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Labeling of multiple HIV-1 proteins with the biarsenical-tetracysteine system.

PloS one (2011-02-25)
Cândida F Pereira, Paula C Ellenberg, Kate L Jones, Tara L Fernandez, Redmond P Smyth, David J Hawkes, Marcel Hijnen, Valérie Vivet-Boudou, Roland Marquet, Iain Johnson, Johnson Mak
RESUMEN

Due to its small size and versatility, the biarsenical-tetracysteine system is an attractive way to label viral proteins for live cell imaging. This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1) structural proteins (matrix, capsid and nucleocapsid), enzymes (protease, reverse transcriptase, RNAse H and integrase) and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus. We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein. Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes. This approach can be adapted to other viral systems for the visualization of the interplay between virus and host cell during infection.

MATERIALES
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Millipore
Nucleasas Benzonase®, ≥250 units/μL, ≥90% (SDS-PAGE), recombinant, expressed in E. coli, buffered aqueous glycerol solution
Sigma-Aldrich
Proteasa from Streptomyces griseus, Type XIV, ≥3.5 units/mg solid, powder
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Proteasa from Streptomyces griseus, powder, BioReagent, suitable for mouse embryo cell culture, ≥3.5 units/mg solid
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1,2-etanoditiol, technical grade, ≥90%
Sigma-Aldrich
1,2-etanoditiol, ≥98.0% (GC)
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Protease from Streptomyces sp., Type XXI, ≥15 units/mg solid