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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

Designof a mutation-resistant antiviral signaling device basedon HIV-1 protease activity. The transcriptional activator Gal4-VP16is anchored to the transmembrane protein domain (CD4 or FAS), whereit is inactive. The activation step occurs via the HIV-1 protease,which cleaves the linker between the transcription factor and membraneanchor, thereby comprising the HIV-1 protease cleavage site. Afterthe irreversible proteolytic step, Gal4-VP16 is released from themembrane and translocates into the nucleus, where it transcribes theselected genes under the control of the GAL4 operator, such as thereporter genes or genes that provide defense against viral infections.
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fig1: Designof a mutation-resistant antiviral signaling device basedon HIV-1 protease activity. The transcriptional activator Gal4-VP16is anchored to the transmembrane protein domain (CD4 or FAS), whereit is inactive. The activation step occurs via the HIV-1 protease,which cleaves the linker between the transcription factor and membraneanchor, thereby comprising the HIV-1 protease cleavage site. Afterthe irreversible proteolytic step, Gal4-VP16 is released from themembrane and translocates into the nucleus, where it transcribes theselected genes under the control of the GAL4 operator, such as thereporter genes or genes that provide defense against viral infections.

Mentions: We engineered a hybrid transcriptionfactor (Gal4-VP16, which iscomposed of the DNA-binding domain [Gal4] and the activation domain[VP16]) to be linked through the HIV-1 protease cleavage site to thecellular membrane by the transmembrane and ectodomains of the CD4or FAS receptors (Figure 1). Yeast regulatoryprotein Gal4,10 which is comprised of aDNA-binding domain, linker, and a coiled-coil dimerization domain,has been used previously as an orthogonal transcription factor inmammalian cells.11 A domain of the herpessimplex virus protein VP16 recruits host-encoded elements of the transcriptionalmachinery upstream from the promoter.12,13 The selectedpeptide linker was comprised of the HIV-1 protease cleavage site withthe amino acid sequence SQVSQNY↓PIVQNLQ, which is also calledthe p17/p24 peptide14 and is used as thesynthetic HIV Protease Substrate 1.15 Foran efficient anchor to the membrane, we selected the nonfunctionaltransmembrane and ectodomains of CD4 and FAS, which are type-I transmembranereceptors that are found on the surface of immune cells, such as T-helpercells, monocytes, macrophages, and dendritic cells.16,17


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

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

Designof a mutation-resistant antiviral signaling device basedon HIV-1 protease activity. The transcriptional activator Gal4-VP16is anchored to the transmembrane protein domain (CD4 or FAS), whereit is inactive. The activation step occurs via the HIV-1 protease,which cleaves the linker between the transcription factor and membraneanchor, thereby comprising the HIV-1 protease cleavage site. Afterthe irreversible proteolytic step, Gal4-VP16 is released from themembrane and translocates into the nucleus, where it transcribes theselected genes under the control of the GAL4 operator, such as thereporter genes or genes that provide defense against viral infections.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487218&req=5

fig1: Designof a mutation-resistant antiviral signaling device basedon HIV-1 protease activity. The transcriptional activator Gal4-VP16is anchored to the transmembrane protein domain (CD4 or FAS), whereit is inactive. The activation step occurs via the HIV-1 protease,which cleaves the linker between the transcription factor and membraneanchor, thereby comprising the HIV-1 protease cleavage site. Afterthe irreversible proteolytic step, Gal4-VP16 is released from themembrane and translocates into the nucleus, where it transcribes theselected genes under the control of the GAL4 operator, such as thereporter genes or genes that provide defense against viral infections.
Mentions: We engineered a hybrid transcriptionfactor (Gal4-VP16, which iscomposed of the DNA-binding domain [Gal4] and the activation domain[VP16]) to be linked through the HIV-1 protease cleavage site to thecellular membrane by the transmembrane and ectodomains of the CD4or FAS receptors (Figure 1). Yeast regulatoryprotein Gal4,10 which is comprised of aDNA-binding domain, linker, and a coiled-coil dimerization domain,has been used previously as an orthogonal transcription factor inmammalian cells.11 A domain of the herpessimplex virus protein VP16 recruits host-encoded elements of the transcriptionalmachinery upstream from the promoter.12,13 The selectedpeptide linker was comprised of the HIV-1 protease cleavage site withthe amino acid sequence SQVSQNY↓PIVQNLQ, which is also calledthe p17/p24 peptide14 and is used as thesynthetic HIV Protease Substrate 1.15 Foran efficient anchor to the membrane, we selected the nonfunctionaltransmembrane and ectodomains of CD4 and FAS, which are type-I transmembranereceptors that are found on the surface of immune cells, such as T-helpercells, monocytes, macrophages, and dendritic cells.16,17

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