a synthetic hiv-1 subtype c backbone generates comparable pr and rt resistance profiles to a subtype b backbone in a recombinant virus assay合成hiv - 1 c亚型骨干产生类似的公关和rt电阻配置文件a亚型b骨干重组病毒化验.pdfVIP

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2017-09-07 发布于上海
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a synthetic hiv-1 subtype c backbone generates comparable pr and rt resistance profiles to a subtype b backbone in a recombinant virus assay合成hiv - 1 c亚型骨干产生类似的公关和rt电阻配置文件a亚型b骨干重组病毒化验.pdf

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a synthetic hiv-1 subtype c backbone generates comparable pr and rt resistance profiles to a subtype b backbone in a recombinant virus assay合成hiv - 1 c亚型骨干产生类似的公关和rt电阻配置文件a亚型b骨干重组病毒化验

A Synthetic HIV-1 Subtype C Backbone Generates Comparable PR and RT Resistance Profiles to a Subtype B Backbone in a Recombinant Virus Assay 1¤ 1 1 1 1 2 David Nauwelaers , Margriet Van Houtte , Bart Winters , Kim Steegen , Kurt Van Baelen , Ellen Chi , 2 2 2 2 1 Mimi Zhou , Derek Steiner , Rachelle Bonesteel , Colin Aston , Lieven J. Stuyver * 1Virco BVBA, Beerse, Belgium, 2 Centocor Research and Discovery, Centocor, San Diego, California, United States of America Abstract In order to determine phenotypic protease and reverse transcriptase inhibitor-associated resistance in HIV subtype C virus, we have synthetically constructed an HIV-1 subtype C (HIV-1-C) viral backbone for use in a recombinant virus assay. The in silico designed viral genome was divided into 4 fragments, which were chemically synthesized and joined together by conventional subcloning. Subsequently, gag-protease-reverse-transcriptase (GPRT) fragments from 8 HIV-1 subtype C- infected patient samples were RT-PCR-amplified and cloned into the HIV-1-C backbone (deleted for GPRT) using In-Fusion reagents. Recombinant viruses (1 to 5 per patient sample) were produced in MT4-eGFP cells where cyto-pathogenic effect (CPE), p24 and Viral Load (VL) were monitored. The resulting HIV-1-C recombinant virus stocks (RVS) were added to MT4- eGFP cells in the presence of serial dilutions of antiretroviral drugs (PI, NNRTI, NRTI) to determine the fold-change in IC50 compared to the IC50 of wild-type HIV-1 virus. Additionally, viral RNA was extracted from the HIV-1-C RVS and the amplified GPRT products we