Limits...
A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

View Article: PubMed Central

ABSTRACT

Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1.

No MeSH data available.


Synthesis of catalytically active HIV-1 PR using the wheat germ cell-free system. (A) Schematic representation of rapid production of HIV-1 PR using a wheat germ cell-free system. A transcriptional template, including the HIV-1 PR open reading frame flanked by N-terminal 168 bases (56 aa) and C-terminal 54 bases (18 aa), produced by split-primer PCR as described in section “Materials and Methods.” Cell-free translation was carried out using products of in vitro transcription. (B) The formation of WT PR (catalytic active) or D25N PR (catalytic inactive) after translation. WT PR generated as a 11 kDa protein by self-cleavage. D25N PR (catalytic inactive) generated as a 19 kDa protein lacking self-cleavage activity. (C,D) WT or D25N PRs (-mRNA and DHFR as a negative control) were separated by SDS-PAGE followed by CBB-staining (C) and immunoblotting using anti-HIV-1 PR antibody that recognizes only mature HIV protease (PR) but not its precursor (D). Arrows depict protein products.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4628118&req=5

Figure 1: Synthesis of catalytically active HIV-1 PR using the wheat germ cell-free system. (A) Schematic representation of rapid production of HIV-1 PR using a wheat germ cell-free system. A transcriptional template, including the HIV-1 PR open reading frame flanked by N-terminal 168 bases (56 aa) and C-terminal 54 bases (18 aa), produced by split-primer PCR as described in section “Materials and Methods.” Cell-free translation was carried out using products of in vitro transcription. (B) The formation of WT PR (catalytic active) or D25N PR (catalytic inactive) after translation. WT PR generated as a 11 kDa protein by self-cleavage. D25N PR (catalytic inactive) generated as a 19 kDa protein lacking self-cleavage activity. (C,D) WT or D25N PRs (-mRNA and DHFR as a negative control) were separated by SDS-PAGE followed by CBB-staining (C) and immunoblotting using anti-HIV-1 PR antibody that recognizes only mature HIV protease (PR) but not its precursor (D). Arrows depict protein products.

Mentions: To synthesize catalytically active HIV-1 PR, we initially generated a transcriptional template of this enzyme by PCR, using the HIV-1NL4-3 clone as a wild-type (WT) reference sample. We designed a transcription template encoding the open reading frame of HIV-1 PR flanked by 56 N-terminal amino acids (Gag p6 region) and 18 C-terminal amino acids (the reverse-transcriptase region), as shown in Figure 1A. The in vitro transcription template for the HIV-1 PR gene was constructed by split-primer PCR using primers encoding the SP6 and E01 sites, as described in section “Materials and Methods.” To generate a catalysis-incompetent PR, we designed a PR mutant harboring the catalytic active site substitution D25N (D25N; Figure 1B). All cDNA templates were subjected to cell-free transcription/translation and then separated by SDS-PAGE. By CBB staining, WT PR migrated at 11 kDa (as the truncated form of PR) due to self-cleavage of the flanking 56 and 18 amino acids at the N- and C-terminal ends, respectively, whereas D25N PR migrated at 19 kDa, corresponding to full-length PR with the flanking sequences (Figures 1C,D). Immunoblot analysis with anti-HIV-1 PR antibody recognizing mature form of HIV PR, only detected the cleaved form of WT PR at the expected size (∼11 kDa; Figure 1D).


A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors
Synthesis of catalytically active HIV-1 PR using the wheat germ cell-free system. (A) Schematic representation of rapid production of HIV-1 PR using a wheat germ cell-free system. A transcriptional template, including the HIV-1 PR open reading frame flanked by N-terminal 168 bases (56 aa) and C-terminal 54 bases (18 aa), produced by split-primer PCR as described in section “Materials and Methods.” Cell-free translation was carried out using products of in vitro transcription. (B) The formation of WT PR (catalytic active) or D25N PR (catalytic inactive) after translation. WT PR generated as a 11 kDa protein by self-cleavage. D25N PR (catalytic inactive) generated as a 19 kDa protein lacking self-cleavage activity. (C,D) WT or D25N PRs (-mRNA and DHFR as a negative control) were separated by SDS-PAGE followed by CBB-staining (C) and immunoblotting using anti-HIV-1 PR antibody that recognizes only mature HIV protease (PR) but not its precursor (D). Arrows depict protein products.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4628118&req=5

Figure 1: Synthesis of catalytically active HIV-1 PR using the wheat germ cell-free system. (A) Schematic representation of rapid production of HIV-1 PR using a wheat germ cell-free system. A transcriptional template, including the HIV-1 PR open reading frame flanked by N-terminal 168 bases (56 aa) and C-terminal 54 bases (18 aa), produced by split-primer PCR as described in section “Materials and Methods.” Cell-free translation was carried out using products of in vitro transcription. (B) The formation of WT PR (catalytic active) or D25N PR (catalytic inactive) after translation. WT PR generated as a 11 kDa protein by self-cleavage. D25N PR (catalytic inactive) generated as a 19 kDa protein lacking self-cleavage activity. (C,D) WT or D25N PRs (-mRNA and DHFR as a negative control) were separated by SDS-PAGE followed by CBB-staining (C) and immunoblotting using anti-HIV-1 PR antibody that recognizes only mature HIV protease (PR) but not its precursor (D). Arrows depict protein products.
Mentions: To synthesize catalytically active HIV-1 PR, we initially generated a transcriptional template of this enzyme by PCR, using the HIV-1NL4-3 clone as a wild-type (WT) reference sample. We designed a transcription template encoding the open reading frame of HIV-1 PR flanked by 56 N-terminal amino acids (Gag p6 region) and 18 C-terminal amino acids (the reverse-transcriptase region), as shown in Figure 1A. The in vitro transcription template for the HIV-1 PR gene was constructed by split-primer PCR using primers encoding the SP6 and E01 sites, as described in section “Materials and Methods.” To generate a catalysis-incompetent PR, we designed a PR mutant harboring the catalytic active site substitution D25N (D25N; Figure 1B). All cDNA templates were subjected to cell-free transcription/translation and then separated by SDS-PAGE. By CBB staining, WT PR migrated at 11 kDa (as the truncated form of PR) due to self-cleavage of the flanking 56 and 18 amino acids at the N- and C-terminal ends, respectively, whereas D25N PR migrated at 19 kDa, corresponding to full-length PR with the flanking sequences (Figures 1C,D). Immunoblot analysis with anti-HIV-1 PR antibody recognizing mature form of HIV PR, only detected the cleaved form of WT PR at the expected size (∼11 kDa; Figure 1D).

View Article: PubMed Central

ABSTRACT

Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1.

No MeSH data available.