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.

Application of Advanced Imaging to the Study of Virus–Host Interactions

“…The review by Mukherjee et al [ 3 ] summarizes strategies to study the early phase of HIV-1 infection by fluorescence microscopy. Quantitative microscopy and multi-color imaging studies have brought to light new data on the dynamics of the early stages of infection, from entry to integration in the nucleus, as well as the role of host cell factors participating in infection.…”

Overlap Intensity: An ImageJ Macro for Analyzing the HIV-1 In Situ Uncoating Assay

“…There are multiple assays available to study uncoating including capsid core stability assays, the fate of the capsid assay, the CsA washout assay, and fluorescence microscopybased uncoating assays [1,3,[12][13][14][15][16][17][18][19][20][21][22][23][24][25]. Each of these assays has different strengths and weaknesses, so often a combination of assays is used to study the factors that influence uncoating.…”

“…Each of these assays has different strengths and weaknesses, so often a combination of assays is used to study the factors that influence uncoating. Several uncoating assays have been developed utilizing fluorescence microscopy with capsid detection by antibody staining or through the use of fluorescent markers [8,[15][16][17][19][20][21][22]. Collectively, fluorescence microscopy techniques allow the process of uncoating to be studied in infected cells with direct visualization of the capsid and the process of uncoating relative to other cellular structures or proteins.…”

“…Most recently, fluorescent microscopy-based uncoating assays have been developed that utilize live cell imaging to track the progression of early steps of replication (uncoating, reverse transcription, cytoplasmic transit, nuclear import) in single virions and, in some assays, infection of the target cell [15][16][17][22][23][24][25]. Despite the importance of these experiments, the equipment needed to maintain live cell imaging can be limiting to research institutions.…”

Rapid detection of φx-174 virus based on synchronous fluorescence of Tryptophan

“…[4] Purification of viruses from their host cell debris and other contaminants was previously done by different methods of density gradient centrifugation [5], ultrafiltration [6], and different chromatographic methods. [7,8] The small sizes of viruses makes them invisible to simple microscopic observation and can be done by transmission electron microscopy (TEM) [9] or by utilization of fluorescent labelling agents under advanced microscopic instrumentation [10]. Determination of viral structures was extensively investigated by X-ray crystallography, NMR and cryo-TEM.…”