Cone-shaped HIV-1 capsids are transported through intact nuclear pores

Abstract

Summary Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrated that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, cone-shaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.

HIV-1 uncoating by release of viral cDNA from capsid-like structures in the nucleus of infected cells

“…Using CLEM-tomography of IN-positive and eGFP.OR3-negative nuclear complexes, we observed morphologically intact cone-shaped structures with internal density representing the nucleoprotein complex, which closely resembled HIV-1 capsids inside the cytosol or in complete virions. This finding is consistent with our recent study showing that the nuclear pore channel is sufficiently large to accommodate the HIV-1 core and apparently intact cone-shaped HIV-1 capsids can enter the nucleus through intact nuclear pores (Zila et al, 2021). Taken together, these results indicate that reverse transcription initiates in the cytoplasm inside a complete or largely complete capsid, and this capsid-encased complex traffics into the nucleus, where reverse transcription is completed; subsequently it must be uncoated for integration to occur.…”

“…Of note, nuclear CA immunofluorescence signals have been detected previously in MDM (Bejarano et al, 2019;Francis et al, 2020), but not or only weakly in T cell lines (Zila et al, 2019) 7e-f). Thus, the failure to detect nuclear CA by IF in CD4 + T cells is due to shielding of epitopes by the accumulation of CPSF6 rather than to CA being lost upon nuclear entry in these cells, in accordance with our recent cryo-ET analyses in SupT1 cells (Zila et al, 2021).…”

“…Next, we generated a non-infectious derivative of HIV ANCH termed NNHIV ANCH to allow for live cell imaging outside the BSL3 facility. NNHIV ANCH is based on the previously reported plasmid NNHIV that carries point mutations in the active site of IN and a deletion in the tat gene (IN D64N/D116N tat Δ33-64bp ) (Zila et al, 2021). This derivative retains reverse transcription and nuclear import ability, whereas integration and transcription are blocked.…”

“…This implies that capsid uncoating should occur -at least partially -prior to nuclear entry, and various publications reported uncoating either in the cytoplasm (Cosnefroy et al, 2016;Hulme et al, 2011;Mamede et al, 2017;Xu et al, 2013) or at the nuclear pore (Arhel et al, 2007;Burdick et al, 2017;Francis & Melikyan, 2018), with some evidence for cell type dependent differences. On the other hand, nuclear HIV-1 pre-integration complexes (PIC) were found to retain varying amounts of CA molecules (Bejarano et al, 2019;Burdick et al, 2020;Chin et al, 2015;Hulme et al, 2015;Stultz et al, 2017;Zila et al, 2019), at least in certain cell types (Zila et al, 2019), and recent reports indicating the presence of intact capsid lattice (Dharan et al, 2020;Selyutina et al, 2020) and capsid-like structures (Zila et al, 2021) inside the nucleus challenged the current models of early HIV-1 replication. Accordingly, the timing, subcellular localization, trigger and mechanism of HIV-1 capsid uncoating are still under debate.…”

SARS-CoV-2 Portrayed Against HIV: Contrary Viral Strategies in Similar Disguise

“…Still within the HIV-1 capsid in the form of a PIC, the dsDNA complex is transported to the nucleus, where it is uncoated and released into the nucleus via the nuclear pore. Within the nucleus, the dsDNA complex is integrated into the host genome by the viral enzyme Integrase [116][117][118]. It is only after integration that the HIV-1 genome is transcribed into mRNAs by host enzymes in the nucleus, which are then transported out and translated in the cytoplasm.…”