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The initial step in human immunodeficiency virus type 1 GagProPol processing can be regulated by reversible oxidation.

Daniels SI, Davis DA, Soule EE, Stahl SJ, Tebbs IR, Wingfield P, Yarchoan R - PLoS ONE (2010)

Bottom Line: Although reversible oxidation can regulate mature protease activity as well as retrovirus maturation, it is possible that the effects of oxidation on viral maturation are mediated in whole, or part, through effects on the initial intramolecular cleavage event of GagProPol.In order assess the effect of reversible oxidation on this event, we developed a system to isolate the first step in protease activation involving GagProPol.Diamide, a sulfhydral oxidizing agent, inhibited processing at p2/NC by >60% for pGPfs-1C and was readily reversed with the reductant, dithiothreitol.

View Article: PubMed Central - PubMed

Affiliation: HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT

Background: Maturation of human immunodeficiency virus type 1 (HIV-1) occurs upon activation of HIV-1 protease embedded within GagProPol precursors and cleavage of Gag and GagProPol polyproteins. Although reversible oxidation can regulate mature protease activity as well as retrovirus maturation, it is possible that the effects of oxidation on viral maturation are mediated in whole, or part, through effects on the initial intramolecular cleavage event of GagProPol. In order assess the effect of reversible oxidation on this event, we developed a system to isolate the first step in protease activation involving GagProPol.

Methodology/principal findings: To determine if oxidation influences this step, we created a GagProPol plasmid construct (pGPfs-1C) that encoded mutations at all cleavage sites except p2/NC, the initial cleavage site in GagProPol. pGPfs-1C was used in an in vitro translation assay to observe the behavior of this initial step without interference from subsequent processing events. Diamide, a sulfhydral oxidizing agent, inhibited processing at p2/NC by >60% for pGPfs-1C and was readily reversed with the reductant, dithiothreitol. The ability to regulate processing by reversible oxidation was lost when the cysteines of the embedded protease were mutated to alanine. Unlike mature protease, which requires only oxidation of cys95 for inhibition, both cysteines of the embedded protease contributed to this inhibition.

Conclusions/significance: We developed a system that can be used to study the first step in the cascade of HIV-1 GagProPol processing and show that reversible oxidation of cysteines of HIV-1 protease embedded in GagProPol can block this initial GagProPol autoprocessing. This type of regulation may be broadly applied to the majority of retroviruses.

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Time course for pGPfs-1C GagProPol processing.In vitro transcription translation with 35S-methionine was carried out for 45, 60, 75 and 90 minutes. Samples were separated by LDS-PAGE and visualized by autoradiography and the percent processing determined using densitometry. The precursors (GagProPol and GagProPolii) and two products (MA-p2, NC-IN) were scanned and the extent of processing calculated as a percent: [(products)/(precursor plus products) x 100]. The LDS-PAGE for this data is shown Figure S1.
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pone-0013595-g002: Time course for pGPfs-1C GagProPol processing.In vitro transcription translation with 35S-methionine was carried out for 45, 60, 75 and 90 minutes. Samples were separated by LDS-PAGE and visualized by autoradiography and the percent processing determined using densitometry. The precursors (GagProPol and GagProPolii) and two products (MA-p2, NC-IN) were scanned and the extent of processing calculated as a percent: [(products)/(precursor plus products) x 100]. The LDS-PAGE for this data is shown Figure S1.

Mentions: The rate of processing at the initial cleavage site was investigated in a time course experiment. Approximately 60% of the pGPfs-1C GagProPol underwent cleavage at the p2/NC within 45 minutes and 90% underwent cleavage within 75 minutes, at which time it reached a plateau (Figure 2 and Figure S1). After two hours of incubation, minor bands below the large product were evident but these represented less than 5% of the total products produced (data not shown). These likely represent alternate, albeit much less favorable, cleavages occurring through the action of the embedded protease within the large product (NC-IN). These data indicate the embedded HIV-1 protease readily cleaves within pGPfs-1C GagProPol at the p2/NC junction yielding MA-p2 and NC-IN products and that the blocking mutations do not adversely affect processing at the initial site. However, the presence of blocking mutations prevents any significant processing of the polyprotein at the other cleavage sites.


The initial step in human immunodeficiency virus type 1 GagProPol processing can be regulated by reversible oxidation.

Daniels SI, Davis DA, Soule EE, Stahl SJ, Tebbs IR, Wingfield P, Yarchoan R - PLoS ONE (2010)

Time course for pGPfs-1C GagProPol processing.In vitro transcription translation with 35S-methionine was carried out for 45, 60, 75 and 90 minutes. Samples were separated by LDS-PAGE and visualized by autoradiography and the percent processing determined using densitometry. The precursors (GagProPol and GagProPolii) and two products (MA-p2, NC-IN) were scanned and the extent of processing calculated as a percent: [(products)/(precursor plus products) x 100]. The LDS-PAGE for this data is shown Figure S1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2962637&req=5

pone-0013595-g002: Time course for pGPfs-1C GagProPol processing.In vitro transcription translation with 35S-methionine was carried out for 45, 60, 75 and 90 minutes. Samples were separated by LDS-PAGE and visualized by autoradiography and the percent processing determined using densitometry. The precursors (GagProPol and GagProPolii) and two products (MA-p2, NC-IN) were scanned and the extent of processing calculated as a percent: [(products)/(precursor plus products) x 100]. The LDS-PAGE for this data is shown Figure S1.
Mentions: The rate of processing at the initial cleavage site was investigated in a time course experiment. Approximately 60% of the pGPfs-1C GagProPol underwent cleavage at the p2/NC within 45 minutes and 90% underwent cleavage within 75 minutes, at which time it reached a plateau (Figure 2 and Figure S1). After two hours of incubation, minor bands below the large product were evident but these represented less than 5% of the total products produced (data not shown). These likely represent alternate, albeit much less favorable, cleavages occurring through the action of the embedded protease within the large product (NC-IN). These data indicate the embedded HIV-1 protease readily cleaves within pGPfs-1C GagProPol at the p2/NC junction yielding MA-p2 and NC-IN products and that the blocking mutations do not adversely affect processing at the initial site. However, the presence of blocking mutations prevents any significant processing of the polyprotein at the other cleavage sites.

Bottom Line: Although reversible oxidation can regulate mature protease activity as well as retrovirus maturation, it is possible that the effects of oxidation on viral maturation are mediated in whole, or part, through effects on the initial intramolecular cleavage event of GagProPol.In order assess the effect of reversible oxidation on this event, we developed a system to isolate the first step in protease activation involving GagProPol.Diamide, a sulfhydral oxidizing agent, inhibited processing at p2/NC by >60% for pGPfs-1C and was readily reversed with the reductant, dithiothreitol.

View Article: PubMed Central - PubMed

Affiliation: HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT

Background: Maturation of human immunodeficiency virus type 1 (HIV-1) occurs upon activation of HIV-1 protease embedded within GagProPol precursors and cleavage of Gag and GagProPol polyproteins. Although reversible oxidation can regulate mature protease activity as well as retrovirus maturation, it is possible that the effects of oxidation on viral maturation are mediated in whole, or part, through effects on the initial intramolecular cleavage event of GagProPol. In order assess the effect of reversible oxidation on this event, we developed a system to isolate the first step in protease activation involving GagProPol.

Methodology/principal findings: To determine if oxidation influences this step, we created a GagProPol plasmid construct (pGPfs-1C) that encoded mutations at all cleavage sites except p2/NC, the initial cleavage site in GagProPol. pGPfs-1C was used in an in vitro translation assay to observe the behavior of this initial step without interference from subsequent processing events. Diamide, a sulfhydral oxidizing agent, inhibited processing at p2/NC by >60% for pGPfs-1C and was readily reversed with the reductant, dithiothreitol. The ability to regulate processing by reversible oxidation was lost when the cysteines of the embedded protease were mutated to alanine. Unlike mature protease, which requires only oxidation of cys95 for inhibition, both cysteines of the embedded protease contributed to this inhibition.

Conclusions/significance: We developed a system that can be used to study the first step in the cascade of HIV-1 GagProPol processing and show that reversible oxidation of cysteines of HIV-1 protease embedded in GagProPol can block this initial GagProPol autoprocessing. This type of regulation may be broadly applied to the majority of retroviruses.

Show MeSH
Related in: MedlinePlus