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Function-based mutation-resistant synthetic signaling device activated by HIV-1 proteolysis.

Majerle A, Gaber R, Benčina M, Jerala R - ACS Synth Biol (2014)

Bottom Line: The HIV-1 protease released the transcriptional activator from the membrane, thereby inducing transcription of the selected genes.The device was still strongly activated by clinically relevant protease mutants that are resistant to protease inhibitors.In the future, a similar principle could be applied to detect also other pathogens and functions.

View Article: PubMed Central - PubMed

Affiliation: †Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.

ABSTRACT
The high mutation rate of the human immunodeficiency virus type 1 (HIV-1) virus is a major problem since it evades the function of antibodies and chemical inhibitors. Here, we demonstrate a viral detection strategy based on synthetic biology principles to detect a specific viral function rather than a particular viral protein. The resistance caused by mutations can be circumvented since the mutations that cause the loss of function also incapacitate the virus. Many pathogens encode proteases that are essential for their replication and that have a defined substrate specificity. A genetically encoded sensor composed of a fused membrane anchor, viral protease target site, and an orthogonal transcriptional activator was engineered into a human cell line. The HIV-1 protease released the transcriptional activator from the membrane, thereby inducing transcription of the selected genes. The device was still strongly activated by clinically relevant protease mutants that are resistant to protease inhibitors. In the future, a similar principle could be applied to detect also other pathogens and functions.

No MeSH data available.


Related in: MedlinePlus

Synthetic anti-HIV-1 signaling devicedetects the activity of HIV-1protease mutations resistant to protease inhibitors. (a, b, c) HEK293Tcells were transfected with a reporter plasmid coding for the fireflyluciferase, a plasmid constitutively expressing the Renilla luciferase, 10 ng of CD4(HA)-hivp-GAL4-VP16, and increasing amountsof pNL4–3.HSA.R–.E– (pNL4–3 in the figure)or HIV-1 protease mutants (G48V/I54T or V82A) in the medium with orwithout saquinavir (SAQ) or saquinavir and ritonavir (SAQ/RTV). Thecells were lysed 24 h after transfection, and the expression of thefirefly and Renilla luciferase reporter genes wasanalyzed. (d, e, f) HEK293T cells were transfected with plasmids expressingthe firefly luciferase reporter and the constitutively expressed Renilla luciferase, CD4(HA)-hivp-GAL4-VP16 (10 ng) and pNL4–3.HSA.R–.E–,G48V/I54T, or V82A (all 120 ng) in the medium with or without theHIV-1 protease inhibitors atazanavir (ATZ), atazanavir and ritonavir(ATZ/RTV), saquinavir (SAQ), or saquinavir and ritonavir (SAQ/RTV).The cells were lysed 24 h after transfection, and the activity ofthe firefly and Renilla luciferase reporters wasmeasured. Error bars represent the SD obtained from four experimentalreplicates. The data are representative of two or three independentexperiments. The two-tailed Student’s t-test(assuming a two-sample equal variance) was used for pairwise comparisons.***p < 0.005.
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fig3: Synthetic anti-HIV-1 signaling devicedetects the activity of HIV-1protease mutations resistant to protease inhibitors. (a, b, c) HEK293Tcells were transfected with a reporter plasmid coding for the fireflyluciferase, a plasmid constitutively expressing the Renilla luciferase, 10 ng of CD4(HA)-hivp-GAL4-VP16, and increasing amountsof pNL4–3.HSA.R–.E– (pNL4–3 in the figure)or HIV-1 protease mutants (G48V/I54T or V82A) in the medium with orwithout saquinavir (SAQ) or saquinavir and ritonavir (SAQ/RTV). Thecells were lysed 24 h after transfection, and the expression of thefirefly and Renilla luciferase reporter genes wasanalyzed. (d, e, f) HEK293T cells were transfected with plasmids expressingthe firefly luciferase reporter and the constitutively expressed Renilla luciferase, CD4(HA)-hivp-GAL4-VP16 (10 ng) and pNL4–3.HSA.R–.E–,G48V/I54T, or V82A (all 120 ng) in the medium with or without theHIV-1 protease inhibitors atazanavir (ATZ), atazanavir and ritonavir(ATZ/RTV), saquinavir (SAQ), or saquinavir and ritonavir (SAQ/RTV).The cells were lysed 24 h after transfection, and the activity ofthe firefly and Renilla luciferase reporters wasmeasured. Error bars represent the SD obtained from four experimentalreplicates. The data are representative of two or three independentexperiments. The two-tailed Student’s t-test(assuming a two-sample equal variance) was used for pairwise comparisons.***p < 0.005.

Mentions: The salient feature of our device is that it should be less sensitiveto the mutations that cause resistance to protease inhibitors. Theviral genome encoding drug-resistant HIV-1 protease mutants G48V/I54Tand V82A exhibited slightly reduced proteolytic activity in comparisonto the wild type for both device constructs, CD4(HA)-hivp-GAL4-VP16or FAS(HA)-hivp-GAL4-VP16 (Supporting InformationFigure S3), probably because of the impaired specific activityof the mutated protease. However, their proteolytic efficiency isstill sufficient for processing the viral polyprotein and viral replication.Both clinically observed HIV-1 protease mutants were significantlyless sensitive to the therapeutic protease inhibitors or their cocktails(atazanavir, SAQ, and RTV) than the wild-type HIV-1 protease (Figure 3d–f). However, our device was able to robustlysense the activity of both HIV-1 protease mutants and trigger genetranscription regardless of the presence of 1 μM SAQ or a cocktailof 1 μM SAQ and 1 μM RTV, as shown in Figure 3a–c for the CD4(HA)-hivp-GAL4-VP16 device.We obtained similar results for FAS(HA)-hivp-GAL4-VP16 (these dataare not shown). These findings demonstrate that the synthetic geneticdevice is still activated by the protease mutations that are resistantto clinically used inhibitors. We have also demonstrated the activationof our device with nontoxic HIV-1 protease precursor fused to greenfluorescent protein (GFP).18 For the activationof the device in HEK293T cells, significantly lower amounts of GFP-PRin comparison to the pNL4–3.HSA.R–.E– were required(Supporting Information Figure S4). Theprotease mutant GFP-PR(G48V/I54T) was also sensed by our device. Wefound that mutant GFP-PR(G48V/I54T) was almost completely resistantto 1 μM SAQ, and we used Western blot to demonstrate the expressionlevels of both precursor proteins (SupportingInformation Figure S5). To demonstrate that our device coulddrive expression of different selected effector genes, we selectedthe gene for human IFN-β1. IFN-β1 is a member of the typeI IFN family; these IFNs are usually secreted as the consequence ofelevated levels of dsRNA in cells and are used in antiviral therapy.19 We found that the expression level of IFN-β1that was produced in HEK293T cells—transfected with our signalingdevice—was dependent on the amount of transfected HIV-1 protease(Figure 4).


Function-based mutation-resistant synthetic signaling device activated by HIV-1 proteolysis.

Majerle A, Gaber R, Benčina M, Jerala R - ACS Synth Biol (2014)

Synthetic anti-HIV-1 signaling devicedetects the activity of HIV-1protease mutations resistant to protease inhibitors. (a, b, c) HEK293Tcells were transfected with a reporter plasmid coding for the fireflyluciferase, a plasmid constitutively expressing the Renilla luciferase, 10 ng of CD4(HA)-hivp-GAL4-VP16, and increasing amountsof pNL4–3.HSA.R–.E– (pNL4–3 in the figure)or HIV-1 protease mutants (G48V/I54T or V82A) in the medium with orwithout saquinavir (SAQ) or saquinavir and ritonavir (SAQ/RTV). Thecells were lysed 24 h after transfection, and the expression of thefirefly and Renilla luciferase reporter genes wasanalyzed. (d, e, f) HEK293T cells were transfected with plasmids expressingthe firefly luciferase reporter and the constitutively expressed Renilla luciferase, CD4(HA)-hivp-GAL4-VP16 (10 ng) and pNL4–3.HSA.R–.E–,G48V/I54T, or V82A (all 120 ng) in the medium with or without theHIV-1 protease inhibitors atazanavir (ATZ), atazanavir and ritonavir(ATZ/RTV), saquinavir (SAQ), or saquinavir and ritonavir (SAQ/RTV).The cells were lysed 24 h after transfection, and the activity ofthe firefly and Renilla luciferase reporters wasmeasured. Error bars represent the SD obtained from four experimentalreplicates. The data are representative of two or three independentexperiments. The two-tailed Student’s t-test(assuming a two-sample equal variance) was used for pairwise comparisons.***p < 0.005.
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Related In: Results  -  Collection

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fig3: Synthetic anti-HIV-1 signaling devicedetects the activity of HIV-1protease mutations resistant to protease inhibitors. (a, b, c) HEK293Tcells were transfected with a reporter plasmid coding for the fireflyluciferase, a plasmid constitutively expressing the Renilla luciferase, 10 ng of CD4(HA)-hivp-GAL4-VP16, and increasing amountsof pNL4–3.HSA.R–.E– (pNL4–3 in the figure)or HIV-1 protease mutants (G48V/I54T or V82A) in the medium with orwithout saquinavir (SAQ) or saquinavir and ritonavir (SAQ/RTV). Thecells were lysed 24 h after transfection, and the expression of thefirefly and Renilla luciferase reporter genes wasanalyzed. (d, e, f) HEK293T cells were transfected with plasmids expressingthe firefly luciferase reporter and the constitutively expressed Renilla luciferase, CD4(HA)-hivp-GAL4-VP16 (10 ng) and pNL4–3.HSA.R–.E–,G48V/I54T, or V82A (all 120 ng) in the medium with or without theHIV-1 protease inhibitors atazanavir (ATZ), atazanavir and ritonavir(ATZ/RTV), saquinavir (SAQ), or saquinavir and ritonavir (SAQ/RTV).The cells were lysed 24 h after transfection, and the activity ofthe firefly and Renilla luciferase reporters wasmeasured. Error bars represent the SD obtained from four experimentalreplicates. The data are representative of two or three independentexperiments. The two-tailed Student’s t-test(assuming a two-sample equal variance) was used for pairwise comparisons.***p < 0.005.
Mentions: The salient feature of our device is that it should be less sensitiveto the mutations that cause resistance to protease inhibitors. Theviral genome encoding drug-resistant HIV-1 protease mutants G48V/I54Tand V82A exhibited slightly reduced proteolytic activity in comparisonto the wild type for both device constructs, CD4(HA)-hivp-GAL4-VP16or FAS(HA)-hivp-GAL4-VP16 (Supporting InformationFigure S3), probably because of the impaired specific activityof the mutated protease. However, their proteolytic efficiency isstill sufficient for processing the viral polyprotein and viral replication.Both clinically observed HIV-1 protease mutants were significantlyless sensitive to the therapeutic protease inhibitors or their cocktails(atazanavir, SAQ, and RTV) than the wild-type HIV-1 protease (Figure 3d–f). However, our device was able to robustlysense the activity of both HIV-1 protease mutants and trigger genetranscription regardless of the presence of 1 μM SAQ or a cocktailof 1 μM SAQ and 1 μM RTV, as shown in Figure 3a–c for the CD4(HA)-hivp-GAL4-VP16 device.We obtained similar results for FAS(HA)-hivp-GAL4-VP16 (these dataare not shown). These findings demonstrate that the synthetic geneticdevice is still activated by the protease mutations that are resistantto clinically used inhibitors. We have also demonstrated the activationof our device with nontoxic HIV-1 protease precursor fused to greenfluorescent protein (GFP).18 For the activationof the device in HEK293T cells, significantly lower amounts of GFP-PRin comparison to the pNL4–3.HSA.R–.E– were required(Supporting Information Figure S4). Theprotease mutant GFP-PR(G48V/I54T) was also sensed by our device. Wefound that mutant GFP-PR(G48V/I54T) was almost completely resistantto 1 μM SAQ, and we used Western blot to demonstrate the expressionlevels of both precursor proteins (SupportingInformation Figure S5). To demonstrate that our device coulddrive expression of different selected effector genes, we selectedthe gene for human IFN-β1. IFN-β1 is a member of the typeI IFN family; these IFNs are usually secreted as the consequence ofelevated levels of dsRNA in cells and are used in antiviral therapy.19 We found that the expression level of IFN-β1that was produced in HEK293T cells—transfected with our signalingdevice—was dependent on the amount of transfected HIV-1 protease(Figure 4).

Bottom Line: The HIV-1 protease released the transcriptional activator from the membrane, thereby inducing transcription of the selected genes.The device was still strongly activated by clinically relevant protease mutants that are resistant to protease inhibitors.In the future, a similar principle could be applied to detect also other pathogens and functions.

View Article: PubMed Central - PubMed

Affiliation: †Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.

ABSTRACT
The high mutation rate of the human immunodeficiency virus type 1 (HIV-1) virus is a major problem since it evades the function of antibodies and chemical inhibitors. Here, we demonstrate a viral detection strategy based on synthetic biology principles to detect a specific viral function rather than a particular viral protein. The resistance caused by mutations can be circumvented since the mutations that cause the loss of function also incapacitate the virus. Many pathogens encode proteases that are essential for their replication and that have a defined substrate specificity. A genetically encoded sensor composed of a fused membrane anchor, viral protease target site, and an orthogonal transcriptional activator was engineered into a human cell line. The HIV-1 protease released the transcriptional activator from the membrane, thereby inducing transcription of the selected genes. The device was still strongly activated by clinically relevant protease mutants that are resistant to protease inhibitors. In the future, a similar principle could be applied to detect also other pathogens and functions.

No MeSH data available.


Related in: MedlinePlus