Multiplex single-cell visualization of nucleic acids and protein during HIV infection

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.

Capsid-CPSF6 Interaction Licenses Nuclear HIV-1 Trafficking to Sites of Viral DNA Integration

“…To assess the generalizability of these observations, we utilized the MICDDRP bDNA hybridization technique, which, at 24 hpi, does not distinguish between unintegrated vDNA and proviral DNA (Puray-Chavez et al, 2017). Cells infected at MOI 0.2 showed the same basic dispersion of vDNA foci as obtained using the Provirus ViewHIV technique (compare Figure 1B to 1A).…”

“…For the experiment reported in Figure S1, HeLa MAGI cells were infected with 10,000 AFUs per cell, yielding an approximate MOI of 70. For MICDDRP experiments, viral titer was determined as the number of blue forming units (BFU) using TZM-bl cells in the presence of 20 μg/mL DEAE-Dextran as described (Puray-Chavez et al, 2017). …”

“…Semi-synchronized infections for ViewHIV (50,000 AFU per cell) and MICDDRP (0.2 BFU per cell) were performed essentially as described (Chin et al, 2015; Puray-Chavez et al, 2017). HEK293T cells were plated onto coverslips precoated with collagen in 24-well plates.…”

“…Pre-amp, amplifier, and label probes were then added to the coverslips as described in (Chin et al, 2015). For the experiment in Figure S1, coverslips were additionally treated with 5 mg/mL RNase A for 30 min at 37°C as described (Puray-Chavez et al, 2017). RNase A treatment was done after protease (PIC assay) or pepsin (provirus assay) treatment.…”

“…Here we analyze the functions of CPSF6 and LEDGF/p75 in HIV-1 intranuclear localization by i) imaging PICs using independently developed ViewHIV and multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP) branched DNA (bDNA) hybridization techniques (Chin et al, 2015; Puray-Chavez et al, 2017), (ii) imaging proviruses using Provirus ViewHIV (Chin et al, 2015) and Single Cell Imaging of HIV-1 Provirus (SCIP) (Di Primio et al, 2013) assays, iii) mapping viral integration sites (Serrao et al, 2016; Sowd et al, 2016), and iv) visualizing the intranuclear positions of some of the genes that are preferred integration targets in wild type (WT) cells or when LEDGF/p75- or CPSF6-dependent targeting pathways are disrupted. Our results fail to support the notion that HIV-1 integration favors the nuclear periphery under baseline infection conditions.…”