Imaging Viral Infection by Fluorescence Microscopy: Focus on HIV-1 Early Stage

Abstract

During the last two decades, progresses in bioimaging and the development of various strategies to fluorescently label the viral components opened a wide range of possibilities to visualize the early phase of Human Immunodeficiency Virus 1 (HIV-1) life cycle directly in infected cells. After fusion of the viral envelope with the cell membrane, the viral core is released into the cytoplasm and the viral RNA (vRNA) is retro-transcribed into DNA by the reverse transcriptase. During this process, the RNA-based viral complex transforms into a pre-integration complex (PIC), composed of the viral genomic DNA (vDNA) coated with viral and host cellular proteins. The protective capsid shell disassembles during a process called uncoating. The viral genome is transported into the cell nucleus and integrates into the host cell chromatin. Unlike biochemical approaches that provide global data about the whole population of viral particles, imaging techniques enable following individual viruses on a single particle level. In this context, quantitative microscopy has brought original data shedding light on the dynamics of the viral entry into the host cell, the cytoplasmic transport, the nuclear import, and the selection of the integration site. In parallel, multi-color imaging studies have elucidated the mechanism of action of host cell factors implicated in HIV-1 viral cycle progression. In this review, we describe the labeling strategies used for HIV-1 fluorescence imaging and report on the main advancements that imaging studies have brought in the understanding of the infection mechanisms from the viral entry into the host cell until the provirus integration step.

Keywords: HIV-1; fluorescence microscopy; virus labeling.

Figure 1

Reported strategies for fluorescent labeling of different Human Immunodeficiency Virus 1 (HIV-1) components (see Table 1 for details).

Figure 2

Fluorescence microscopy imaging of viral fusion: (A) Schematic of doubly labeled HIV-1 viral particles entering into the cell by fusion with the plasma membrane (left) or with the endosomal membrane (right). (B) A complete fusion of HIV-1 (JRFL strain) with endosomal membrane is evidenced by the loss of the green fluorescence signal due to the content release, and the incorporation of the red membrane stain into the endosomal membrane. The blue trace on the last image represents the intracellular trajectory of a viral particle. (Reproduced with permission from [45], copyright 2009, Elsevier).

Figure 3

Fluorescence-based monitoring of the cytoplasmic release of viral proteins: (A) Principle of FlAsH fluorescence quenching at high concentrations. A decrease in the concentration of labeled NCp7 first results in an increase in fluorescence emission due to the reduction in quenching, followed by a decrease when the quenching effect is no longer present and the number of NCp7 molecules decreases further in the complexes. (B) Confocal images of HeLa cells infected by NCp7-TC/FlAsH-containing VSV-G pseudotyped HIV-1 particles at 2, 8, and 16 h.p.i., Scale bar: 10µm (C) Fluorescence intensity of individual cytoplasmic viral complexes detected at 2, 8, and 16 h.p.i. The fluorescence increase reflects the loss of quenching during the release of NCp7-TC/FlAsH molecules. (The box-plot represents SD values, the line and the square represent the median and the mean value, respectively (adapted from [22], copyright 2019, Springer Nature)). (D) Confocal images of C18166 T cells infected by VSV-G pseudotyped HIV-1 containing IN-GFP (green spots), lamin immunostaining (blue); red arrows indicate viral particles located in cell nuclei (E) Intensity of fluorescent spots detected in the cytoplasm and the nucleus (box-plot whiskers represent 5th and 95th percentile, the line and the square depict median and mean value, respectively) (F) Mean FRET ratio of HIV-1 pseudoviruses containing IN-mTFP and IN-mVenus in the cytoplasm and the nucleus, ** p-value < 0.05 (reproduced with permission from the Ref. [21] Copyright 2016, Springer Nature).

Figure 4

Two conflicting examples of uncoating studies based on live-cell imaging of the loss of capsid labeling. (A) Imaging of uncoating at the nuclear pore by time lapse imaging of HeLa-derived cells expressing EBFP2-Lamin and infected with INsfGFP/CypA-DsRed-labeled HIV-1 pseudoviruses. The red signal (CypA-DsRed) is lost upon nuclear entry. (B) Fluorescence intensity time trace and (C) trajectory of a viral particle revealing the capsid loss during docking at the nuclear envelope characterized by confined movements (reproduced with permission from [13], copyright 2018, Cell Press). (D) Time lapse images of CA-eGFP labeled viral particles in HeLa cells expressing BglG-mCherry (located in the cell nucleus) from 6.5 h.p.i. to 7.5 h.p.i. (scale bar 2 µm) (E) The normalized mean intensity time trace of CA-eGFP from the viral particle indicate that most CA-eGFP molecules remain associated with the viral core upon nuclear entry (reproduced with permission from [18], copyright 2020, PNAS).

Figure 5

Monitoring of HIV-1 in the nucleus. (A) 2D projection of the 3D tracking of an IN-YFP labeled HIV-1 complex in HeLa cells expressing POM121-mCherry as an NPC marker. After a long initial docking at the nuclear envelope, the complex enters the nucleus. Scale bar: 5 μm. (B) Trajectory of the imaged particle (C) Representative images of nuclear entry followed by a phase of rapid movements and a second phase of reduced mobility. Scale bars: 2 μm. (D) Schematic of the method of HIV-1 pseudogenome labeling (E) Confocal image of the sites of BglG-YFP accumulation in the nucleus of an infected HeLa cell. The black arrows show the viral RNA and the white arrow shows the transcription site. Scale bar: 5 µm. (Reproduced with permission from the Ref. [12] Copyright 2017, PLOS).