Abstract
Retroviral infection involves the reverse transcription of the viral RNA genome into DNA, which is subsequently integrated into the host cell genome. Human immunodeficiency virus type 1 (HIV-1) and other lentiviruses mediate the infection of non-dividing cells through the ability of the capsid protein1 to engage the cellular nuclear import pathways of the target cell and mediate their nuclear translocation through components of the nuclear pore complex2,3,4. Although recent studies have observed the presence of the capsid protein in the nucleus during infection5,6,7,8, reverse transcription and disassembly of the viral core have conventionally been considered to be cytoplasmic events. Here, we use an inducible nuclear pore complex blockade to monitor the kinetics of HIV-1 nuclear import and define the biochemical staging of these steps of infection. Surprisingly, we observe that nuclear import occurs with relatively rapid kinetics (<5 h) and precedes the completion of reverse transcription in target cells, demonstrating that reverse transcription is completed in the nucleus. We also observe that HIV-1 remains susceptible to the capsid-destabilizing compound PF74 following nuclear import, revealing that uncoating is completed in the nucleus. Additionally, we observe that certain capsid mutants are insensitive to a Nup62-mediated nuclear pore complex blockade in cells that potently block infection by wild-type capsid, demonstrating that HIV-1 can use distinct nuclear import pathways during infection. These studies collectively define the spatio-temporal staging of critical steps of HIV-1 infection and provide an experimental system to separate and thereby define the cytoplasmic and nuclear stages of infection by other viruses.
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Data availability
The Nup62-DmrB-GFP plasmids used in this study have been deposited with Addgene; relevant information is provided in the Methods. All data generated or analysed during this study are presented in the paper, in the supplementary information or as source data. All data are available from the corresponding author upon reasonable request. Correspondence and requests for materials should be addressed to E.M.C. Source data for Figs. 3 and 4 and Extended Data Figs. 5, 6 and 8 are included with this paper.
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Acknowledgements
We thank K. Verhey for providing us with the constructs required for the study. We thank the anonymous blood donors who contributed leukocytes to this study. We thank S. Zack for proofreading the article. This work was supported by NIH grant no. R21AI139009 to E.M.C.
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A.D. and E.M.C. designed the experiments. A.D., N.B., S.T. and V.Z. conducted the experiments. A.D., N.B., S.T. and E.M.C. analysed the data. A.D. and E.M.C. wrote the manuscript. E.M.C. supervised the study.
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Extended data
Extended Data Fig. 1 Expression of the Nup62 dimerization construct in cells does not alter normal cell physiology.
a, HeLa cells were transduced simultaneously with lentiviral vectors driving expression of Nup62DG or mCherry-estrogen receptor alpha (ERα). Imaging fields were selected that contain cells expressing only ERα (marked by asterisk) or both ERα and Nup62DG. Live cell imaging was performed after addition of estradiol (E2) to mediate nuclear translocation of ERα. Images acquired every four minutes for a total of one hour. b, Similar experiment as in (a) except the cells were treated with the homodimerizing drug (HD) to block NPC transport along with E2 treatment. c, Accumulation of mCherry-ERα fluorescence in the nuclear region was monitored in Nup62DG+ and Nup62DG- cells treated with E2 using ImageJ plugin Time Series Analyzer V3. Depicted mean values from three independent experiments (±SEM). d, similar quantification as in (c) in cells treated with E2 and HD. Error bar represents SEM. Statistical significance was assessed using Two-way ANOVA and Bonferroni post test. P<0.05 was considered significant in our experiments.
Extended Data Fig. 2 Quantification strategy employed to quantify nuclear, perinuclear and cytoplasmic signal using Imaris.
Schematic representation of the quantification technique used in this manuscript with an example image of a cell expressing the Nup62DG and infected with HIV-1. Cells were stained for the capsid protein p24 and DAPI. a, Nuclear and perinuclear signal was quantified using a DAPI mask generated using the surface function in Imaris that exceeded the boundary of DAPI stain to include the perinuclear signal. As depicted, all signal within this mask was considered nuclear and perinuclear signal. Similarly, all signal outside of this mask was considered cytoplasmic signal. b, To focus on exclusively nuclear signal, an algorithm which reliably identified the nuclear boundary, as indicated by a DAPI stain, was generated in Imaris. Sections close to the upper and lower boundary of the nucleus (in Z) were removed to focus analysis on nuclear events. As in (a) signal within this mask was considered nuclear and all signal outside this mask was considered cytoplasmic and perinuclear.
Extended Data Fig. 3 HIV-1 infection induces Nup62 relocalization and colocalization with HIV-1 cores in the cytoplasm.
a, Nup62 localization in uninfected HeLa cells. b, HeLa cells synchronously infected with VSVg- R7ΔEnvmCherry at 3 h post infection, fixed and stained for Nup62 (green), HIV-1 capsid protein p24 (red), and DAPI (blue) for cell nuclei. Enlarged view of colocalization (boxed region) between Nup62 and HIV-1 capsid protein p24 shown in the bottom panel and indicated by arrows. c, Quantification of cytoplasmic Nup62, as described in methods section and Supplementary Fig. 2a. 20 or more cells analyzed per sample. Data averaged from three independent experiments. Error bar represents SEM. d, Quantification of the percent p24 colocalizing with Nup62 in HeLa cells. 20 or more cells analyzed per sample. Data averaged from three independent experiments. Error bar represents SEM. Statistical significance was assessed using Two-way ANOVA and Bonferroni post test. P<0.05 was considered significant in our experiments.
Extended Data Fig. 4 HIV-1 particles retained at the NPC over extended period upon Nup62 dimerization.
HeLa cells expressing Nup62DG synchronously infected with Gag-Integrase-Ruby (GIR) labeled HIV-1 particles. 1 hour following synchronized infection, cells were treated with homodimerizing drug (HD) and imaged every 4 minutes for 1 hour. Snapshot of a GIR labeled virus particle (boxed region) at the indicated times during acquisition is shown. Similar pattern observed across 5 independent experiments.
Extended Data Fig. 5 Monitoring reverse transcription with a low dose of RT inhibitor nevirapine to mirror the level of inhibition induced by Nup62DG blockade.
THP1 cells expressing the Nup62DG were differentiated to macrophages and infected with VSVg- R7ΔEnvmCherry and NIK monitored by HD addition (red) as described in Fig. 2. To monitor replication kinetics, cells incubated with HIV-1 reverse transcriptase inhibitor Nevirapine (NVP, 1.6 µM) at different time’s post infection as depicted (green) and washed off at 24 h. Infection measured as described in Fig. 2. Bar graphs depict data from a single experiment and line graphs depict normalized and average data (±SEM) as described in Fig. 2 from three independent experiments. NT represents no treatment.
Extended Data Fig. 6 HIV-1 reverse transcription completes in the nucleus of infected T cells.
a-b, CEM and SupT1 expressing the Nup62DG infected with VSVg- R7ΔEnvmCherry and NIK monitored by HD addition (red) as described in Fig. 2. To monitor replication kinetics, cells incubated with HIV-1 reverse transcriptase inhibitor Nevirapine (NVP, 5µM) at different time’s post infection as depicted (green) and washed off at 24 h. Infection measured as described in Fig. 2. c, Similar experiment as above done on SupT1 cells expressing Nup62DG after infection with R7ΔEnvmCherry pseudotyped with HIV-1 envelope from the HXB2 strain. All bar graphs depict data from a single experiment and line graphs depict normalized and average data (±SEM) as described in Fig. 2 from three independent experiments. NT represents no treatment.
Extended Data Fig. 7 Positive strand HIV-1 vDNA colocalize with Negative strand only in the nucleus of infected THP1 differentiated macrophages and primary macrophages.
a,b, THP1 differentiated macrophages (a) and monocyte derived macrophages (b) synchronously infected with VSVg- R7ΔEnvmCherry and fixed at different times post synchronized infection. Cells treated with RNase A and stained for (–) VDNA (orange) and (+) vDNA (red) using specific sense and antisense probes. Upon probe staining, these cells were also stained for HIV-1 capsid protein p24 (green) and nuclear Lamin A/C (blue). Depicted a representative image at the indicated time points. Data shown here is representative of three independent experiments. Quantification provided in Fig. 3d.
Extended Data Fig. 8 Nup358 relocalization induced upon HIV-1 infection absent upon inhibition of HIV-1 reverse transcription.
a, HeLa cells expressing the Nup62DG infected with VSVg- R7ΔEnvmCherry and NIK monitored by HD addition alone (red) or in the presence of NVP treatment for the first 6 hours (blue) following synchronized infection. Bar graphs depict data from a single experiment and line graphs depict normalized and average data (±SEM) as described in Fig. 2 from three independent experiments. b, HeLa cells and primary human macrophages (MDM) synchronously infected with HIV-1 (WT) and treated with HIV-1 reverse transcriptase inhibitor NVP. Cells fixed 0, 1 and 3 h (shown) post infection and stained for Nup358 (green), HIV-1 capsid protein p24 (red), and DAPI (blue). Middle panel depicts colocalization between Nup358 and p24. Data shown here is representative of three independent experiments. c,d, Percent Nup358 in the cytoplasm and percent p24 colocalizing with Nup358 at the different times post infection in HeLa and MDM. 20 or more cells analyzed in each experiment. Data averaged (mean) from three independent experiment. e, Proximity ligation assay performed in HeLa and MDM after HIV-1 infection and cells fixed 3h post infection. Each red puncta represents a positive PLA signal generated by interaction of Nup358 and p24. f, Average fold increase in PLA signal, relative to uninfected control, from three independent experiments. 20 or more cells analyzed in each experiment. Error bar represents SEM. All statistical significance was assessed using Two-way ANOVA and Bonferroni post test. P<0.05 was considered significant in our experiments.
Extended Data Fig. 9 Nuclear p24 signal upon inhibition of reverse transcription.
a, HeLa cells expressing Nup62DG infected with VSVg- R7ΔEnvmCherry and treated with HIV-1 reverse transcriptase inhibitor NVP for 7 hours following synchronized infection. Cells fixed and stained for HIV-1 capsid protein p24 (red), and DAPI (blue). b, Quantification of nuclear HIV-1 p24 signal, performed as described in methods and Supplementary Fig. 2b. 20 or more cells analyzed in each experiment. Data averaged from three independent experiments. Error bar represents SEM. Statistical significance was assessed using Two-way ANOVA and Bonferroni post test. P<0.05 was considered significant in our experiments.
Extended Data Fig. 10 Insensitivity of the capsid mutants N74D and P90A to the Nup62 mediated artificial nuclear pore block suggests heterogeneity in the nuclear pores and usage by WT virus.
HeLa cells stably expressing the Nup62DG infected with HIV-1 harboring either the WT capsid (blue) or capsid mutants N74D (red) and P90A (orange). NIK measured in these cells by HD addition as described in Fig. 2. a, Shown a bar graph from a single experiment. b, Percent inhibition attained upon blocking the nuclear pore for the first 24 h. Data averaged from three independent experiments. Error bar represents SEM.
Supplementary information
Supplementary Information
Supplementary Figs. 1 and 2.
Supplementary Fig. 1
Unprocessed western blots.
Supplementary Video 1
HeLa cells expressing Nup62DG synchronously infected with Gag-Integrase-Ruby-labelled HIV-1 particles. 1 h following synchronized infection, cells were treated with HD and imaged every 4 min for 1 h using deltavision microscope. Video depicts (shown by arrow) a HIV-1 particle (red) retained at the NPC (green) for an extended period. A similar pattern was observed across five independent experiments.
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Source Data Extended Data Fig. 5
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Dharan, A., Bachmann, N., Talley, S. et al. Nuclear pore blockade reveals that HIV-1 completes reverse transcription and uncoating in the nucleus. Nat Microbiol 5, 1088–1095 (2020). https://doi.org/10.1038/s41564-020-0735-8
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DOI: https://doi.org/10.1038/s41564-020-0735-8
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