Limits...
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.

Show MeSH

Related in: MedlinePlus

Boxplot illustrating the effect of RAM 184V on the NRTI FC in a subtype B and C backbone.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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3101197&req=5

pone-0019643-g005: Boxplot illustrating the effect of RAM 184V on the NRTI FC in a subtype B and C backbone.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.

Mentions: Clone 3 of Sample 3 (Table 1) enabled us to investigate the effect of a single RAM (M184V in RT) on the FC of viruses with the subtype C GPRT sequence inserted in the HIV-1 Subtype B and C backbones. In RT, a change at position 184 from methionine to valine results in an increase in FC for 3TC [11], [12] and FTC [13] while it decreases the FC for AZT, d4T and TDF [14]. This effect was observed with both types of backbone as shown in Fig. 5. The increase in FC is most pronounced and highly significant (p = <0.0001) for both 3TC and FTC and for both subtype backbones. The resensitizing effect for AZT was highly significant (p<0.0001) in both subtype backbones while it was significant (p<0.05) only in the subtype C backbone for d4T (pC = 0.046; pB = 0.2576) and TDF (pC = 0.0267; pB = 0.1257). FCs for ddI and ABC were not significantly affected by the presence of 184V.


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)

Boxplot illustrating the effect of RAM 184V on the NRTI FC in a subtype B and C backbone.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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101197&req=5

pone-0019643-g005: Boxplot illustrating the effect of RAM 184V on the NRTI FC in a subtype B and C backbone.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.
Mentions: Clone 3 of Sample 3 (Table 1) enabled us to investigate the effect of a single RAM (M184V in RT) on the FC of viruses with the subtype C GPRT sequence inserted in the HIV-1 Subtype B and C backbones. In RT, a change at position 184 from methionine to valine results in an increase in FC for 3TC [11], [12] and FTC [13] while it decreases the FC for AZT, d4T and TDF [14]. This effect was observed with both types of backbone as shown in Fig. 5. The increase in FC is most pronounced and highly significant (p = <0.0001) for both 3TC and FTC and for both subtype backbones. The resensitizing effect for AZT was highly significant (p<0.0001) in both subtype backbones while it was significant (p<0.05) only in the subtype C backbone for d4T (pC = 0.046; pB = 0.2576) and TDF (pC = 0.0267; pB = 0.1257). FCs for ddI and ABC were not significantly affected by the presence of 184V.

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