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Development of a new phenotypic assay based on the C2-V3 region of gp120 of Human Immunodeficiency Virus type 1 (HIV-1) for the determination of viral tropism


HIV-1 entry into the host cells is a complex process, involving HIV-1 envelope proteins; gp120 and gp41, a cellular receptor, CD4 and cellular coreceptor, either CCR5 or CXCR4. Several studies have associated different regions in env gene as the determinants of coreceptor tropism. But V3 region of gp120 is the major determinant of coreceptor specificity. New antiretroviral drugs targeting different aspects of HIV-1 entry have recently been approved. Maraviroc is the only commercially available CCR5 antagonist. Since CCR5 antagonist activity has only been demonstrated in patients harboring exclusively R5 variants, viral tropism determination has been strongly recommended before CCR5 antagonist prescription. Here a new phenotypic assay has been proposed based on 450 bp C2-V3 region of envelope gp120 of HIV-1. Using site directed mutagenesis technique, C2-V3 region of gp120 of pNL4-3 will be deleted to get the vector pNL4-3V3. 750 bp fragments will be amplified from patient samples encompassing deleted C2-V3 region with approximately 150 bp extensions on both sides to allow homologous recombination during transfection. PCR amplified patient samples and pNL4-3V3 will be cotransfected in 293-T cells to get the recombinant viral particles and then allowed to infect U373.MAGI.CD4 cell lines expressing either CCR5 or CXCR4 to determine tropism. This assay will further reduce the phenotypic tropism determining region to 450 bp C2-V3 region of gp120. TRT phenotypic assay developed by Trouplin et al (2001) will also be developed to validate our tropism assay.V3 regions of all the patients will also be sequenced and genotypic algorithms will then be used to predict tropism on the basis of these sequences. Results of the phenotypic and genotypic assays will be compared to find a correlation between them. (AU)

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(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
ARIF, MUHAMMAD SHOAIB; HUNTER, JAMES; LEDA, ANA RACHEL; LOPES ZUKUROV, JEAN PAULO; SAMER, SADIA; CAMARGO, MICHELLE; GALINSKAS, JULIANA; KALLAS, ESPER GEORGES; KOMNINAKIS, SHIRLEY VASCONCELOS; JANINI, LUIZ MARIO; et al. Pace of Coreceptor Tropism Switch in HIV-1-Infected Individuals after Recent Infection. Journal of Virology, v. 91, n. 19, . (11/17334-3)

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