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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

HIV-1protease triggers the release of the membrane-bound reporter.In cells without HIV-1 protease, the membrane localization of (a)CFP and (c) mCherry-2xNLS was observed. In the presence of HIV-1 protease,(b) CFP was released to the cytosol and (d) mCherry was released fromthe membrane and translocated to the nucleus. An overlay of the imageshowing the nucleus stained with Hoechst 33342 and mCherry is shownon the left (d). The microscopic images are representative of fiveseparate observations. Scale bar = 10 μm. The HEK293T cellswere transfected with (a, b) 40 ng CD4(HA)-hivp-CFP-6xHis or (c, d)40 ng CD4(HA)-hivp-mCherry-2xNLS, as well as with the (b, d) HIV-1genome-encoding plasmid pNL4–3.HSA.R–.E– (320or 360 ng).
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fig2: HIV-1protease triggers the release of the membrane-bound reporter.In cells without HIV-1 protease, the membrane localization of (a)CFP and (c) mCherry-2xNLS was observed. In the presence of HIV-1 protease,(b) CFP was released to the cytosol and (d) mCherry was released fromthe membrane and translocated to the nucleus. An overlay of the imageshowing the nucleus stained with Hoechst 33342 and mCherry is shownon the left (d). The microscopic images are representative of fiveseparate observations. Scale bar = 10 μm. The HEK293T cellswere transfected with (a, b) 40 ng CD4(HA)-hivp-CFP-6xHis or (c, d)40 ng CD4(HA)-hivp-mCherry-2xNLS, as well as with the (b, d) HIV-1genome-encoding plasmid pNL4–3.HSA.R–.E– (320or 360 ng).

Mentions: Activation of the device by theviral protease was observed withfluorescence confocal microscopy, where we used the fluorescent proteinscyan florescent protein (CFP) or mCherry linked by a cleavable linkerpeptide (a HIV-1 protease cleavage site [hivp]) to the cytosolic sideof the transmembrane domain. Human embryonic kidney (HEK)293T cellswere transfected with the plasmid coding fusion proteins CD4(HA)-hivp-CFP-6xHisor FAS(HA)-hivp-mCherry-2xNLS and cotransfected with a replication-incompetentHIV-1 pseudoviral genome on a pNL4–3.HSA.R–.E–plasmid, thereby encoding the functional viral protease. In the absenceof the HIV-1 genome, the fluorescence was clearly localized to theplasma membrane (Figure 2a and c). In the cellscotransfected with an HIV-1 genome, the fluorescent CFP was releasedfrom the membrane into the cytosol (Figure 2b), while mCherry, which is comprised of two copies of SV40 largeT-antigen nuclear localization sequence (NLS), was translocated tothe nucleus (Figure 2d). The expression ofthe transmembrane proteins CD4(HA)-hivp-GAL4-VP16 or FAS(HA)-hivp-GAL4-VP16at the surface of the cell membrane was also confirmed by stainingthe HEK293T cells with anti-HA antibodies, as the HA-tag was positionedat the extracellular side of the receptors (SupportingInformation Figure S1). The incorporation of the transmembranereceptors into the membrane demonstrates the appropriate protein foldingand localization, suggesting that the sensor could be functional.


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

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

HIV-1protease triggers the release of the membrane-bound reporter.In cells without HIV-1 protease, the membrane localization of (a)CFP and (c) mCherry-2xNLS was observed. In the presence of HIV-1 protease,(b) CFP was released to the cytosol and (d) mCherry was released fromthe membrane and translocated to the nucleus. An overlay of the imageshowing the nucleus stained with Hoechst 33342 and mCherry is shownon the left (d). The microscopic images are representative of fiveseparate observations. Scale bar = 10 μm. The HEK293T cellswere transfected with (a, b) 40 ng CD4(HA)-hivp-CFP-6xHis or (c, d)40 ng CD4(HA)-hivp-mCherry-2xNLS, as well as with the (b, d) HIV-1genome-encoding plasmid pNL4–3.HSA.R–.E– (320or 360 ng).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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fig2: HIV-1protease triggers the release of the membrane-bound reporter.In cells without HIV-1 protease, the membrane localization of (a)CFP and (c) mCherry-2xNLS was observed. In the presence of HIV-1 protease,(b) CFP was released to the cytosol and (d) mCherry was released fromthe membrane and translocated to the nucleus. An overlay of the imageshowing the nucleus stained with Hoechst 33342 and mCherry is shownon the left (d). The microscopic images are representative of fiveseparate observations. Scale bar = 10 μm. The HEK293T cellswere transfected with (a, b) 40 ng CD4(HA)-hivp-CFP-6xHis or (c, d)40 ng CD4(HA)-hivp-mCherry-2xNLS, as well as with the (b, d) HIV-1genome-encoding plasmid pNL4–3.HSA.R–.E– (320or 360 ng).
Mentions: Activation of the device by theviral protease was observed withfluorescence confocal microscopy, where we used the fluorescent proteinscyan florescent protein (CFP) or mCherry linked by a cleavable linkerpeptide (a HIV-1 protease cleavage site [hivp]) to the cytosolic sideof the transmembrane domain. Human embryonic kidney (HEK)293T cellswere transfected with the plasmid coding fusion proteins CD4(HA)-hivp-CFP-6xHisor FAS(HA)-hivp-mCherry-2xNLS and cotransfected with a replication-incompetentHIV-1 pseudoviral genome on a pNL4–3.HSA.R–.E–plasmid, thereby encoding the functional viral protease. In the absenceof the HIV-1 genome, the fluorescence was clearly localized to theplasma membrane (Figure 2a and c). In the cellscotransfected with an HIV-1 genome, the fluorescent CFP was releasedfrom the membrane into the cytosol (Figure 2b), while mCherry, which is comprised of two copies of SV40 largeT-antigen nuclear localization sequence (NLS), was translocated tothe nucleus (Figure 2d). The expression ofthe transmembrane proteins CD4(HA)-hivp-GAL4-VP16 or FAS(HA)-hivp-GAL4-VP16at the surface of the cell membrane was also confirmed by stainingthe HEK293T cells with anti-HA antibodies, as the HA-tag was positionedat the extracellular side of the receptors (SupportingInformation Figure S1). The incorporation of the transmembranereceptors into the membrane demonstrates the appropriate protein foldingand localization, suggesting that the sensor could be functional.

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