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SCIENCE CHINA Life Sciences, https://doi.org/10.1007/s11427-020-1716-x

HIV-1 viral cores enter the nucleus collectively through the nuclear endocytosis-like pathway

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  • ReceivedApr 2, 2020
  • AcceptedApr 28, 2020
  • PublishedMay 14, 2020

Abstract

It is recognized that HIV-1 capsid cores are disassembled in the cytoplasm, releasing their genomes into the nucleus through nuclear pores, but there is also evidence showing the capsid (CA) exists in the nucleus. Whether HIV-1 enters the nucleus and how it enters the nucleus through the undersized nuclear pore remains mysterious. Based on multicolor labeling and real-time imaging of the viral and cellular components, our observations via light and electron microscopy suggest that HIV-1 selectively gathered at the microtubule organization center (MTOC), leading the nearby nuclear envelope (NE) to undergo deformation, invagination and restoration to form a nuclear vesicle in which the viral particles were wrapped; then, the inner membrane of the nuclear vesicle ruptured to release HIV-1 into the nucleus. This unexpected discovery expands our understanding of the complexity of HIV-1 nuclear entry, which may provide new insights to HIV-1 virology.


Funded by

the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB29050100)


Acknowledgment

We would like to thank the Center for Biological Imaging (CBI), IBP-CAS, particularly Shuoguo Li, Yun Feng and Can Peng, for technical support with the SIM and TEM work. This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB29050100).


Interest statement

The author(s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

The supporting information is available online at https://doi.org/10.1007/s11427-020-1716-x. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    HIV-1 gradually aggregated near the MTOC. A, Confocal microscopy analyses of HIV-1TC.ReAsH/IN.eGFP (NL4-3(KFS)) in TZM-bl cells at different time points (5 min/9 h/24 h/36 h). As time progressed, the distribution of viral particles in the cell changed from scattered to aggregated and from cytoplasmic to nuclear. Samples were stained for laminb1 (blue). Scale bar, 5  μm. In this article, fluorescent images were obtained by confocal microscopy, and the cells were TZM-bl cells unless otherwise stated. B, TEM analysis of HIV-1-infected cells. Here, the nonlabeled HIV-1 strain NL4-3 (KFS) was used. The cells were infected with the virions at an MOI of 0.7 for 24 h in all experiments, unless otherwise stated. The cells were then fixed and processed for TEM analysis. TEM image indicated that viral particles (white arrow) grouped near the outer nuclear membrane. The solid triangle and the open triangle indicate the NE region with virus aggregation and the NE region without virus, respectively. Scale bar, 200 nm. C, Confocal microscopic imaging of primary human macrophages infected with HIV-1TC.ReAsH (NL4-3(KFS)) for 24 h showed that the virus aggregated near the deformed region of the nucleus. Staining for nuclei (blue), NL4-3CA (red). Scale bar, 5 μm. D and E, Fluorescence analyses of fixed HIV-1-infected cells showing HIV-1 aggregated near the MTOC. HIV-1 strains AD8 (D) and NL4-3 (E) shared the same behavior involving the aggregation effect. The suspected MTOC in (D) was identified by γ-tubulin (turquoise) labeling in (E). Staining for laminb1 (blue), MT (green), γ-tubulin (turquoise), AD8CA and NL4-3Vpr (red). Scale bar, 5  μm. F, Time-lapse fluorescence snapshots of HIV-1Vpr-mCherry (NL4-3(KFS)) in TZM-bl cells. The aggregation of viral particles in the MTOC and their transport into the nucleus were dynamically traced. G, Same as (F) but highlights the signals of the MTOC, nucleus and virus. Stained for DNA (blue), MT (green), and γ-tubulin (turquoise). Scale bar, 10 μm.

  • Figure 2

    Formation of intranuclear vesicles containing HIV-1. A and B, Time-lapse imaging of HIV-1Vpr-mCherry (NL4-3(KFS)) infection of cells. After HIV-1 aggregated at the NE, the NE underwent a series of morphological changes. B, NM channel. Scale bar, 10  μm. C and D, Time-lapse imaging of HIV-1TC.ReAsH-IN.eGFP (NL4-3(KFS)) infection of cells showed a similar phenomenon. The long arrows indicate the extracellular direction. D, NM channel. Scale bar, 5 μm. E and F, Fluorescence intensity of the NE deformation regions in C and D, quantitatively indicating the regeneration and extension of new NE structures. G, MICDDRP bDNA hybridization. The results indicated that HIV-1 DNA locally aggregated in the nucleus. Staining for nuclear DNA (blue) and viral DNA (red). Scale bar, 5  μm. H, TEM images revealed the accumulation and existence of viral particles in the intra-NE vesicles. The inserts in (H) is enlarged images of the mentioned region.

  • Figure 3

    Vesicles ruptured to release HIV-1 into the nucleus. A, Time-lapse imaging of vesicle rupture and release of HIV-1 into the nucleus. Staining for viral CA (red), lamin B1 (blue), NLS (green). B, Quantitative fluorescence intensity of the labeled components in (A). The measurements indicate that after the formation of intranuclear vesicles, the NLS-GFP signal increasingly accumulated in the vesicles, with rise and fall of the NE debris signal, indicating the rupture of the vesicles. Staining for viral CA (red), lamin B1 (blue) and NLS (green). C and D, TEM images of the rupture and dissolution of the intranuclear vesicles. The inserted enlarged images show some debris-associated viral particles in the ruptured vesicle region. E, Time-lapse imaging of viral infection and the formation of NE vesicles. F, Quantitative fluorescence intensity measurement in (E). No significant NLS-GFP signal leaked into the cytoplasm from the nucleus during the deformation of NE and formation of the intranuclear vesicles. Staining for viral CA (red), lamin B1 (blue) and NLS (green). Scale bar,5 μm.

  • Figure 4

    ESCRT-III is involved in vesicle formation. A, Recruitment of ESCRT-III in the NE deformation region where viral particles aggregated. Staining for the ESCRT-III subunit CHMP4B (green), viral CA (AD8) (red), and laminb1 (blue). Scale bar, 5 μm. B, Similar results were obtained using the NL4-3 strain with labeled Vpr. C, Time-lapse imaging shows that formation of the intranuclear vesicle was accompanied by diffusion of some ESCRT-III into the vesicle, implying that ESCRT-III plays a role in repairing and stabilizing the NE system. Scale bar, 5 μm. D, Quantitative measurement of fluorescence intensity at the corresponding time in (C).

  • Figure 5

    Model of HIV-1 nuclear entry. After entering the cell, HIV-1 moves along the microtubules and gradually aggregates near the MTOC outside the NE, causing deformation of the adjacent NE; the viral particles enter the deformed area, and with the participation of ESCRT-III, the ends of the concave edge extend to the middle to form a new NE. In turn, an intranuclear vesicle structure is formed, and the HIV-1 are encapsulated; the inner membrane of the vesicle is subsequently ruptured, and the HIV-1 are released into the nucleus.

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