Labeling of Multiple HIV-1 Proteins with the Biarsenical-Tetracysteine System

Cândida F Pereira,Redmond P Smyth,Kate L Jones,Valérie Vivet-Boudou,David J Hawkes,Iain Johnson,Tara L Fernandez,Paula C Ellenberg,Roland Marquet,Johnson Mak,Marcel Hijnen,Ashok Aiyar

doi:10.1371/journal.pone.0017016

Abstract

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.

Highlights

Fluorescent viral fusion proteins have been instrumental in the visualization of the intracellular behavior of viruses during infection [1,2,3]
To assess the effect of these modifications on the viral life-cycle, the replication kinetics of HIVMA-C, HIVCA-N, HIVCA-C, HIVNC-N, HIVNC-C, HIVPR-C, HIVRT-N, HIVRN-N, HIVRN-C and HIVIN-N were assessed in peripheral blood mononuclear cells (PBMCs), which are the natural target cells of human immunodeficiency virus type 1 (HIV-1) (Figure 1C)
The imaging of fluorescent-labeled viruses is limited to the visualization of a small number of viral proteins that can be fluorescently labeled without compromising virus infectivity

Summary

Introduction

Fluorescent viral fusion proteins have been instrumental in the visualization of the intracellular behavior of viruses during infection [1,2,3]. The incorporation of these 27 kDa fluorescent proteins (FPs) into most viral proteins has limited application due to its potential impact on viral protein function and infectivity [4,5,6,7] This problem has been partially overcome with the development of the biarsenical-tetracysteine labeling system [8], in which the protein of interest is fused to a six to twelve amino acids tetracysteine motif (TC tag), which is only 0.5– 1 kDa and less likely to interfere with the structure or biological activity of the protein. Another recent study has elegantly shown that the loop/linker regions, but not the a-helix regions of influenza A virus non-structural protein 1, can accommodate TC tags without affecting virus infectivity [16]

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Conclusion