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Title: Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells}
Article:
Abstract:
Disassembly of the cone-shaped HIV-1 capsid in target cells is a prerequisite for establishing a life-long infection. This step in HIV-1 entry, referred to as uncoating, is critical yet poorly understood. Here we report a novel strategy to visualize HIV-1 uncoating using a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A( CypA-DsRed), which is specifically packaged into virions through the high-avidity binding to capsid( CA). Single virus imaging reveals that CypA-DsRed remains associated with cores after permeabilization/removal of the viral membrane and that CypA-DsRed and CA are lost concomitantly from the cores in vitro and in living cells. The rate of loss is modulated by the core stability and is accelerated upon the initiation of reverse transcription. We show that the majority of single cores lose CypA-DsRed shortly after viral fusion, while a small fraction remains intact for several hours. Single particle tracking at late times post-infection reveals a gradual loss of CypA-DsRed which is dependent on reverse transcription. Uncoating occurs both in the cytoplasm and at the nuclear membrane. Our novel imaging assay thus enables time-resolved visualization of single HIV-1 uncoating in living cells, and reveals the previously unappreciated spatio-temporal features of this incompletely understood process.
Introduction:
Mature HIV-1 particles contain a cone-shape capsid core made of a hexagonal lattice of the capsid protein( CA) that encases the viral genomic RNA, nucleocapsid( NC), reverse transcriptase( RT) and integrase( IN) proteins. After HIV-1 fuses with a target cell, the released viral cores go through a series of carefully orchestrated steps that ultimately lead to productive infection. A key early step of HIV-1 entry—referred to as uncoating—is generally defined as( full or partial) shedding of CA from the viral core( reviewed in[1–3]). Several lines of genetic and functional evidence support the importance of this incompletely understood process in regulating reverse transcription and nuclear import of pre-integration complexes( PICs)( reviewed in[1–3]). Given its critical role in productive entry, the HIV-1 capsid is considered an attractive target for the development of new antiviral drugs[1, 4]. HIV-1 uncoating has been traditionally studied by biochemical assays[5–8], which revealed a general correlation between capsid stability and infectivity, supporting the importance of timely uncoating[5]. Establishment of the fate-of-capsid assay[6, 7, 9], which determines the fraction of particulate capsid recovered from cells shortly after infection, allowed assessment of the effects of host factors and pharmacological agents on the core stability. However, most of these assays examine the bulk population of viruses, many of which are not infectious( reviewed in[1]). The more recently developed cyclosporine A( CsA) washout assay[8] enables indirect measurements of HIV-1 uncoating based upon the virus escape from the host restriction factor, TRIMCyp. However, this assay does not provide information regarding the sites of HIV-1 uncoating, and the interpretation of the obtained results is complex[1]. To elucidate the sites of HIV-1 uncoating, a complementary in-situ uncoating assay has been introduced[8]. This assay visualizes the loss of immunolabeled CA/p24 from the eGFP-Vpr labeled HIV-1 reverse transcription complexes( RTC) /PICs in fixed cells after infection. The inability to follow the dynamics of HIV-1 uncoating in fixed cells underscores the need for live cell imaging techniques. Direct visualization of capsid uncoating in living cells requires the labeling of both RTC/PIC and CA. Whereas PICs have been visualized by incorporating an integrase-GFP( IN-GFP) chimera into pseudoparticles[10, 11], attempts to label CA were not successful. CA labeling with a fluorescent protein or a smaller tetracysteine tag[12] adversely affects virus infectivity. Even point mutations in CA can compromise infectivity through altering capsid assembly and/or stability( e. g.,[5]). A recently introduced microscopy assay that may indirectly monitor HIV-1 uncoating live cells is based upon the assertion that a fraction of GFP molecules produced upon cleavage of Gag-iGFP precursor[13] is trapped within an intact mature core and is released at the time of uncoating[14, 15]. However, further validation of the notion that a detectable number of GFP molecules is trapped in a considerable fraction of mature cores is needed. Here, we developed a novel assay to visualize single HIV-1 uncoating in living cells using the oligomeric cyclophilin A-DsRed chimera( CypA-DsRed) as a novel CA probe. Single virus imaging reveals that this probe efficiently and specifically incorporates into HIV-1 particles without noticeably compromising viral infectivity. Unlike the monomeric version of CypA tagged with a fluorescent protein, CypA-DsRed tightly binds to CA and reports the loss of CA during HIV-1 uncoating. The loss of CypA-DsRed from single cores is delayed upon inhibition of reverse transcription and correlates well with the core stability in vitro and in living cells. Importantly, kinetic measurements revealed that the majority of cores uncoat shortly after viral fusion, whereas a small fraction of incoming cores retain CypA-DsRed for several hours. Collectively, our results show that CypA-DsRed enables time-resolved visualization of single HIV-1 uncoating, thus providing an important new tool for understanding the early steps in HIV-1 infection.
Discussion:
In this study, we developed a novel approach to detect the loss of CA from incoming HIV-1 cores in living cells, using the oligomeric CypA-DsRed marker. Previous attempts to label HIV-1 CA with a monomeric fluorescently tagged CypA were unsuccessful[20], most likely because of its low affinity for CA, estimated to be in the micromolar range[22–24, 52, 53]. In contrast, CypA-DsRed specifically incorporates into virions and remains bound to CA in the context of a mature core, enabling the visualization of single virus entry/uncoating without the need for genetic or pharmacologic manipulations that could perturb multiple functions of the CA protein. Importantly, the CypA-DsRed binding to the core is sufficiently tight to prevent its release after removal of the viral membrane in vitro or within several hours after virus fusion with cells. Except when displaced by CsA, CypA-DsRed is lost from IN-labeled RTC/PICs concurrently with loss of CA/p24. CypA has been implicated in multiple steps of HIV-1 entry, including:( i) CA uncoating[54, 55];( ii) virus restriction by rhesus TRIM5α[56];( iii) evasion of innate immune sensing[57];( iv) reverse transcription[58]; and( v) post-nuclear entry events[22, 59]. These functions have been attributed to CypA expressed in target cells, whereas the producer cell-expressed CypA that is incorporated into virions has been demonstrated to be inconsequential for infection[30, 31]. Remarkably, however, the virus-incorporated CypA-DsRed was able to functionally complement the lack of CypA in the target Jurkat CypA-/- cell line( Fig 1D). This important result supports the notion that, contrary to the virus-incorporated CypA that is quickly shed from post-fusion cores and replaced by CypA in target cells, the oligomeric CypA-DsRed remains tightly associated with the core. Interestingly, unlike the artificial dimeric and trimeric CypA constructs that bind more tightly to CA and inhibit HIV-1 infection when expressed in target cells[25], CypA-DsRed expressed in target 293T cells did not affect HIV-1 infection( S4 Fig). The basis for this difference is currently unclear. We surmise that a long flexible linker between CypA and DsRed allows for multivalent interactions with the core by adapting to the CA lattice rather than disrupting it. Whereas we cannot rule out an effect of CypA-DsRed on the stability of the HIV-1 core, as has been reported for monomeric CypA[33, 54, 58, 60], the lack of a negative effect on HIV-1 infectivity when expressed in producer or target cells, along with the existence of apparently intact post-fusion cores( that shed CypA-DsRed upon CsA addition) in the cytoplasm several hours after synchronized infection, show the utility of CypA-DsRed as a marker for HIV-1 uncoating. Our conclusion that CypA-DsRed reports the dissociation of CA from the viral core is also strongly supported by the excellent correlation between the HIV-1 capsid stability and loss of this marker, along with p24, from IN-labeled cores in vitro. Unlike other reported in vitro uncoating assays[5], the CypA-based assay enables the visualization of CA on mature native HIV-1 cores without the need for additional manipulations( e. g., ultracentrifugation) known to destabilize the fragile cores. In agreement with a previous study of HIV-1 core stability[33], we also observed that cytosolic extract from human cells inhibited the loss of CypA-DsRed/CA in vitro, thus enabling biochemical approaches to identify host factors that regulate the HIV-1 uncoating. Although the loss of CypA-DsRed from IN-labeled puncta in living cells exhibited more complex phenotypes than in in vitro experiments, we were able to demonstrate correlation between loss of this marker and the core stability using CA mutants. We emphasize that an observed loss of CypA-DsRed should not necessarily be interpreted as complete dissociation of CA. Due to the relatively high background signal in living cells, single virus imaging is only possible with a threshold number of fluorescent protein molecules in these particles. Thus, a small fraction of CA could remain associated with pre-integration complexes, as recently reported in fixed cell imaging studies[38, 61, 62]. Three principal models for HIV-1 uncoating in target cells have been proposed( Fig 7, reviewed in[1]). Uncoating may occur:( 1) immediately after virus fusion;( 2) gradually, during cytoplasmic trafficking of post-fusion cores; and( 3) at the nuclear pore. In this study, we observed intracellular particles that appear to represent all three types of uncoating. We found that, surprisingly, the majority of HIV-1 cores lost CypA-DsRed soon after release into the cytoplasm. Since HIV-1 cores are quite stable in vitro in the presence of cytosolic extract( Fig 2D), such a dramatic loss of integrity could be caused by innate host factors that induce HIV-1 uncoating and/or by initiation of reverse transcription( Fig 3F), which is unlikely to occur in our in vitro experiments. In stark contrast to the rapidly-uncoating cores, a small fraction of particles( <5%) retained CypA-DsRed for several hours after infection. The very slow loss of CypA-DsRed signal from these cores, together with inhibition of CypA-DsRed loss by Nevirapine, supports the gradual uncoating model[8, 45]. Finally, a fraction of late-uncoating cores that approached the nuclear membrane exhibited restricted movement( Fig 6B and 6C), which is consistent with HIV-1 uncoating at the nuclear pore[63–65]. Additional single particle tracking experiments using CypA-DsRed, combined with CA mutants exhibiting nuclear entry defects, will be useful for validating this model. Note that our findings apply to HeLa-derived cells that differ from other cell types in their uncoating/infection requirements[45, 57]. An important question that remains to be answered is which of the observed uncoating events—early or late—lead to productive infection. There is biochemical and virological evidence supporting early HIV-1 uncoating[8, 66]. The functional CsA washout assay[8], which relies on the kinetics of HIV-1 escape from the host restriction factor TRIMCyp, yielded a half-time of ~25–45 min for HIV-1 uncoating in owl monkey cells and ~9 min in HeLa-derived cells[8, 45, 67]. The latter value is close to our estimate of the half-time of post-fusion HIV-1 uncoating in HeLa-derived cells( ~13 min), which is delayed upon inhibition of reverse transcription( Fig 3F). However, since the structural basis for HIV-1 escape from TRIMCyp restriction in the CsA washout assay has not been fully elucidated[1], the rare post-fusion cores which survive for several hours in the cytoplasm may also contribute to productive entry. This notion is supported by the slow kinetics of reverse transcription observed in functional assays([38] and Fig 6A) and by inhibition of the late CypA-DsRed release events by Nevirapine( Figs 4E and 5G). In conclusion, we have developed a novel method for elucidating the HIV-1 uncoating process in living cells and in vitro, using CypA-DsRed as a marker for CA. This technological advance provides a powerful means to elucidate the spatial and temporal details of HIV-1 uncoating and the role of host factors in regulating this critical step of virus entry.
HIV-1 genome and key enzymes required for establishing productive infection are encased in a cone-shaped shell made of the capsid protein ( CA ) . After being released into the cytosol of target cells , the cone-shaped core complex undergoes a series of carefully orchestrated steps , including uncoating ( loss of CA ) . HIV-1 uncoating remains poorly understood , due in part to the lack of direct assays enabling studies of this process in living cells . Here , we introduce a novel strategy for labeling the HIV-1 capsid without genetically modifying the CA protein . We designed a novel fluorescent cyclophilin A construct that binds the capsid with an extremely high avidity and ( 1 ) efficiently incorporates into virions without compromising infectivity; ( 2 ) remains bound to cores after viral fusion; and ( 3 ) is lost from post-fusion cores along with CA . The novel imaging assay provides new insights into the kinetics and spatial distribution of HIV-1 uncoating in living cells .