Quantitative Live-Cell Imaging of Human Immunodeficiency Virus (HIV-1) Assembly

Abstract

Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site.

Modeling Viral Capsid Assembly

“…Recent experiments that visualize or otherwise monitor the formation of individual viruses (e.g. [24, 108, 132, 285]) can provide information about distributions of capsid growth times and the rates at which individual subunits associate to or dissociate from partial capsids of different sizes. This information would provide additional constraints with which to refine or extend current models.…”

“…19). This correspondence could be tested experimentally by comparing elongation times measured by single molecule experiments [24, 131, 132] with lag times measured by bulk experiments.…”

“…Furthermore, fluorescent labeling of capsid proteins [131] and in some cases RNA has enabled measuring assembly timescales for capsids in vivo (reviewed in Refs. [24, 132]).…”

How HIV-1 Gag assembles in cells: Putting together pieces of the puzzle

“…In recent years, live imaging has been used to study many aspects of the HIV-1 life cycle, including genome packaging [reviewed in (Moore and Hu, 2009)], budding and release [reviewed in (Baumgartel et al, 2012)], and cell-to-cell transmission [reviewed in (Campbell et al, 2008)]. Here we will confine our discussion to how live cell imaging has been used to study assembly of Gag in cells.…”

MAS NMR of HIV-1 protein assemblies

“…Both immature and mature virions are visualized here. Reprinted from open access V. Baumgartel et al, Viruses 4 2012 777–779 [112]. …”