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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.

Nauwelaers D, Van Houtte M, Winters B, Steegen K, Van Baelen K, Chi E, Zhou M, Steiner D, Bonesteel R, Aston C, Stuyver LJ - PLoS ONE (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Virco BVBA, Beerse, Belgium.

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.

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Subcloning strategy of the vector containing the HIV-1 subtype C-Δgprt backbone.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).
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pone-0019643-g001: Subcloning strategy of the vector containing the HIV-1 subtype C-Δgprt backbone.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).

Mentions: The final design of the subtype C sequence was divided into 4 fragments (flanked by EcoRI and BamHI restriction sites for cloning purposes), three of which were destined for synthesis (Fig. 1, fragments I, II, III). The synthesis of the 3 DNA fragments was performed at Centocor, CA, USA [9], [10] as follows: padded sequences were parsed into contiguous segments of equal length on both the forward and reverse strands; each segment was chemically synthesized as an oligonucleotide using GENEWRITER™ instrumentation (Centocor) and purified by reversed phase HPLC (Dionex, Sunnyvale, CA); purified oligonucleotides were assembled using proprietary gene assembly technology (Centocor, [9], [10] and cloned into a pGEM-3z vector (2743 bp) using EcoRI and BamHI (Fig. 1). Vector Fragment-I (Fig. 1-A) contained an EcoRI-BamHI flanking fragment of HIV-1 5′-LTR and GAG, as well as an inserted BstEII restriction site and a small downstream part of POL (2205 bp). Vector Fragment-II (Fig. 1-B) contained an EcoRI-BamHI flanking fragment of HIV-1 GAG, as well as an inserted BstEII restriction site, the 3′ part of POL, a fragment of ENV (mostly deleted and replaced with a NotI-containing sequence) and the 3′-LTR (3460 bp). Vector Fragment-III (Fig. 1-D) contained an EcoRI-BamHI flanking fragment of the complete HIV-1 ENV and the upstream part of the 3′LTR (3412 bp). While the V3 envelope region of AB023804 was predicted to be R5-tropic according to the Geno2Pheno prediction tool (http://coreceptor.bioinf.mpi-inf.mpg.de/index.php) and Position Specific Scoring Matrices (PSSM, http://indra.mullins.microbiol.washington.edu), an R4-tropic virus was needed for the transfection assay in MT4 host cells. An envelope sequence retrieved from Los Alamos (subtype C clone C.ZA.01.01ZARP1) was predicted to be X4-tropic and was used to design Vector Fragment-III (Fig. 1-D) The fragment containing the protease and reverse transcriptase region was not synthesized but PCR-amplified from clinical samples as described above.


A synthetic HIV-1 subtype C backbone generates comparable PR and RT resistance profiles to a subtype B backbone in a recombinant virus assay.

Nauwelaers D, Van Houtte M, Winters B, Steegen K, Van Baelen K, Chi E, Zhou M, Steiner D, Bonesteel R, Aston C, Stuyver LJ - PLoS ONE (2011)

Subcloning strategy of the vector containing the HIV-1 subtype C-Δgprt backbone.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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101197&req=5

pone-0019643-g001: Subcloning strategy of the vector containing the HIV-1 subtype C-Δgprt backbone.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).
Mentions: The final design of the subtype C sequence was divided into 4 fragments (flanked by EcoRI and BamHI restriction sites for cloning purposes), three of which were destined for synthesis (Fig. 1, fragments I, II, III). The synthesis of the 3 DNA fragments was performed at Centocor, CA, USA [9], [10] as follows: padded sequences were parsed into contiguous segments of equal length on both the forward and reverse strands; each segment was chemically synthesized as an oligonucleotide using GENEWRITER™ instrumentation (Centocor) and purified by reversed phase HPLC (Dionex, Sunnyvale, CA); purified oligonucleotides were assembled using proprietary gene assembly technology (Centocor, [9], [10] and cloned into a pGEM-3z vector (2743 bp) using EcoRI and BamHI (Fig. 1). Vector Fragment-I (Fig. 1-A) contained an EcoRI-BamHI flanking fragment of HIV-1 5′-LTR and GAG, as well as an inserted BstEII restriction site and a small downstream part of POL (2205 bp). Vector Fragment-II (Fig. 1-B) contained an EcoRI-BamHI flanking fragment of HIV-1 GAG, as well as an inserted BstEII restriction site, the 3′ part of POL, a fragment of ENV (mostly deleted and replaced with a NotI-containing sequence) and the 3′-LTR (3460 bp). Vector Fragment-III (Fig. 1-D) contained an EcoRI-BamHI flanking fragment of the complete HIV-1 ENV and the upstream part of the 3′LTR (3412 bp). While the V3 envelope region of AB023804 was predicted to be R5-tropic according to the Geno2Pheno prediction tool (http://coreceptor.bioinf.mpi-inf.mpg.de/index.php) and Position Specific Scoring Matrices (PSSM, http://indra.mullins.microbiol.washington.edu), an R4-tropic virus was needed for the transfection assay in MT4 host cells. An envelope sequence retrieved from Los Alamos (subtype C clone C.ZA.01.01ZARP1) was predicted to be X4-tropic and was used to design Vector Fragment-III (Fig. 1-D) The fragment containing the protease and reverse transcriptase region was not synthesized but PCR-amplified from clinical samples as described above.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Virco BVBA, Beerse, Belgium.

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.

Show MeSH
Related in: MedlinePlus