The HIV-1 capsid is a fullerene cone composed of hexameric and pentameric capsid proteins (CA) that packages the viral genome and mediates nuclear entry. Lenacapavir (LEN), a potent molecular long-acting inhibitor developed by Gilead, disrupts capsid morphogenesis by binding a phenylalanine-glycine (FG) pocket at the interface between adjacent CA subunits. Interestingly, cellular polyanion inositol hexakisphosphate (IP6) promotes conical capsid assembly by coordinating the central pore, which is allosterically coupled to the FG pocket. Because LEN and IP6 engage overlapping structural elements, they can compete to influence the capsid assembly pathway and outcomes. Using coarse-grained molecular simulations, we show that LEN accelerates hexamer formation while suppressing pentamer incorporation, yielding malformed, multilayered, and incomplete capsids. Simulations incorporating a ribonucleoprotein model further reveal that LEN-treated capsids often fail to encapsulate RNA, indicating impaired maturation. Our calculations confirm that LEN impairs the formation of high-curvature CA lattice regions necessary for closure, supporting a model of off-pathway assembly as a mechanism of viral inhibition. These findings define the core mechanism by which a small-molecule inhibitor disrupts the much larger-scale HIV-1 morphogenesis and underscore general principles for targeting self-assembling multi-protein complexes.

AP-1 transcription factors are a network of cellular regulators that combine in different dimer pairs to control a range of pathways involved in differentiation, growth, and cell death. They dimerize via leucine zipper coiled-coil domains that are preceded by a basic DNA binding domain. Depending on which AP-1 transcription factors dimerize, different DNA sequences will be recognized resulting in differential gene expression. The affinity of AP-1 transcription factors for each other dictates which dimers form. The relative concentration of AP-1 transcription factors varies with tissue type and environment, adding another layer of control to this integral network of cellular regulation. The development of artificial intelligence (AI)-based protein structure prediction methods gives us a new technique to investigate or predict how dimerization affects combinatorial control. All versions of AlphaFold2 and AlphaFold3 are AI/deep learning programs that predict 3D structures of proteins from an amino acid sequence and multiple sequence alignments of homologous proteins. To fully realize the potential of AI for structural biology, it is essential to understand its current capabilities and limitations. In this study, we used the classical example of an AP-1 dimer: Fos and Jun, and an array of over 2000 experimentally tested human leucine zippers to interrogate how AlphaFold models leucine zipper domains and if AlphaFold can be used to differentiate between probable and improbable dimer interfaces. We found that AlphaFold predicts highly confident leucine zipper dimers, even for dimer pairs such as the FosB homodimer, for which electrostatics are known to prevent their formation in vivo. This is an important case study concerning high-confidence but low-accuracy protein structure prediction.

The HIV-1 capsid is a fullerene-like shell composed of hexamer and pentamer arrangements of the capsid (CA) proteins. The cone shape of the capsid is particularly important for packaging the viral genome and coordinating nuclear entry. Lenacapavir (LEN), a potent long-acting inhibitor, has been shown to disrupt capsid morphogenesis by binding at the FG-binding pocket located between neighboring CA subunits. Interestingly, inositol hexakisphosphate (IP6), a cellular polyanion, binds within the central pore of capsid pentamers and some hexamers while playing a key role in regulating the hexamer/pentamer switch. As LEN and IP6 interact with overlapping structural elements, they can compete to influence the capsid assembly pathway and outcomes. Using coarse-grained molecular simulations, we examined capsid assembly across varying IP6 and LEN conditions. Our results reveal a concentration-dependent shift in assembly outcomes: LEN accelerates hexamer assembly and reduces pentamer incorporation, leading to malformed, multilayered, or incomplete capsids. Simulations including a model for the viral ribonucleoprotein (RNP) complex further show that LEN-treated capsids frequently fail to encapsidate the RNA genome, indicating impaired maturation. Our calculations confirm that LEN impairs the formation of high-curvature CA lattice regions necessary for closure, supporting a model of off-pathway assembly as a mechanism of viral inhibition.

The nuclear pore complex (NPC) facilitates macromolecular exchange between the cytoplasm and nucleus; the selective nature of which is vital to proper cell functioning. Many DNA and some RNA viruses must also access the host nucleus for successful replication and have evolved numerous strategies for this purpose, the most common of which is mimicry of cellular cargoes. Recent biochemical studies have also identified an alternative strategy, mimicry of nuclear transport receptors (NTRs). In this Review, we summarize the multiple ways in which viral protein complexes and capsids access the nucleus and discuss how studying these interactions has reshaped our understanding of the NPC and the nature of nuclear cytoplasmic transport.

The protective capsid encasing the genetic material of Human Immunodeficiency Virus (HIV) has been shown to traverse the nuclear pore complex (NPC) intact, despite exceeding the passive diffusion threshold by over three orders of magnitude. This remarkable feat is attributed to the properties of the capsid surface, which confer solubility within the NPC's phase-separated, condensate-like barrier. In this context, we apply the classical framework of wetting and capillarity – integrating analytical methods with sharp- and diffuse-interface numerical simulations – to elucidate the physical underpinnings of HIV nuclear entry. Our analysis captures several key phenomena: the reorientation of incoming capsids due to torques arising from asymmetric capillary forces; the role of confinement in limiting capsid penetration depths; the classification of translocation mechanics according to changes in topology and interfacial area; and the influence of (spontaneous) rotational symmetry-breaking on energetics. These effects are all shown to depend critically on capsid geometry, arguing for a physical basis for HIV's characteristic capsid shape.

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 nucleus 1 , 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 pore 3 . 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 transport 4 . 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-1 capsid has emerged as a tractable target for antiretroviral therapy. Lenacapavir, developed by Gilead Sciences, is the first capsid-targeting drug approved for medical use. Here we investigate the effect of Lenacapavir on HIV capsid stability and uncoating. We employ a single particle approach that simultaneously measures capsid content release and lattice persistence. We demonstrate that Lenacapavir's potent antiviral activity is predominantly due to lethal hyperstabilisation of the capsid lattice and resultant loss of compartmentalisation. This study highlights that disrupting capsid metastability is a powerful strategy for the development of novel antivirals.

Summary ParagraphHIV 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 nucleus. Remarkably, the intact HIV capsid is over one thousand times greater than the size-limit prescribed by the nuclear pore’s diffusion barrier. This barrier is a phase-separated condensate in the central channel of the nuclear pore and is comprised of intrinsically-disordered nucleoporin domains enriched in phenylalanine-glycine (FG) dipeptides. Through multivalent FG-interactions, cellular karyopherins and their bound cargoes solubilise in this phase to drive nucleocytoplasmic transport. By performing anin vitrodissection 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 licenses capsids to penetrate nucleoporin condensates. This karyopherin mimicry model resolves a key conceptual challenge for the role of the HIV capsid in nuclear entry, and explains how an exogenous entity much larger than any known cellular cargo can non-destructively breach the nuclear envelope.

Human T cell leukaemia virus 1 (HTLV-1) is a retrovirus belonging to the family of Retroviridae and causes adult T cell leukaemia (ATL) and HTLV-1-associated myelopathy (HAM).HTLV-1 subtype C is highly prevalent in the indigenous peoples of central Australia with over 40% adults were testing seropositive in 2018 [1].The viral capsid is essential for the maturation of virions and protects the RNA genome from hydrolysis by cytosolic enzymes.Here, we report novel structural information of HTLV-1 capsid protein (CA) by crystallising the N-terminal domain (NTD), C-terminal domain (CTD) and full-length, respectively.Intriguingly, three crystal forms with different space groups of NTD were obtained: 1) triclinic P 1 with an ultra-high resolution of 0.87 Å that offers unambiguous atomic information of each residue, including the N-terminus β-hairpin which is important in HIV-1 CA for nucleotides transport[2]; 2) hexagonal P 6 2 2 which diffracted to 2.05 Å showing that crystallographic six-fold symmetry appears in the hexagonal capsid lattice, indicating HTLV-1 CA could potentially assemble to a hexameric conformation that is canonical in HIV-1 CA hexamer; 3) orthorhombic P 212121 diffracted to 1.47 Å displaying that a sulfate occupies the positively charged pocket, enclosed by H71/72, R98 and W117, implying HTLV-1 CA might interplay with new cellular factors that are different from the cofactors interacting with the HIV-1 capsid.The HTLV-1 CA-CTD was also solved to 1.47 Å and reveals the dimerisation packing of the capsid lattice.The crystal of HTLV-1 CA-full-length diffracted to 2.25 Å belonging to the F 2 2 2 space group, which also contains a six-fold crystallographic symmetry to generate a hexameric lattice.With the insight these structures have given us, we can predict how the HTLV-1 CA protein self-assembles and begin exploring how to disrupt the capsid pharmaceutically.

Human immunodeficiency virus (HIV) is the most extensively researched human pathogen. Despite this massive scientific endeavour, several fundamental viral processes remain enigmatic. One such critical process is uncoating-the event that releases the viral genome from the proteinaceous shell of the capsid during infection. While this process is conceptually simple, the molecular underpinnings, timing, regulation, and cellular location of uncoating remain contentious. This review describes the hurdles that have limited our understanding in this area and presents recently deployed in vitro and in cellulo techniques that have been developed expressly with the aim of directly visualising capsid uncoating at the single-particle level and understanding the mechanics behind this essential aspect of HIV infection.

SUMMARYPandemic viruses remain a global threat to health and economics but how they adapt to become pandemic remains poorly understood. Here we compare pandemic HIV-1(M) and non-pandemic HIV-(O) and HIV-2 strains finding that non-pandemic HIV replicate poorly in myeloid cell models due to activation of cGAS and TRIM5, and ensuing antiviral responses. We use phylogenetics and viral capsid structural biology to define specific differences between pandemic and non-pandemic HIV capsids and demonstrate that their genetic reversal in HIV-1(M) mutants causes TRIM5, cGAS and innate immune activation. We propose a model in which the parental lineage of pandemic HIV-1(M) has uniquely evolved a dynamic capsid that avoids activation of cGAS and TRIM5 to establish cloaked replication in myeloid cells. The unique adaptations of the pandemic virus lineage suggests a role in effective human-to-human transmissibility and highlight the importance of avoiding innate immune activation during pandemic human-to-human viral transmission.