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Sec24C is an HIV-1 host dependency factor crucial for virus replication

Nature Microbiology volume 6, pages 435–444 (2021)Cite this article

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Abstract

Early events of the human immunodeficiency virus 1 (HIV-1) lifecycle, such as post-entry virus trafficking, uncoating and nuclear import, are poorly characterized because of limited understanding of virus–host interactions. Here, we used mass spectrometry-based proteomics to delineate cellular binding partners of curved HIV-1 capsid lattices and identified Sec24C as an HIV-1 host dependency factor. Gene deletion and complementation in Jurkat cells revealed that Sec24C facilitates infection and markedly enhances HIV-1 spreading infection. Downregulation of Sec24C in HeLa cells substantially reduced HIV-1 core stability and adversely affected reverse transcription, nuclear import and infectivity. Live-cell microscopy showed that Sec24C co-trafficked with HIV-1 cores in the cytoplasm during virus ingress. Biochemical assays demonstrated that Sec24C directly and specifically interacted with hexameric capsid lattices. A 2.3-Å resolution crystal structure of Sec24C228–242 in the complex with a capsid hexamer revealed that the Sec24C FG-motif bound to a pocket comprised of two adjoining capsid subunits. Combined with previous data1,2,3,4, our findings indicate that a capsid-binding FG-motif is conserved in unrelated proteins present in the cytoplasm (Sec24C), the nuclear pore (Nup153; refs. 3,4) and the nucleus (CPSF6; refs. 1,2). We propose that these virus–host interactions during HIV-1 trafficking across different cellular compartments are crucial for productive infection of target cells.

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Fig. 1: Identification of Sec24C as a cellular interacting partner of HIV-1 cores.
Fig. 2: A role of Sec24C during early steps of HIV-1 infection.
Fig. 3: A role of Sec24C in spreading of replication-competent HIV-1 in Jurkat cells.
Fig. 4: Structural basis for Sec24C228–242 interaction with the CA hexamer.

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Data availability

The datasets generated and analysed during the current study are included in this article or are available in the Protein Data Bank under the accession number 6PU1 (structural data) and via ProteomeXchange with identifier PXD020970 (proteomics data). All other data are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

Purified recombinant Sec23A/Sec24C and Sec23A/Sec24D proteins were a gift from R. Lesch, University of California Berkeley. We are grateful to M. Dzieciatkowska, University of Colorado School of Medicine Biological Mass Spectrometry Facility, for her help with proteomics studies. We thank P. Bieniasz, the Rockefeller University, for insightful discussions and advice. We are grateful to P. Koneru, S. Bester, K.-E. Lee, Y. Pan and other members of the participating laboratories for their help with data analysis, providing some reagents and valuable suggestions. This work was supported by the National Institutes of Health grant nos. R01 AI062520 and R01 AI157802 (to M.K.), U54 GM103368 (to M.K., A.C.F. and G.B.M.), R01 AI129862 (to G.B.M.), KL2 TR001068 (to R.C.L.) and R01 AI77344 (to E.M.P).

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Author notes

  1. These authors contributed equally: S. V. Rebensburg, G. Wei.

Authors and Affiliations

  1. Division of Infectious Diseases, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Stephanie V. Rebensburg, Guochao Wei, Jared Lindenberger, Arun S. Annamalai, James Morrison, Nikoloz Shkriabai, Eric M. Poeschla & Mamuka Kvaratskhelia

  2. College of Pharmacy, The Ohio State University, Columbus, OH, USA

    Ross C. Larue

  3. Department of Pediatrics, Infectious Diseases, Emory University, Atlanta, GA, USA

    Ashwanth C. Francis & Gregory B. Melikyan

  4. Center for Cancer Research, National Cancer Institute, Frederick, MD, USA

    Szu-Wei Huang & Vineet KewalRamani

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  1. Stephanie V. Rebensburg
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Contributions

S.V.R., G.W., R.C.L., J.L., A.C.F., A.S.A., J.M., N.S. and S.W. H. performed the experiments and/or analysed the experimental results. V.K.R., E.M.P., G.B.M. and M.K. designed and supervised separate sections of the study. M.K., together with S.V.R. and G.W., conceived the entire study and wrote the manuscript with contributions from all other authors.

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Correspondence to Mamuka Kvaratskhelia.

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Peer review information Nature Microbiology thanks Edward M. Campbell, David Melville and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Identification of Sec24C as HIV-1 CA-interacting partner.

a, Experimental design of the MS-based proteomics approach (See Online Methods for details). b, Representative immunoblots showing CA tubes mediated pull-downs of endogenous Sec24C from THP1 cells without (lanes 1–5) or with (lanes 6–10) IFN treatments. Lanes 1 and 6: lysates, 2 and 7: supernatant or unbound fraction after pulldown in the absence of CA tubes, 3 and 8: supernatant or unbound fraction after pulldown in the presence of CA tubes; 4 and 9: pelleted or bound fraction in the absence of CA tubes; 5 and 10: pelleted or bound fraction in the presence of CA tubes.

Extended Data Fig. 2 Effects of Trim-Sec24C196–314 on different retroviruses.

(a) Representative immunoblot showing the stable expression of Trim-HA (blue line), Trim-CPSF6261–358 (orange line) and Trim- Sec24C196–314 (grey line) in HeLa cells. (b-h) Infections of HeLa cells stably expressing indicated Trim-fusion constructs with following VSV-G pseudotyped viruses: HIV-1NL4-3 (b), SIVmac239 (c), SIVstm (d), SIVsmE041 (e), FIV (f), EIAV (g), MLV (h). Infection levels of HIV-1-scarlet, SIVstm-eGFP, SIVmac239- eGFP, SIVsmE041-eGFP, FIV-eGFP, EIAV-eGFP, and MLV-RFP were determined by FACS. The averaged data (mean values ± SD) from three independent experiments are shown with exception of FIV infection in Trim-HA overexpressing and EIAV infection in Trim-CPSF6261–358 overexpressing Hela cells which were obtained from two independent experiments.

Extended Data Fig. 3 Comparative analysis of corresponding protein regions from Sec24C and Sec24D.

a, Sequence alignment of corresponding N-terminal regions of Sec24C and Sec24D are shown. The full-length proteins (NCBI ref sequences: NP_004913.2 and NP_055637.2) were aligned using Clustal Omega program from EMBL-EBI. The CA-binding FG-motif in Sec24C is highlighted in yellow and the FG residues are underlined with red. b, Effects of stably expressing Trim-Sec24C196–314 and Trim-Sec24D136–256 on HIV-1 infection in HeLa cells. The data (mean values +/− SD) from three independent experiments are shown.

Extended Data Fig. 4 Comparative analysis of full-length Sec23A/Sec24C and Sec23A/Sec24D for their interactions with CA tubes in vitro.

Interactions of purified recombinant Sec23A/Sec24C and Sec23A/Sec24D heterodimers with pre-assembled HIV-1 CA(A92E) tubes were analysed. Since Sec23A/Sec24C and Sec23A/Sec24D heterodimers were not sufficiently soluble in a 2 M NaCl containing buffer needed for assembly WT CA tubes, we used CA(A92E) which allowed us to effectively assemble the tubes with 1 M NaCl. In turn, the lower ionic strength conditions enabled us to avoid a background precipitation of the Sec23A/Sec24C and Sec23A/Sec24D heterodimers. a, A representative SDS–PAGE image of stock solutions of Sec23A/Sec24C, Sec23A/Sec24D and CA(A92E) proteins visualized by BlueFast Protein Staining Solution. b-c, Representative immunoblotting images of Sec23A/Sec24C (b) and Sec23A/Sec24D (c) interactions with CA(A92E) tubes. The Sec23A/Sec24C and Sec23A/Sec24D heterodimers were diluted to 0.03 mg/ml for the pulldown assays to avoid background protein precipitation. Lanes 1: load of Sec23A/Sec24C (b) or Sec23A/Sec24D (c); Lanes 2: pull-downs in the absence of CA; Lanes 3: pre-assembled CA(A92E) tubes were incubated with Sec23A/Sec24C (b) or Sec23A/Sec24D (c) in the assembly buffer. The mixture was centrifuged and washed three times with the buffer containing 50 mM Tris- HCl, pH 7.5, 1 M NaCl, 0.01% NP40, 2% glycerol. The pelleted fractions were run on SDS–PAGE and visualized by Sec24C (b) or Sec24D (c) antibody.

Extended Data Fig. 5 Analysis of cytoplasmic HIV-1 core colocalization with Sec24C.

TZM-bl cells transiently expressing Sec24C-mCherry were infected with INmNG-labelled HIV-1 bearing WT CA or the N74D CA mutant. At 2 hpi, cells were analysed for colocalization of INmNG-labelled WT or mutant N74D virus with Sec24C. (a) Representative images and (b) quantification of Sec24C-mCherry signal associated with INmNG-labelled puncta. The results are from n = 1202 WT/CA puncta from 43 cells and n = 1202 N74D/CA puncta from 25 cells. Colocalization (%) of the Sec24C-mCherry signal with INmNG puncta is shown in the graph. The averaged data are representative of two independent experiments. Statistical significance was determined by two-tailed student t-test. Arrows in (a) point to Sec24C-mCherry puncta colocalized with INmNG spots. Scale bar is 5 µm.

Extended Data Fig. 6 Effects of Sec24C KD on infectivity of HIV-1 (WT CA) and HIV-1 (N74D CA).

a, Representative immunoblot for the siRNA-mediated KD of Sec24C in HeLa cells. b and c, Infectivity (normalized to negative control KD) of VSV-G pseudotyped HIV-1 virus in WT and Sec24C KD HeLa cells without (b) and with (c) 1 µg/ml Aphidicolin. d-e, Infectivity (normalized to negative control KD) of VSV-G pseudotyped HIV-1 N74D CA mutant virus in WT and Sec24C KD HeLa cells without (d) and with (e) 1 µg/ml Aphidicolin. The data (mean values ± SD) from three independent experiments are shown. Statistical significance was determined by two-tailed student t-test.

Extended Data Fig. 7 Effects of GST-Sec24C196–314 on stability of isolated native HIV-1 cores.

VSV- G pseudotyped HIV-1 particles fluorescently labelled with INmNG (green) were bound to a poly-lysine-coated coverslip and treated with Saponin (100 μg/ml, 1 min) to expose the viral cores. Cores were either fixed immediately after saponin treatment (0 min) or after 10 min incubation at 37 °C with 25 μM of GST- Sec24C196–314, GST-Sec24C196–314(ΔFG) or with buffer (mock). After PFA fixation the cores were incubated with 100 nM of recombinant CypA-DsRed to bind the mature viral cores. (a) Representative images show INmNG and CypA-DsRed labelled HIV-1 cores after 0 and 10 min of incubation in the presence of GST-Sec24C196–314, GST-Sec24C196–314(ΔFG) or buffer. Dashed white circles show INmNG and CypA-DsRed co-localized puncta. Scale bar is 2 μm. (b) The data (mean values ± SD) from three independent experiments are shown. Statistical significance was determined by two-tailed student t-test.

Extended Data Fig. 8 Effects of GST-Sec24C196–314 on the stability of CA tubes.

(a-c) Stability of CA tubes as a function of time. CA tubes were preformed at 2 M NaCl, pelleted and then resuspended in a 1 M NaCl containing buffer to induce disassembly of the tubes (a). GST-Sec24C196–314(ΔFG) (b) or GST-Sec24C196–314 (c) was added to pre-formed CA tubes and then reactions were diluted to 1 M NaCl buffer. The mixtures were centrifuged at indicated time points and pellets were analysed by SDS–PAGE. (d) Quantification of the results in (a–c). The data (mean values ± SD) from three independent experiments are shown.

Extended Data Fig. 9 Effects of Sec24C KO and repletion on infection of different retroviruses.

Single-cycle replication assays were performed in WT, Sec24C KO, KO + Sec24C, or KO + Sec24C(ΔFG) Jurkat cells. Infection levels (normalized to WT Jurkat cells) of VSV-G pseudotyped eGFP reporter viruses: HIV-1 (a), SIVmac239 (b), SIVstm (c), SIVsmE041 (d), FIV (e), EIAV (f), MLV (g) were measured by FACS. The data (mean values ± SD) from three independent experiments are shown. (h) P-values. Statistically significant changes were observed for all lentiviruses, when their infection levels were compared for KO vs WT, KO + WT vs KO and KO + ΔFG vs KO + WT. In contrast, no statistically significant differences were seen for FIV or MLV across these cell lines. Sec24C KO resulted in slight but statistically significant increase of EIAV infection (KO vs WT). This effect is opposite to Sec24C KO effects on primate lentiviruses. Furthermore, in complete contrast with primate lentiviruses, no detectable differences were observed for EIAV in KO + ΔFG vs KO + WT cells. Statistical significance was determined by two-tailed student t-test.

Extended Data Fig. 10 Comparative analyses of interactions of cognate cellular proteins and PF74 inhibitor with CA.

a–c, Superimposition of the x-ray crystal structures of the Sec24C peptide (magenta) with CPSF6 peptide (blue) (a), Nup153 peptide (red) (b) and PF74 (black) (c) binding sites. d-f, Competition assays to show CPSF6 (d) and Nup153 (e) pull-downs from HeLa cell lysates by CA tubes in the absence (lane 3) and presence (lanes 4) of GST-Sec24C196–314. Representative immunoblots for CPSF6 and Nup153 are shown for the following samples. Lanes: 1, cellular lysates; 2, pelleting of cellular lysates in the absence of CA tubes; 3, pelleting of cellular lysates with CA tubes; 4, pelleting of cellular lysates with CA tubes + 2- fold excess GST-Sec24C196–314. (f), PF74 dose-dependent inhibition of interactions of GST-Sec24C196–314 with HIV-1 cores. The data (mean values +/− SD) from three independent experiments are shown. The results were analysed by Origin 2019 software to determine the IC50 value.

Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Tables 3–7.

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Supplementary Tables

Supplementary Table 1. Total spectrum counts of protein hits. Supplementary Table 2. Quantitative values for protein hits.

Supplementary Video 1

HIV-1 cores interact with Sec24C in living cells. TZM-bl cells transiently expressing mCherry-Sec24C fusion protein were infected with INsfGFP-labelled HIVeGFP pseudoviruses. Live-cell imaging of a single virus co-trafficking with Sec24C was performed for 45 min, starting from 1 hpi using a Zeiss LSM880 confocal microscope. Individual tiles of images corresponding to INsfGFP (green), mCherry-Sec24C (red), EBFP2-Lamin labelled nuclear membrane (blue) and merged channel are shown. The single particle track is highlighted. Scale bar, 5 µm.

Supplementary Video 2

HIV-1 cores interact with Sec24C in living cells. TZM-bl cells transiently expressing SNAP-Sec24C fusion protein were infected with CypA-DsRed-labelled HIVeGFP pseudoviruses. Live-cell imaging of a single virus co-trafficking with Sec24C at the perinuclear area was performed for 45 min, starting from 2 hpi using a Zeiss LSM880 confocal microscope. Individual tiles of images corresponding to SNAP-Sec24C (green), CypA-DsRed (red), EBFP2-Lamin labelled nuclear membrane (blue) and merged channel are shown. The multiple colocalized single particle tracks are highlighted. Scale bar, 5 µm.

Supplementary Video 3

HIV-1 cores interact with Sec24C in living cells. TZM-bl cells transiently expressing SNAP-Sec24C fusion protein were infected with HIVeGFP pseudoviruses co-labelled with INsfGFP and CypA-DsRed. Live-cell imaging of multiple HIV-1 cores co-trafficking with Sec24C was performed for 60 min, starting at 1 hpi using a Zeiss LSM880 confocal microscope. Individual tiles of images corresponding to INsfGFP (green), CypA-DsRed (red), SNAP-Sec24C (grey), EBFP2-Lamin labelled nuclear membrane (blue) and merged channel are shown. The single particle track is highlighted. Scale bar, 5 µm.

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Source Data Fig. 1

Unprocessed gels and western blots.

Source Data Fig. 2

Statistical data.

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Rebensburg, S.V., Wei, G., Larue, R.C. et al. Sec24C is an HIV-1 host dependency factor crucial for virus replication. Nat Microbiol 6, 435–444 (2021). https://doi.org/10.1038/s41564-021-00868-1

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