Where in the Cell Are You? Probing HIV-1 Host Interactions through Advanced Imaging Techniques

Abstract

Viruses must continuously evolve to hijack the host cell machinery in order to successfully replicate and orchestrate key interactions that support their persistence. The type-1 human immunodeficiency virus (HIV-1) is a prime example of viral persistence within the host, having plagued the human population for decades. In recent years, advances in cellular imaging and molecular biology have aided the elucidation of key steps mediating the HIV-1 lifecycle and viral pathogenesis. Super-resolution imaging techniques such as stimulated emission depletion (STED) and photoactivation and localization microscopy (PALM) have been instrumental in studying viral assembly and release through both cell-cell transmission and cell-free viral transmission. Moreover, powerful methods such as Forster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) have shed light on the protein-protein interactions HIV-1 engages within the host to hijack the cellular machinery. Specific advancements in live cell imaging in combination with the use of multicolor viral particles have become indispensable to unravelling the dynamic nature of these virus-host interactions. In the current review, we outline novel imaging methods that have been used to study the HIV-1 lifecycle and highlight advancements in the cell culture models developed to enhance our understanding of the HIV-1 lifecycle.

Keywords: HIV-1; cell culture; host-pathogen interactions; microscopy.

Figure 1

Fluorescent human immunodeficiency virus type 1 (HIV-1) vectors enable the visualization of alternative methods of HIV-1 cell-free entry. (A) Dialkylcarbocyanine analogue (DiD)-labeled HIV-1 expressing green fluorescent protein (GFP)-tagged nucleocapsid identifies viral fusion at the plasma membrane. Plasma membrane fusion corresponds to a loss of red fluorescence due to diffusion of the DiD probe in the plasma membrane and the subsequent presence of green fluorescence; (B) Endosomal entry of HIV-1 is marked by red and green puncta within the cell as DiD probe diffusion is limited to the smaller endosomal location. Viral entry through an endosome is detected by the visualization of a green signal (GFP-tagged nucleocapsid) migrating away from a red signal (DiD-labeled endosome); (C) pH-dependent HIV-1 entry can be measured by incorporation of an intracellular adhesion molecule 1 (ICAM)-pHlourin-labeled HIV-1. The decrease of pHlourin fluorescence corresponds to the presence of HIV-1 within the increasingly acidic endosomal compartment.

Figure 2

Techniques to visualize cell–cell transmission of HIV-1 through virological synapses and tunneling nanotubules. Upon engagement of Env with cluster of differentiation 4 (CD4) and C-C chemokine receptor type 4/ C-X-C chemokine receptor type 5 (CCR4/CXCR5), there is a recruitment of host cellular factors including ICAM-1 and leukocyte function-associated antigen 1 (LFA-1) to form the virological synapse. (A) 3D-confocal microscopy enables the visualization of GFP-Gag (green) particles across the virological synapse; (B) Super-resolution imaging of ATTO-647N (red) demonstrated that labeled HIV-1 can be taken up by a neighbouring cell though protruding membrane extensions within the virological synapse; (C) Fluorescence recovery after photobleaching (FRAP) studies enable imaging of the dynamics of membrane proteins in live cells to investigate the structure of tunneling nanotubules. Hexagons represent membrane proteins; bleached: black, fluorescent: green; (D) Co-culture of infected primary cells with TZM-Bl cells can be used to detect cell–cell transfer of HIV-1 by β-galactosidase activity, which is expressed under the regulation of the HIV-1 promoter; LTRs (long terminal repeats) are DNA sequences used by retroviruses to integrate into the host genome.

Figure 3

Selected imaging techniques and vectors used to identify key steps in HIV-1 trafficking to the nucleus. (A) yellow fluorescent protein-apolipoprotein B mRNA editing enzyme catalytic subunit 3G (YFP-APOBEC3G)-labeled HIV-1 virions (represented by yellow circles) demonstrate the trafficking of the nucleocapsid to the nuclear pore; (B) Click-labelling allows detection of reverse transcription in the infected cell, where the incorporation of 5’-ethynyl 2’-deoxyuridine into newly synthesized viral DNA results in detectable fluorescence, represented by the yellow star; (C) The use of fluorescence in situ hybridization (FISH) probes (black with green fluorophore) identified that preferential integration sites (blue) are located near nuclear pores.

Figure 4

Fluorescence-based methods to monitor virus–host interactions required for the HIV-1 accessory protein negative factor (Nef) to hijack the host cell. (A) Key interactions between Nef and the Src-family kinase Hck are detectable in cells by Forster resonance energy transfer (FRET). Co-expression of Hck–CFP (cyan fluorescent protein) and Nef–YFP (yellow fluorescent protein) generates a FRET signal demonstrating their interaction. This interaction activates the signaling pathways required to internalize major histocompatability complex-I (MHC-I) from the cell surface to prevent cytotoxic killing of HIV-1 infected cells; (B) Bimolecular fluorescence complementation (BiFC) reports the interaction between the PACS1 and PACS2 proteins and Nef, localizing these interactions to early and late endosomal compartments, respectively. Yn: N-terminal of YFP, Yc: C-terminal of YFP, which reconstitute upon protein–protein interactions.

Figure 5

Tools to study HIV-1 assembly and budding. (A) Gag and viral RNA has been localized to the centriolar region by visualizing FRET between a FISH probe (tetramethylrhodamine (TRITC):Red) targeted towards viral RNA and AlexaFluor-488 immunostained Gag (Green); (B) Gag multimers assemble on the plasma membrane prior to viral RNA localization. RNA dynamics can be visualized by exploiting the high affinity interaction between the major capsid protein (GFP (Green)–major capsid protein (MCP; Brown)) and an MS2 bacteriophage stem-loop engineered onto the HIV-1 genome (C) Super-resolution interferometric photoactivation and localization microscopy (iPALM) imaging demonstrates how endosomal sorting complexes required for transport (ESCRT) proteins assemble around HIV-1 budding sites. Charged multivesicular body protein 2a/4b (CHMP2A/4B) (Red and Green) assemble within the neck of the budding virion to enable pinching of the plasma membrane to form single virions.