of the International AIDS Society, USA.

[12]  L. Mofenson,et al.  Identification of the K103N resistance mutation in Ugandan women receiving nevirapine to prevent HIV-1 vertical transmission , 2000, AIDS.

[13]  Matthew J. Gonzales,et al.  Human Immunodeficiency Virus Reverse Transcriptase and Protease Sequence Database: an expanded data model integrating natural language text and sequence analysis programs , 2001, Nucleic Acids Res..

[14]  Thomas Lengauer,et al.  Characterization and Structural Analysis of Novel Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Involved in the Regulation of Resistance to Nonnucleoside Inhibitors , 2007, Journal of Virology.

[15]  Martin A. Nowak,et al.  Antibody neutralization and escape by HIV-1 , 2003, Nature.

[16]  N. Ford,et al.  Scaling up of highly active antiretroviral therapy in a rural district of Malawi: an effectiveness assessment , 2006, The Lancet.

[17]  J. Alcamí,et al.  A new strategy based on recombinant viruses as a tool for assessing drug susceptibility of human immunodeficiency virus type 1 , 2007, Journal of medical virology.

[18]  R. Siliciano,et al.  Novel Single-Cell-Level Phenotypic Assay for Residual Drug Susceptibility and Reduced Replication Capacity of Drug-Resistant Human Immunodeficiency Virus Type 1 , 2004, Journal of Virology.

[19]  Colombe Chappey,et al.  Broad nucleoside reverse-transcriptase inhibitor cross-resistance in human immunodeficiency virus type 1 clinical isolates. , 2003, The Journal of infectious diseases.

[20]  J. Kappes,et al.  Emergence of Resistant Human Immunodeficiency Virus Type 1 in Patients Receiving Fusion Inhibitor (T-20) Monotherapy , 2002, Antimicrobial Agents and Chemotherapy.

[21]  A. Mohammadpour,et al.  HIV-infected patients' adherence to highly active antiretroviral therapy: a phenomenological study. , 2010, Nursing & health sciences.

[22]  Brendan Larder,et al.  A Rapid Method for Simultaneous Detection of Phenotypic Resistance to Inhibitors of Protease and Reverse Transcriptase in Recombinant Human Immunodeficiency Virus Type 1 Isolates from Patients Treated with Antiretroviral Drugs , 1998, Antimicrobial Agents and Chemotherapy.

[23]  A. Vandamme,et al.  Novel Recombinant Virus Assay for Measuring Susceptibility of Human Immunodeficiency Virus Type 1 Group M Subtypes To Clinically Approved Drugs , 2009, Journal of Clinical Microbiology.

[24]  J. Kappes,et al.  Sensitivity of Human Immunodeficiency Virus Type 1 to the Fusion Inhibitor T-20 Is Modulated by Coreceptor Specificity Defined by the V3 Loop of gp120 , 2000, Journal of Virology.

[25]  B. G. Gutsche.  Intrapartum Exposure to Nevirapine and Subsequent Maternal Responses to Nevirapine-Based Antiretroviral Therapy , 2005 .

[26]  D. Katzenstein,et al.  Drug resistance in plasma and breast milk after single-dose nevirapine in subtype C HIV type 1: population and clonal sequence analysis. , 2007, AIDS research and human retroviruses.

[27]  H. Vahaboğlu,et al.  Activities of cefepime and five other antibiotics against nosocomial PER-1-type and/or OXA-10-type beta-lactamase-producing Pseudomonas aeruginosa and Acinetobacter spp. , 1998, The Journal of antimicrobial chemotherapy.

[28]  R. Shafer,et al.  Genotypic predictors of human immunodeficiency virus type 1 drug resistance , 2006, Proceedings of the National Academy of Sciences.

[29]  R. Siliciano,et al.  New approaches for quantitating the inhibition of HIV-1 replication by antiviral drugs in vitro and in vivo , 2009, Current opinion in infectious diseases.

[30]  Y. Takeuchi,et al.  Identification of Gammaretroviruses Constitutively Released from Cell Lines Used for Human Immunodeficiency Virus Research , 2008, Journal of Virology.

[31]  B. Masquelier,et al.  Resistance mutations in subtype C HIV type 1 isolates from Indian patients of Mumbai receiving NRTIs plus NNRTIs and experiencing a treatment failure: resistance to AR. , 2007, AIDS research and human retroviruses.