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      HIV-1 uncoats in the nucleus near sites of integration

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          Significance

          For several decades, retroviral core uncoating has been thought to occur in the cytoplasm in coordination with reverse transcription, and while some recent studies have concluded that HIV-1 uncoating occurs at the nuclear envelope during nuclear import, none have concluded that uncoating occurs in the nucleus. Here, we developed methods to study HIV-1 uncoating by direct labeling and quantification of the viral capsid protein associated with infectious viral cores that produced transcriptionally active proviruses. We find that infectious viral cores in the nuclei of infected cells are largely intact and uncoat near their integration sites just before integration. These unexpected findings fundamentally change our understanding of HIV-1 postentry replication events.

          Abstract

          HIV-1 capsid core disassembly (uncoating) must occur before integration of viral genomic DNA into the host chromosomes, yet remarkably, the timing and cellular location of uncoating is unknown. Previous studies have proposed that intact viral cores are too large to fit through nuclear pores and uncoating occurs in the cytoplasm in coordination with reverse transcription or at the nuclear envelope during nuclear import. The capsid protein (CA) content of the infectious viral cores is not well defined because methods for directly labeling and quantifying the CA in viral cores have been unavailable. In addition, it has been difficult to identify the infectious virions because only one of ∼50 virions in infected cells leads to productive infection. Here, we developed methods to analyze HIV-1 uncoating by direct labeling of CA with GFP and to identify infectious virions by tracking viral cores in living infected cells through viral DNA integration and proviral DNA transcription. Astonishingly, our results show that intact (or nearly intact) viral cores enter the nucleus through a mechanism involving interactions with host protein cleavage and polyadenylation specificity factor 6 (CPSF6), complete reverse transcription in the nucleus before uncoating, and uncoat <1.5 h before integration near (<1.5 μm) their genomic integration sites. These results fundamentally change our current understanding of HIV-1 postentry replication events including mechanisms of nuclear import, uncoating, reverse transcription, integration, and evasion of innate immunity.

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          Most cited references20

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          The Nuclear Pore Complex as a Flexible and Dynamic Gate.

          Nuclear pore complexes (NPCs) perforate the nuclear envelope and serve as the primary transport gates for molecular exchange between nucleus and cytoplasm. Stripping the megadalton complex down to its most essential organizational elements, one can divide the NPC into scaffold components and the disordered elements attached to them that generate a selective barrier between compartments. These structural elements exhibit flexibility, which may hold a clue in understanding NPC assembly and function. Here we review the current status of NPC research with a focus on the functional implications of its structural and compositional heterogeneity.
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            Complementary assays reveal a relationship between HIV-1 uncoating and reverse transcription.

            During the early stages of HIV-1 replication the conical capsid composed of p24(CA) protein dissociates from the rest of the cytoplasmic viral complex by a process called uncoating. Although proper uncoating is known to be required for HIV-1 infection, many questions remain about the timing and factors involved in the process. Here we have used two complementary assays to study the process of uncoating in HIV-1-infected cells, specifically looking at the timing of uncoating and its relationship to reverse transcription. We developed a fluorescent microscopy-based uncoating assay that detects the association of p24(CA) with HIV-1 viral complexes in cells. We also used an owl monkey kidney (OMK) cell assay that is based on timed TRIM-CypA-mediated restriction of HIV-1 replication. Results from both assays indicate that uncoating is initiated within 1 h of viral fusion. In addition, treatment with the reverse transcriptase inhibitor nevirapine delayed uncoating in both assays. Analysis of reverse transcription products in OMK cells revealed that the generation of early reverse transcription products coincides with the timing of uncoating in these assays. Collectively, these results suggest that some aspect of reverse transcription has the ability to influence the kinetics of uncoating.
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              Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1.

              The assembly of infectious human immunodeficiency virus (HIV) requires that Gag transport and oligomerization be coordinated with its association with other viral proteins, viral RNAs, and cellular membranes. We have developed a replication-competent HIV type 1 molecular clone that carries a Gag-internal or interdomain green fluorescent protein (iGFP) fusion to reveal a physiologically accurate temporal sequence of Gag localization and oligomerization during the formation of infectious HIV. This recombinant HIV is as infectious as native HIV in single-round infectivity assays, validating its use for trafficking studies. It replicates robustly in permissive MT4 cells and is infectious, yet it spreads poorly in other T-cell lines. Immunofluorescence of Gag-iGFP showed a pattern very similar to that of native Gag. However, the intense plasma membrane Gag-iGFP fluorescence contrasts markedly with its immunofluorescence at this site, indicating that many Gag epitopes can be masked by oligomerization. Consistent with this, fluorescence resonance energy transfer studies visualized intense Gag oligomerization at the plasma membrane and weaker oligomerization at cytoplasmic sites. Four-dimensional, time-lapse confocal imaging reveals a temporal progression of Gag distribution over hours in which Gag is initially diffusely localized within the cytoplasm. Plasma membrane signals then accumulate as Gag levels increase and vesicular association appears late, only after plasma membrane site signals have reached high intensity. Lastly, the cell rounds up and HIV protease activation induces diffuse fluorescence throughout the cell. These distinct phases reveal a natural progression of Gag trafficking during the viral gene expression program. HIV Gag-iGFP is a useful tool for dissecting mechanisms of viral assembly and transmission.
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                Author and article information

                Journal
                Proc Natl Acad Sci U S A
                Proc. Natl. Acad. Sci. U.S.A
                pnas
                pnas
                PNAS
                Proceedings of the National Academy of Sciences of the United States of America
                National Academy of Sciences
                0027-8424
                1091-6490
                10 March 2020
                24 February 2020
                24 February 2020
                : 117
                : 10
                : 5486-5493
                Affiliations
                [1] aViral Mutation Section, HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute at Frederick , Frederick, MD 21702;
                [2] bViral Recombination Section, HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute at Frederick , Frederick, MD 21702;
                [3] cElectron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research (FNLCR) , Frederick, MD 21702
                Author notes
                1To whom correspondence may be addressed. Email: pathakv@ 123456mail.nih.gov .

                Edited by Stephen P. Goff, Columbia University Medical Center, New York, NY, and approved January 29, 2020 (received for review November 22, 2019)

                Author contributions: R.C.B. and V.K.P. designed research; R.C.B., C.L., M.M., and K.N. performed research; J.M.O.R. and W.-S.H. contributed new reagents/analytic tools; R.C.B., C.L., M.M., K.N., W.-S.H., and V.K.P. analyzed data; and R.C.B. and V.K.P. wrote the paper.

                The authors declare no competing interest.

                Author information
                http://orcid.org/0000-0003-2789-967X
                Article
                201920631
                10.1073/pnas.1920631117
                7071919
                32094182
                4ea433e0-bea3-42cf-8f4c-8f8c6a3b5f7a
                Copyright © 2020 the Author(s). Published by PNAS.

                This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

                History
                Page count
                Pages: 8
                Funding
                Funded by: HHS | NIH | National Cancer Institute (NCI) 100000054
                Award ID: Intramural
                Award Recipient : Ryan C Burdick Award Recipient : Chenglei Li Award Recipient : MohamedHusen Munshi Award Recipient : Jonathan M.O. Rawson Award Recipient : Kunio Nagashima Award Recipient : Wei-Shau Hu Award Recipient : Vinay K Pathak
                Funded by: HHS | NIH | National Cancer Institute (NCI) 100000054
                Award ID: HHSN26120080001E
                Award Recipient : Ryan C Burdick Award Recipient : Chenglei Li Award Recipient : MohamedHusen Munshi Award Recipient : Jonathan M.O. Rawson Award Recipient : Kunio Nagashima Award Recipient : Wei-Shau Hu Award Recipient : Vinay K Pathak
                Categories
                Biological Sciences
                Microbiology
                From the Cover

                hiv-1,capsid,uncoating,integration,transcription
                hiv-1, capsid, uncoating, integration, transcription

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