Abstract
Technical limitations in simultaneous microscopic visualization of RNA, DNA, and proteins of HIV have curtailed progress in this field. To address this need we develop a microscopy approach, multiplex immunofluorescent cell-based detection of DNA, RNA and Protein (MICDDRP), which is based on branched DNA in situ hybridization technology. MICDDRP enables simultaneous single-cell visualization of HIV (a) spliced and unspliced RNA, (b) cytoplasmic and nuclear DNA, and (c) Gag. We use MICDDRP to visualize incoming capsid cores containing RNA and/or nascent DNA and follow reverse transcription kinetics. We also report transcriptional “bursts” of nascent RNA from integrated proviral DNA, and concomitant HIV-1, HIV-2 transcription in co-infected cells. MICDDRP can be used to simultaneously detect multiple viral nucleic acid intermediates, characterize the effects of host factors or drugs on steps of the HIV life cycle, or its reactivation from the latent state, thus facilitating the development of antivirals and latency reactivating agents.
Highlights
Technical limitations in simultaneous microscopic visualization of RNA, DNA, and proteins of HIV have curtailed progress in this field
fluorescence in situ hybridization (FISH) techniques have been established for detection of nucleic acids in cells, but lack the sensitivity required for some applications, and are often incompatible with immunofluorescent labeling
BDNA-FISH techniques[6] have been developed to enhance the sensitivity and specificity of RNA detection, (e.g., PrimeFlow[7], ViewRNA (Affymetrix) and RNAscope8) and permit co-staining by immunofluorescence. branched DNA (bDNA)-FISH approaches have been adapted for imaging of HIV-1 nucleic acids[5, 9]
Summary
Technical limitations in simultaneous microscopic visualization of RNA, DNA, and proteins of HIV have curtailed progress in this field. One of the first approaches to allow visualization of integrated HIV-1 proviruses exploited the recruitment of specific histones to sites of DNA damage, in combination with a reporter virus containing a rare restriction site[1] This single-cell imaging of HIV-1 provirus (SCIP) approach provided sensitive labeling of integrated provirus, but not unintegrated vDNA, in apparent contrast to later techniques. For nucleic acid labeling in dividing cells, several groups have applied variations of fluorescence in situ hybridization (FISH); either immuno-DNA FISH4 or branched DNA (bDNA)-FISH5 These FISH approaches allowed investigators to examine the vDNA localization at various points during infection, and to identify the number and position of viral integration sites in the host genome. The ability to visualize these nucleic acid intermediates in the context of viral or host proteins will advance efforts to elucidate mechanisms of antiviral inhibition by small molecules or host restriction factors, enhance our understanding of latency reactivation, and further efforts for novel drug development
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