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.

HIV-1 Phenotypic Reverse Transcriptase Inhibitor Drug Resistance Test Interpretation Is Not Dependent on the Subtype of the Virus Backbone

“…The resulting HIV-1 genotype was used to select for resistant samples for this study. Secondly, a 1.9 kb GPRT fragment was amplified (One-Step SuperscriptIII High Fidelity, Invitrogen, CA, USA) using the “3′-RT” and “5′-OUT” primers [13] , with a 10 µl RNA input in a total volume of 35 µl. Nested PCR was performed using the Expand High Fidelity PCR System (Roche Diagnostics GmbH, Manheim, Germany), with 8 µl of first round amplicon and primers 3′IN, and 5′IN in a final volume of 100 µl [13] resulting in a final amplicon encompassing nucleotides 2012 to 3879 in pol (according to HXB2 numbering – genbank: AF033829).…”

“…Sequencing was performed using the Big Dye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems, CA, USA) as described previously [13] . Cycle sequencing purification was performed using the DyeEX (Qiagen, Hilden, Germany) purification kit, according to manufacturer guidelines.…”

“…(1998) [12] . Generation of the subtype C recombinant viruses was performed in MT4/eGFP cells [13] , whereas subtype B recombinant viruses were generated in MT4 cell lines, respectively using eGFP expression and Cytopathic Effect (CPE) scoring, respectively to monitor adequate viral growth. One hundred (100) µl of harvested subtype B and C recombinant viruses were then titrated in MT4/eGFP cells.…”

“…Wild type subtype B (HXB2) and C [13] cell line adapted viruses were used as reference viruses for the subtype B- and C- backbone experiments, respectively. All 50% inhibitory concentration (IC 50 ) values were calculated from 8 readings for each recombinant virus against the different ARV drugs.…”

“…Until recently, it remained, however, unclear whether a recombinant virus assay using a subtype B backbone would correctly measure drug resistance when the patient-derived sequences are of subtype C. The Antivirogram® assay [11] recombines patient-derived PR and RT sequences into an HIV-1 subtype B (HXB2) backbone deleted for these sequences [12] . Nauwelaers et al (2011) [13] constructed an HIV-1 subtype C-backbone within the Antivirogram® assay setting, and tested eight subtype C samples on a clonal level within both an HIV-1 subtype B- and C- backbone. Resistance profiles generated were similar in both backbones.…”