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

After cell entry, human immunodeficiency virus type 1 (HIV-1) replication involves reverse transcription of the RNA genome, nuclear import of the subviral complex without nuclear envelope breakdown, and integration of the viral complementary DNA into the host genome. Here, we discuss recent evidence indicating that completion of reverse transcription and viral genome uncoating occur in the nucleus rather than in the cytoplasm, as previously thought, and suggest a testable model for nuclear import and uncoating. Multiple recent studies indicated that the cone-shaped capsid, which encases the genome and replication proteins, not only serves as a reaction container for reverse transcription and as a shield from innate immune sensors but also may constitute the elusive HIV-1 nuclear import factor. Rupture of the capsid may be triggered in the nucleus by completion of reverse transcription, by yet-unknown nuclear factors, or by physical damage, and it appears to occur in close temporal and spatial association with the integration process. Expected final online publication date for the Annual Review of Virology, Volume 9 is September 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Nuclear Capsid Uncoating and Reverse Transcription of HIV-1.

Abstract Of the 13 known independent zoonoses of simian immunodeficiency viruses to humans, only one, leading to human immunodeficiency virus (HIV) type 1(M) has become pandemic, causing over 80 million human infections. To understand the specific features associated with pandemic human-to-human HIV spread, we compared replication of HIV-1(M) with non-pandemic HIV-(O) and HIV-2 strains in myeloid cell models. We found that non-pandemic HIV lineages replicate less well than HIV-1(M) owing to activation of cGAS and TRIM5-mediated antiviral responses. We applied phylogenetic and X-ray crystallography structural analyses to identify differences between pandemic and non-pandemic HIV capsids. We found that genetic reversal of two specific amino acid adaptations in HIV-1(M) enables activation of TRIM5, cGAS and innate immune responses. We propose a model in which the parental lineage of pandemic HIV-1(M) evolved a capsid that prevents cGAS and TRIM5 triggering, thereby allowing silent replication in myeloid cells. We hypothesize that this capsid adaptation promotes human-to-human spread through avoidance of innate immune response activation. 

/pdf/evasion-of-cgas-and-trim5-defines-pandemic-hiv-1poxihio.pdf

Evasion of cGAS and TRIM5 defines pandemic HIV

HIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus1,2. Remarkably, the intact HIV capsid is more than 1,000 times larger than the size limit prescribed by the diffusion barrier of the nuclear pore3. This barrier in the central channel of the nuclear pore is composed of intrinsically disordered nucleoporin domains enriched in phenylalanine-glycine (FG) dipeptides. Through multivalent FG interactions, cellular karyopherins and their bound cargoes solubilize in this phase to drive nucleocytoplasmic transport4. By performing an in vitro dissection of the nuclear pore complex, we show that a pocket on the surface of the HIV capsid similarly interacts with FG motifs from multiple nucleoporins and that this interaction licences capsids to penetrate FG-nucleoporin condensates. This karyopherin mimicry model addresses a key conceptual challenge for the role of the HIV capsid in nuclear entry and offers an explanation as to how an exogenous entity much larger than any known cellular cargo may be able to non-destructively breach the nuclear envelope. 

The HIV capsid mimics karyopherin engagement of FG-nucleoporins.

The Human Immunodeficiency Virus type 1 (HIV-1) virion contains a conical shell, termed capsid, encasing the viral RNA genome. After cellular entry of the virion, the capsid is released and ensures the protection and delivery of the HIV-1 genome to the host nucleus for integration. The capsid relies on many virus–host factor interactions which are regulated spatiotemporally throughout the course of infection. In this paper, we will review the current understanding of the highly dynamic HIV-1 capsid–host interplay during the early stages of viral replication, namely intracellular capsid trafficking after viral fusion, nuclear import, uncoating, and integration of the viral genome into host chromatin. Conventional anti-retroviral therapies primarily target HIV-1 enzymes. Insights of capsid structure have resulted in a first-in-class, long-acting capsid-targeting inhibitor, GS-6207 (Lenacapavir). This inhibitor binds at the interface between capsid protein subunits, a site known to bind host factors, interferes with capsid nuclear import, HIV particle assembly, and ordered assembly. Our review will highlight capsid structure, the host factors that interact with capsid, and high-throughput screening techniques, specifically genomic and proteomic approaches, that have been and can be used to identify host factors that interact with capsid. Better structural and mechanistic insights into the capsid–host factor interactions will significantly inform the understanding of HIV-1 pathogenesis and the development of capsid-centric antiretroviral therapeutics.

Interactions of HIV-1 Capsid with Host Factors and Their Implications for Developing Novel Therapeutics

When viruses infect human cells, they hijack the cell’s machinery to produce the proteins they need to replicate. Retroviruses like HIV-1 do this by entering the nucleus and inserting their genetic information into the genome of the infected cell. This requires HIV-1 to convert its genetic material into DNA, which is then released from the protective shell surrounding it (known as the capsid) via a process called uncoating. The nucleus is enclosed within an envelope containing pores that molecules up to a certain size can pass through. Until recently these pores were thought to be smaller than the viral capsid, which led scientists to believe that the HIV-1 genome must shed this coat before penetrating the nucleus. However, recent studies have found evidence for HIV-1 capsid proteins and capsid structures inside the nucleus of some infected cells. This suggests that the capsid may not be removed before nuclear entry or that it may even play a role in helping the virus get inside the nucleus. To investigate this further, Muller et al. attached fluorescent labels to the newly made DNA of HIV-1 and some viral and cellular proteins. Powerful microscopy tools were then used to monitor the uncoating process in various cells that had been infected with the virus. Muller et al. found large amounts of capsid protein inside the nuclei of all the infected cells studied. During the earlier stages of infection, the capsid proteins were mostly associated with viral DNA and the capsid structure appeared largely intact. At later time points, the capsid structure had been broken down and the viral DNA molecules were gradually separating themselves from these remnants. These findings suggest that the HIV-1 capsid helps the virus get inside the nucleus and may protect its genetic material during conversion into DNA until right before integration into the cell’s genome. Further experiments studying this process could lead to new therapeutic approaches that target the capsid as a way to prevent or treat HIV-1.

/pdf/hiv-1-uncoating-by-release-of-viral-cdna-from-capsid-like-1kagpd8ir8.pdf

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

HIV-1 replication commences inside the cone-shaped viral capsid, but timing, localization and mechanism of uncoating are under debate. We adapted a strategy to visualize individual reverse-transcribed HIV-1 cDNA molecules and their association with viral and cellular proteins using fluorescence and correlative-light-and-electron-microscopy (CLEM). We specifically detected HIV-1 cDNA inside nuclei, but not in the cytoplasm. Nuclear cDNA initially co-localized with a fluorescent integrase fusion (IN-FP) and the viral CA (capsid) protein, but cDNA-punctae separated from IN-FP/CA over time. This phenotype was conserved in primary HIV-1 target cells, with nuclear HIV-1 complexes exhibiting strong CA-signals in all cell types. CLEM revealed cone-shaped HIV-1 capsid-like structures and apparently broken capsid-remnants at the position of IN-FP signals and elongated chromatin-like structures in the position of viral cDNA punctae lacking IN-FP. Our data argue for nuclear uncoating by physical disruption rather than cooperative disassembly of the CA-lattice, followed by physical separation from the pre-integration complex.

https://biorxiv.org/content/10.1101/2020.11.13.380030v1.full.pdf

The cone-shaped mature HIV-1 capsid is the main orchestrator of early viral replication. After cytosolic entry, it transports the viral replication complex along microtubules towards the nucleus. Capsid uncoating from the viral genome apparently occurs beyond the nuclear pore. Observation of post-entry events via microscopic detection of HIV-1 capsid protein (CA) is challenging, since epitope shielding limits immunodetection, and the genetic fragility of CA hampers other labeling approaches. Here, we present a minimally invasive strategy based on genetic code expansion and click chemistry that allows for site-directed fluorescent labeling of HIV-1 CA, while retaining virus morphology and infectivity. Thereby, we could directly visualize virions and subviral complexes using advanced microscopy, including nanoscopy and correlative imaging. Quantification of signal intensities of subviral complexes showed that the amount of CA associated with nuclear complexes in HeLa-derived cells and primary T cells is consistent with a complete capsid and revealed that treatment with the small molecule inhibitor PF74 did not result in capsid dissociation from nuclear complexes. Cone-shaped objects detected in the nucleus by electron tomography were clearly identified as capsid-derived structures by correlative microscopy. High-resolution imaging revealed dose-dependent clustering of nuclear capsids, suggesting that incoming particles may follow common entry routes.

/pdf/direct-capsid-labeling-of-infectious-hiv-1-by-genetic-code-2ih527qdxr.pdf

Direct capsid labeling of infectious HIV-1 by genetic code expansion allows detection of largely complete nuclear capsids and suggests nuclear entry of HIV-1 complexes via common routes

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

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

Direct capsid labeling of infectious HIV-1 by genetic code expansion allows detection of largely complete nuclear capsids and suggests nuclear entry of HIV-1 complexes via common routes