9 research outputs found
A Synthetic HIV-1 Subtype C Backbone Generates Comparable PR and RT Resistance Profiles to a Subtype B Backbone in a Recombinant Virus Assay
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
Scatter plots of FC of subtype C amplicons recombined in subtype B and subtype C backbones.
<p>(A–D) X-axis: Subtype B and Y-axis: subtype C for 1346 pairs; Black line x = y; (A) all drug classes (R<sup>2</sup> = 0.88); (B) NRTIs (R<sup>2</sup> = 0.88); (C) NNRTIs (R<sup>2</sup> = 0.90); (D) PIs (R<sup>2</sup> = 0.87); (E) Analysis of the pair-wise comparison of differences in FCs per clone and per drug, Ratio FC<sub>Subtype B</sub>/FC<sub>Subtype C</sub> (Average, Red squares) and P-value (Black diamonds).</p
Kinetics of HIV-1 subtype C virus production.
<p>A. viral load and pictures of fluorescence (day 4(1); day 6 (2); day 11 (3)); B: p24 measurements for the three clones of sample 4.</p
Experimental flow.
<p>Flow of the testing of the subtype C GPRT amplicons in the pGEM-HIV-1-C-Δgprt-BstEII-V (pHIV-1-C-Δgprt) and the pGEM-HXB2-Δgprt-BstEII (pHIV-1-B-Δgprt) backbones. “TRF”: transfection (Amaxa); “FC”: Fold Change; Red boxes: phenotypes; Green boxes: genotypes.</p
Boxplot illustrating the effect of RAM 184V on the NRTI FC in a subtype B and C backbone.
<p>Blue = HIV-1 subtype B backbone; Green = HIV-1 subtype C backbone; “+” = mutation 184V is present in RT; number under block = number of observed FC. P values have been calculated for each subtype for FC with mutation vs. FC without mutation.</p
Subcloning strategy of the vector containing the HIV-1 subtype C-Δgprt backbone.
<p>Fragment I (A) and Fragment II (B) were digested using BstEII and EcoRI and religated resulting in an HIV-1 subtype C clone lacking a part of GAG, protease and reverse Transcriptase and most of ENV (Fragment I-II (C)). Fragment I-II was linearized using PacI and AccIII to insert the Env region from Fragment III (D) resulting in a final clone, pGEM-HIV-1-C-Δgprt-BstEII-V, that can be linearized using BstEII/EcoRV, ready for In-Fusion cloning with the 1.7 kb GPRT amplicon. • pGEM-HIV-1-C-Δgprt-BstEII-V+GPRT (wild type sequence).</p