Imaging HIV-1 Genomic DNA from Entry through Productive Infection

Abstract

In order to track the fate of HIV-1 particles from early entry events through productive infection, we developed a method to visualize HIV-1 DNA reverse transcription complexes by the incorporation and fluorescent labeling of the thymidine analog 5-ethynyl-2=-deoxyuridine (EdU) into nascent viral DNA during cellular entry. Monocyte-derived macrophages were chosen as natural targets of HIV-1, as they do not divide and therefore do not incorporate EdU into chromosomal DNA, which would obscure the detection of intranuclear HIV-1 genomes. Using this approach, we observed distinct EdU puncta in the cytoplasm of infected cells within 12 h postinfection and subsequent accumulation of puncta in the nucleus, which remained stable through 5 days. The depletion of the restriction factor SAMHD1 resulted in a markedly increased number of EdU puncta, allowing efficient quantification of HIV-1 reverse transcription events. Analysis of HIV-1 isolates bearing defined mutations in the capsid protein revealed differences in their cytoplasmic and nuclear accumulation, and data from quantitative PCR analysis closely recapitulated the EdU results. RNA fluorescence in situ hybridization identified actively transcribing, EdUlabeled HIV-1 genomes in productively infected cells, and immunofluorescence analysis confirmed that CDK9, phosphorylated at serine 175, was recruited to RNApositive HIV-1 DNA, providing a means to directly observe transcriptionally active HIV-1 genomes in productively infected cells. Overall, this system allows stable labeling and monitoring of HIV genomic DNA within infected cells during cytoplasmic transit, nuclear import, and mRNA synthesis. IMPORTANCEThe fates of HIV-1 reverse transcription products within infected cells are not well understood. Although previous imaging approaches identified HIV-1 intermediates during early stages of infection, few have connected these events with the later stages that ultimately lead to proviral transcription and the production of progeny virus. Here we developed a technique to label HIV-1 genomes using modified nucleosides, allowing subsequent imaging of cytoplasmic and nuclear HIV-1 DNA in infected monocyte-derived macrophages. We used this technique to track the efficiency of nuclear entry as well as the fates of HIV-1 genomes in productively and nonproductively infected macrophages. We visualized transcriptionally active HIV-1 DNA, revealing that transcription occurs in a subset of HIV-1 genomes in productively infected cells. Collectively, this approach provides new insights into the nature of transcribing HIV-1 genomes and allows us to track the entire course of infection in macrophages, a key target of HIV-1 in infected individuals.KEYWORDS fluorescent-image analysis, human immunodeficiency virus, macrophages H uman immunodeficiency virus type 1 (HIV-1) infection can be viewed as occurring in early (entry) stages, including reverse transcription (RT), nuclear import, and the integration of the viral genome, and late (productive) stages, including viral transcr...

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

“…Regardless, we failed to detect preferential PN localization using either the Provirus ViewHIV assay, which predominantly detects integrated HIV-1 (Figure S1), or the SCIP assay, which exclusively scores for integration (Figure 2B). We also note that visual inspection of images of MDM-infected cells are consistent with the notion that HIV-1 disperses fairly randomly throughout cell nuclei (Stultz et al, 2017). …”

Early cytoplasmic uncoating is associated with infectivity of HIV-1

“…However, it is not clear that this is how uncoating proceeds in other cell types. For example, it was recently reported that reverse transcription, nuclear localization, and loss of capsid took place at a slower rate in primary macrophages, as monitored by specific labeling of the viral DNA (38). Other circumstances in vivo may also show different kinetics of uncoating.…”

“…The HIV-1 capsid uncoating dependence on microtubule stability also supports a cytoplasmic uncoating (25,26,(30)(31)(32)(33)(34). Recently, the importance and dependence on CA of most of the early steps of infection have been partially explained by the identification of a small amount of CA that associates with the PIC in the nucleus (35)(36)(37)(38). Because of the differences between these models, the kinetics, dynamics, and cellular location of uncoating of infectious HIV-1 particles have remained controversial topics (4, 25-29, 37, 39, 40).…”

Localized Phosphorylation of a Kinesin-1 Adaptor by a Capsid-Associated Kinase Regulates HIV-1 Motility and Uncoating

“…Given the size of the viral core, the capsid (CA) shell has to disassemble or “uncoat” to allow the genome to pass through nuclear pores (Campbell and Hope, 2015). Although some CA can be detected in the nucleus and functions in processes after nuclear entry (Chen et al, 2016; Hulme et al, 2015; Koh et al, 2013; Peng et al, 2014; Schaller et al, 2011; Stultz et al, 2017), whether reverse transcription and uncoating occur during transport along MTs in the cytoplasm or at nuclear pores remains a matter of debate (Arhel et al, 2007; Fassati and Goff, 2001; Le Sage et al, 2014; McDonald et al, 2002; Miller et al, 1997; Rasaiyaah et al, 2013; Zhou et al, 2011). However, recent live cell imaging supports the notion that uncoating begins in the cytoplasm (Francis et al, 2016).…”

“…However, recent live cell imaging supports the notion that uncoating begins in the cytoplasm (Francis et al, 2016). Notably, uncoating and reverse transcription are intricately coupled processes (Cosnefroy et al, 2016; Hulme et al, 2011; Rankovic et al, 2017; Stultz et al, 2017; Yang et al, 2013) that are further intertwined with the bi-directional movement of HIV-1 particles. Interfering with MT or dynein function suppresses HIV-1 trafficking and delays uncoating during early infection (Arhel et al, 2006; Delaney et al, 2017; Lukic et al, 2014; Malikov et al, 2015; McDonald et al, 2002; Pawlica and Berthoux, 2014; Sabo et al, 2013).…”