Live cell imaging of the HIV-1 life cycle

Abstract

Technology developed in the past 10 years has dramatically increased the ability of researchers to directly visualize and measure various stages of the HIV type 1 (HIV-1) life cycle. In many cases, imaging-based approaches have filled critical gaps in our understanding of how certain aspects of viral replication occur in cells. Specifically, live cell imaging has allowed a better understanding of dynamic, transient events that occur during HIV-1 replication, including the steps involved in viral fusion, trafficking of the viral nucleoprotein complex in the cytoplasm and even the nucleus during infection and the formation of new virions from an infected cell. In this review, we discuss how researchers have exploited fluorescent microscopy methodologies to observe and quantify these events occurring during the replication of HIV-1 in living cells.

Figure 1

Trafficking of HIV-1 viral complexes on microtubules. In this time lapse series, human osteosarcoma cells were microinjected with fluorescently labeled tubulin protein and infected with VSV-g pseudotyped HIV-1 virus. The image sequence shows an individual viral complex (green) trafficking on the microtubule network (red). Images were taken every 10 seconds in this movie series.

Figure 2

Visualizing the cell-to-cell contacts that mediate viral transfer. (a) The virological synapse visualized by Jolly and coworkers [47]. Cell-to-cell contact mediates the recruitment of CD4 receptor (red) on the target cell and viral envelope in the infected cell (green) to the VS. Scale bar, 1 μm. (b) The recruitment of individual HIV-1 virions (green) in the dendritic cell (bottom) to regions of the cell in contact with a T cell (top cell), as described by McDonald et al. [50]. Cells are visible by nuclear (blue) and actin (red) staining. (c) The recruitment of FlAsH labeled Gag protein (green) expressed in primary macrophages (M) to the point of cell-to-cell contact. Scale bar, 15 μm [51]. The left panel shows the Gag fluorescence, the middle panel shows the macrophages, and the right panel combines these images and the point of cell-to-cell contact is highlighted the boxed region. Panel (a) is reprinted with permission from Ref. [46], panel (b) is reprinted with permission from Ref. [48], and panel (c) is reprinted with permission from Ref. [49].

Figure I

The power of the OMX imaging system. (a) Polystyrene fluorescent beads approximately the size of virions (0.100 μm) were allowed to puddle on a #1.5 coverslip before imaging on a deconvolution fluorescence microscope. (b) Images were processed using constrained iterative deconvolution. (c) The same field of view imaged by the OMX superresolution microscope. Scale bar, 2.5 μm. Image courtesy of Applied Precision.