title: A synthetic HIV-1 subtype C backbone generates comparable PR and RT resistance profiles to a subtype B backbone in a recombinant virus assay.
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 were used to generate recombinant virus in a subtype B backbone. Phenotypic resistance profiles in a subtype B and subtype C backbone were compared. The following observations were made: i) functional, infectious HIV-1 subtype C viruses were generated, confirmed by VL and p24 measurements; ii) their rate of infection was slower than viruses generated in the subtype B backbone; iii) they did not produce clear CPE in MT4 cells; and iv) drug resistance profiles generated in both backbones were very similar, including re-sensitizing effects like M184V on AZT.
authors.affiliation: Virco BVBA, Beerse, Belgium.
authors.forename: David
authors.initials: D
authors.lastname: Nauwelaers
authors.forename: Margriet
authors.initials: M
authors.lastname: Van Houtte
authors.forename: Bart
authors.initials: B
authors.lastname: Winters
authors.forename: Kim
authors.initials: K
authors.lastname: Steegen
authors.forename: Kurt
authors.initials: K
authors.lastname: Van Baelen
authors.forename: Ellen
authors.initials: E
authors.lastname: Chi
authors.forename: Mimi
authors.initials: M
authors.lastname: Zhou
authors.forename: Derek
authors.initials: D
authors.lastname: Steiner
authors.forename: Rachelle
authors.initials: R
authors.lastname: Bonesteel
authors.forename: Colin
authors.initials: C
authors.lastname: Aston
authors.forename: Lieven J
authors.initials: LJ
authors.lastname: Stuyver
mesh_terms: D024882:Drug Resistance, Viral / Q000235:genetics*; D005838:Genotype; D054303:HIV Reverse Transcriptase / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D012367:RNA, Viral / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000627:therapeutic use; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase