Lentiviruses like HIV-1 infect non-dividing cells by traversing the nuclear pore, but studying this process has been challenging due to its scarcity and dynamic nature in infected cells. Here, we developed a robust cell-permeabilization system that recapitulates HIV-1 nuclear import and established an integrated cryo-correlative workflow combining cryo-CLEM, cryo-FIB, and cryo-ET for targeted imaging of this process. These advancements enabled the successful capture of 1,899 HIV-1 cores at various stages of nuclear import. Statistical and structural analyses of native wild-type and mutant cores revealed that HIV-1 nuclear import depends on both capsid elasticity and nuclear pore adaptability, as well as nuclear factors such as CPSF6. Brittle cores fail to enter the nuclear pore complex (NPC), while CPSF6-binding-deficient cores stall inside the NPC, resulting in impaired nuclear import. Intriguingly, nuclear pores function as selective filters favoring the import of smaller, tube-shaped cores. Our study opens new avenues for dissecting the biochemistry and structural biology of HIV-1 nuclear import as well as downstream events including core uncoating and potentially integration, with unprecedented detail.

Lentiviruses, such as HIV-1, infect non-dividing cells by traversing the nuclear pore complex (NPC); however, the detailed molecular processes remain unclear. Here we reconstituted functional HIV-1 nuclear import using permeabilized T cells and isolated HIV-1 cores, which significantly increases import events, and developed an integrated three-dimensional cryo-correlative workflow to specifically target and image 1,489 native HIV-1 cores at 4 distinct nuclear import stages using cryo-electron tomography. We found HIV-1 nuclear import depends on both capsid elasticity and nuclear pore adaptability. The NPC acts as a selective filter, preferentially importing smaller cores, while expanding and deforming to accommodate their passage. Brittle mutant cores fail to enter the NPC, while CPSF6-binding-deficient cores enter but stall within the NPC, leading to impaired nuclear import. This study uncovers the interplay between the HIV-1 core and the NPC and provides a framework to dissect HIV-1 nuclear import and downstream events, such as uncoating and integration.

HIV-1 uses the microtubule cytoskeleton to reach the host cell nucleus during replication, yet the molecular basis for microtubule-dependent HIV-1 motility is poorly understood. Using in vitro reconstitution biochemistry and single-molecule imaging, we found that HIV-1 binds to the retrograde microtubule-associated motor, dynein, directly and not via a cargo adaptor, as has been previously suggested. The HIV-1 capsid lattice binds to accessory chains on dynein's tail domain. Further, we demonstrate that multiple dynein motors tethered to rigid cargoes, such as HIV-1 capsids, display reduced motility, distinct from the behavior of multiple motors on membranous cargoes. Our results provide an updated model of HIV-1 trafficking wherein HIV-1 binds to dynein directly to "hijack" the dynein transport machinery for microtubule motility.

Cleavage and polyadenylation specificity factor 6 (CPSF6) is part of the cellular cleavage factor I mammalian (CFIm) complex that regulates mRNA processing and polyadenylation. CPSF6 also functions as an HIV-1 capsid (CA) binding host factor to promote viral DNA integration targeting into gene-dense regions of the host genome. However, the effects of CPSF6 on the activity of the HIV-1 preintegration complex (PIC)-the sub-viral machinery that carries out viral DNA integration-are unknown. To study CPSF6's role in HIV-1 PIC function, we extracted PICs from cells that are either depleted of CPSF6 or express a mutant form that cannot bind to CA. These PICs exhibited significantly lower viral DNA integration activity when compared to the control PICs. The addition of purified recombinant CPSF6 restored the integration activity of PICs extracted from the CPSF6-mutant cells, suggesting a direct role of CPSF6 in PIC function. To solidify CPSF6's role in PIC function, we inoculated CPSF6-depleted and CPSF6-mutant cells with HIV-1 particles and measured viral DNA integration into the host genome. A significant reduction in integration in these cells was detected, and this reduction was not a consequence of lower reverse transcription or nuclear entry. Additionally, mutant viruses deficient in CA-CPSF6 binding showed no integration defect in CPSF6-mutant cells. Finally, sequencing analysis revealed that HIV-1 integration into CPSF6-mutant cell genomes was significantly redirected away from gene-dense regions of chromatin compared to the control cells. Collectively, these results suggest that the CPSF6-CA interaction promotes PIC function both in vitro and in infected cells.IMPORTANCEHIV-1 infection is dependent on the interaction of the virus with cellular host factors. However, the molecular details of HIV-host factor interactions are not fully understood. For instance, the HIV-1 capsid provides binding interfaces for several host factors. CPSF6 is one such capsid-binding host factor, whose cellular function is to regulate mRNA processing and polyadenylation. Initial work identified a truncated cytosolic form of CPSF6 to restrict HIV infection by blocking viral nuclear entry. However, it is now established that the full-length CPSF6 primarily promotes HIV-1 integration targeting into gene-dense regions of the host genome. Here, we provide evidence that CPSF6-CA interaction stimulates the activity of HIV-1 preintegration complexes (PICs). We also describe that disruption of CPSF6-CA binding in target cells significantly reduces viral DNA integration and redirects integration targeting away from gene-dense regions into regions of low transcriptional activity. These findings identify a critical role for the CPSF6-CA interaction in PIC function and integration targeting.

The structural and mechanical properties of the HIV-1 core are critical for successful infection, balancing stability for early replication and controlled disassembly for genome release. Recent studies have highlighted the role of core elasticity in nuclear entry, yet the molecular determinants regulating this property remain poorly understood. Here, atomic force microscopy (AFM) was used to investigate the relationship between reverse transcription progression, genome length, core elasticity, and disassembly. The results demonstrate that reverse transcription induces a gradual loss of elasticity, rendering the core increasingly brittle as DNA synthesis progresses. Cores containing shorter genomes remained highly elastic, whereas those with longer genomes exhibited increased brittleness, structural damage, and a higher degree of disassembly, after 4 hours of reverse transcription. Additionally, cores from an RNase H-deficient HIV-1 mutant retained high elasticity. These findings provide insight into the interplay between genome synthesis, core integrity, and nuclear entry, supporting a model in which reverse transcription-generated mechanical stress facilitates uncoating. Furthermore, early-stage reverse transcription preserved core elasticity, suggesting a temporal window for successful nuclear import before structural destabilization compromises infectivity.

Binding of the host protein cyclophilin A (CypA) to the HIV-1 capsid exerts a variety of effects on infection, including enhancement of reverse transcription, stabilization of the capsid, and promotion of nuclear entry. For several HIV-1 mutants, CypA binding inhibits nuclear entry by an unknown mechanism. We recently demonstrated that HIV-1 cores are elastic and that HIV-1 mutants with inelastic capsids are impaired for nuclear entry and infection of nondividing cells. Here we show that CypA prevents infection of nondividing cells by such mutants and inhibits their entry into the nucleus. CypA binding to mutant cores further reduced their elasticity in vitro, and this effect was reversed by suppressor mutations that restored nuclear entry. We suggest that HIV-1 nuclear entry involves temporal modulation of capsid elasticity by host proteins prior to and during traversal of the nuclear pore.

HIV-1 infection requires passage of the viral core through the nuclear pore of the cell, a process that depends on functions of the viral capsid. Recent studies have shown that HIV-1 cores enter the nucleus prior to capsid disassembly. Interactions of the viral capsid with the nuclear pore complex are necessary but not sufficient for nuclear entry, and the mechanism by which the viral core traverses the comparably sized nuclear pore is unknown. Here we show that the HIV-1 core is highly elastic and that this property is linked to nuclear entry and infectivity. Using atomic force microscopy-based approaches, we found that purified wild type cores rapidly returned to their normal conical morphology following a severe compression. Results from independently performed molecular dynamic simulations of the mature HIV-1 capsid also revealed its elastic property. Analysis of four HIV-1 capsid mutants that exhibit impaired nuclear entry revealed that the mutant viral cores are brittle. Adaptation of two of the mutant viruses in cell culture resulted in additional substitutions that restored elasticity and rescued infectivity and nuclear entry. We also show that capsid-targeting compound PF74 and the antiviral drug Lenacapavir reduce core elasticity and block HIV-1 nuclear entry at concentrations that preserve interactions between the viral core and the nuclear envelope. Our results indicate that elasticity is a fundamental property of the HIV-1 core that enables nuclear entry, thereby facilitating infection. These results provide new insights into the role of the capsid in HIV-1 nuclear entry and the antiviral mechanisms of HIV-1 capsid inhibitors.

HIV-1 inhibitors, such as Bevirimat (BVM) and Lenacapavir (LEN), block the production and maturation of infectious virions. However, their mechanisms remain unclear due to the absence of high-resolution structures for BVM complexes and LEN's structural data being limited to the mature capsid. Utilizing perforated virus-like particles (VLPs) produced from mammalian cells, we developed an approach to determine in situ cryo-electron microscopy (cryo-EM) structures of HIV-1 with inhibitors. This allowed for the first structural determination of the native immature HIV-1 particle with BVM and LEN bound inside the VLPs at high resolutions. Our findings offer a more accurate model of BVM engaging the Gag lattice and, importantly, demonstrate that LEN not only binds the mature capsid but also targets the immature lattice in a distinct manner. The binding of LEN induces a conformational change in the capsid protein (CA) region and alters the architecture of the Gag lattice, which may affect the maturation process. These insights expand our understanding of the inhibitory mechanisms of BVM and LEN on HIV-1 and provide valuable clues for the design of future inhibitors.

Cyclophilin A (CypA) binds to the HIV-1 capsid to facilitate reverse transcription and nuclear entry and counter the antiviral activity of TRIM5 alpha. Interestingly, recent studies suggest that the capsid enters the nucleus of an infected cell and uncoats prior to integration. We have previously reported that the capsid protein regulates HIV-1 integration. Therefore, we probed whether CypA-capsid interaction also regulates this post-nuclear entry step. First, we challenged CypA-expressing (CypA(+/+)) and CypA-depleted (CypA(-/-)) cells with HIV-1 and quantified the levels of provirus. CypA-depletion significantly reduced integration, an effect that was independent of CypA's effect on reverse transcription, nuclear entry, and the presence or absence of TRIM5 alpha. In addition, cyclosporin A, an inhibitor that disrupts CypA-capsid binding, inhibited proviral integration in CypA(+/+) cells but not in CypA(-/-) cells. HIV-1 capsid mutants (G89V and P90A) deficient in CypA binding were also blocked at the integration step in CypA(+/+) cells but not in CypA(-/-) cells. Then, to understand the mechanism, we assessed the integration activity of the HIV-1 preintegration complexes (PICs) extracted from acutely infected cells. PICs from CypA(-/-) cells retained lower integration activity in vitro compared to those from CypA(+/+) cells. PICs from cells depleted of both CypA and TRIM5 alpha also had lower activity, suggesting that CypA's effect on PIC was independent of TRIM5 alpha. Finally, CypA protein specifically stimulated PIC activity, as this effect was significantly blocked by CsA. Collectively, these results provide strong evidence that CypA directly promotes HIV-1 integration, a previously unknown role of this host factor in the nucleus of an infected cell.

Many viral pathogens evolved mechanisms for hijacking host transport machinery during infection. One example is the human immunodeficiency virus type 1 (HIV-1). Using in vitro reconstitution biochemistry, we established that HIV-1 directly binds to dynein motor. The direct attachment of HIV-1 to dynein allows HIV-1 to leverage microtubule-driven motility of that many divergent dynein cargo adaptors. Throughout our studies, we observed significantly reduced velocities of motile HIV-1 compared to the velocity of the dynein-dynactin-cargo adaptors alone.

We have investigated the function of inositol hexakisphosphate (IP6) and inositol pentakisphosphate (IP5) in the replication of murine leukemia virus (MLV). While IP6 is known to be critical for the life cycle of HIV-1, its significance in MLV remains unexplored. We find that IP6 is indeed important for MLV replication. It significantly enhances endogenous reverse transcription (ERT) in MLV. Additionally, a pelleting-based assay reveals that IP6 can stabilize MLV cores, thereby facilitating ERT. We find that IP5 and IP6 are packaged in MLV particles. However, unlike HIV-1, MLV depends upon the presence of IP6 and IP5 in target cells for successful infection. This IP6/5 requirement for infection is reflected in impaired reverse transcription observed in IP6/5-deficient cell lines. In summary, our findings demonstrate the importance of capsid stabilization by IP6/5 in the replication of diverse retroviruses; we suggest possible reasons for the differences from HIV-1 that we observed in MLV.IMPORTANCEInositol hexakisphosphate (IP6) is crucial for the assembly and replication of HIV-1. IP6 is packaged in HIV-1 particles and stabilizes the viral core enabling it to synthesize viral DNA early in viral infection. While its importance for HIV-1 is well established, its significance for other retroviruses is unknown. Here we report the role of IP6 in the gammaretrovirus, murine leukemia virus (MLV). We found that like HIV-1, MLV packages IP6, and as in HIV-1, IP6 stabilizes the MLV core thus promoting reverse transcription. Interestingly, we discovered a key difference in the role of IP6 in MLV versus HIV-1: while HIV-1 is not dependent upon IP6 levels in target cells, MLV replication is significantly reduced in IP6-deficient cell lines. We suggest that this difference in IP6 requirements reflects key differences between HIV-1 and MLV replication.

The viral capsid performs critical functions during HIV-1 infection and is a validated target for antiviral therapy. Previous studies have established that the proper structure and stability of the capsid are required for efficient HIV-1 reverse transcription in target cells. Moreover, it has recently been demonstrated that permeabilized virions and purified HIV-1 cores undergo efficient reverse transcription in vitro when the capsid is stabilized by addition of the host cell metabolite inositol hexakisphosphate (IP6). However, the molecular mechanism by which the capsid promotes reverse transcription is undefined. Here we show that wild type HIV-1 virions can undergo efficient reverse transcription in vitro in the absence of a membrane-permeabilizing agent. This activity, originally termed “natural endogenous reverse transcription” (NERT), depends on expression of the viral envelope glycoprotein during virus assembly and its incorporation into virions. Truncation of the gp41 cytoplasmic tail markedly reduced NERT activity, suggesting that gp41 licenses the entry of nucleotides into virions. By contrast to reverse transcription in permeabilized virions, NERT required neither the addition of IP6 nor a mature capsid, indicating that an intact viral membrane can substitute for the function of the viral capsid during reverse transcription in vitro. Collectively, these results demonstrate that the viral capsid functions as a nanoscale container for reverse transcription during HIV-1 infection.

Delivering the virus genome into the host nucleus through the nuclear pore complex (NPC) is pivotal in human immunodeficiency virus 1 (HIV-1) infection. The mechanism of this process remains mysterious owing to the NPC complexity and the labyrinth of molecular interactions involved. Here we built a suite of NPC mimics-DNA-origami-corralled nucleoporins with programmable arrangements-to model HIV-1 nuclear entry. Using this system, we determined that multiple cytoplasm-facing Nup358 molecules provide avid binding for capsid docking to the NPC. The nucleoplasm-facing Nup153 preferentially attaches to high-curvature regions of the capsid, positioning it for tip-leading NPC insertion. Differential capsid binding strengths of Nup358 and Nup153 constitute an affinity gradient that drives capsid penetration. Nup62 in the NPC central channel forms a barrier that viruses must overcome during nuclear import. Our study thus provides a wealth of mechanistic insight and a transformative toolset for elucidating how viruses like HIV-1 enter the nucleus.

The HIV-1 genome encodes a small number of proteins with structural, enzymatic, regulatory, and accessory functions. These viral proteins interact with a number of host factors to promote the early and late stages of HIV-1 infection. During the early stages of infection, interactions between the viral proteins and host factors enable HIV-1 to enter the target cell, traverse the cytosol, dock at the nuclear pore, gain access to the nucleus, and integrate into the host genome. Similarly, the viral proteins recruit another set of host factors during the late stages of infection to orchestrate HIV-1 transcription, translation, assembly, and release of progeny virions. Among the host factors implicated in HIV-1 infection, Cyclophilin A (CypA) was identified as the first host factor to be packaged within HIV-1 particles. It is now well established that CypA promotes HIV-1 infection by directly binding to the viral capsid. Mechanistic models to pinpoint CypA's role have spanned from an effect in the producer cell to the early steps of infection in the target cell. In this review, we will describe our understanding of the role(s) of CypA in HIV-1 infection, highlight the current knowledge gaps, and discuss the potential role of this host factor in the post-nuclear entry steps of HIV-1 infection.

Increasing evidence has suggested that the HIV-1 capsid enters the nucleus in a largely assembled, intact form. However, not much is known about how the cone-shaped capsid interacts with the nucleoporins (NUPs) in the nuclear pore for crossing the nuclear pore complex. Here, we elucidate how NUP153 binds HIV-1 capsid by engaging the assembled capsid protein (CA) lattice. A bipartite motif containing both canonical and noncanonical interaction modules was identified at the C-terminal tail region of NUP153. The canonical cargo-targeting phenylalanine-glycine (FG) motif engaged the CA hexamer. By contrast, a previously unidentified triple-arginine (RRR) motif in NUP153 targeted HIV-1 capsid at the CA tri-hexamer interface in the capsid. HIV-1 infection studies indicated that both FG- and RRR-motifs were important for the nuclear import of HIV-1 cores. Moreover, the presence of NUP153 stabilized tubular CA assemblies in vitro. Our results provide molecular-level mechanistic evidence that NUP153 contributes to the entry of the intact capsid into the nucleus.

HIV-1 capsid (CA) stability is important for viral replication. E45A and P38A mutations enhance and reduce core stability, thus impairing infectivity. Second-site mutations R132T and T216I rescue infectivity. Capsid lattice stability was studied by solving seven crystal structures (in native background), including P38A, P38A/T216I, E45A, E45A/R132T CA, using molecular dynamics simulations of lattices, cryo-electron microscopy of assemblies, time-resolved imaging of uncoating, biophysical and biochemical characterization of assembly and stability. We report pronounced and subtle, short- and long-range rearrangements: (1) A38 destabilized hexamers by loosening interactions between flanking CA protomers in P38A but not P38A/T216I structures. (2) Two E45A structures showed unexpected stabilizing CANTD-CANTD inter-hexamer interactions, variable R18-ring pore sizes, and flipped N-terminal β-hairpin. (3) Altered conformations of E45Aa α9-helices compared to WT, E45A/R132T, WTPF74, WTNup153, and WTCPSF6 decreased PF74, CPSF6, and Nup153 binding, and was reversed in E45A/R132T. (4) An environmentally sensitive electrostatic repulsion between E45 and D51 affected lattice stability, flexibility, ion and water permeabilities, electrostatics, and recognition of host factors.

The mature HIV-1 capsid is stabilized by host and viral determinants. The capsid protein CA binds to the cellular metabolites inositol hexakisphosphate (IP6) and its precursor inositol (1, 3, 4, 5, 6) pentakisphosphate (IP5) to stabilize the mature capsid. In target cells, capsid destabilization by the antiviral compounds lenacapavir and PF74 reveals a HIV-1 infectivity defect due to IP5/IP6 (IP5/6) depletion. To test whether intrinsic HIV-1 capsid stability and/or host factor binding determines HIV-1 insensitivity to IP5/6 depletion, a panel of CA mutants was assayed for infection of IP5/6-depleted T cells and wildtype cells. Four CA mutants with unstable capsids exhibited dependence on host IP5/6 for infection and reverse transcription (RTN). Adaptation of one such mutant, Q219A, by spread in culture resulted in Vpu truncation and a capsid three-fold interface mutation, T200I. T200I increased intrinsic capsid stability as determined by in vitro uncoating of purified cores and partially reversed the IP5/6-dependence in target cells for each of the four CA mutants. T200I further rescued the changes to lenacapavir sensitivity associated with the parental mutation. The premature dissolution of the capsid caused by the IP5/6-dependent mutations imparted a unique defect in integration targeting that was rescued by T200I. Collectively, these results demonstrate that T200I restored other capsid functions after RTN for the panel of mutants. Thus, the hyperstable T200I mutation stabilized the instability defects imparted by the parental IP5/6-dependent CA mutation. The contribution of Vpu truncation to mutant adaptation was linked to BST-2 antagonization, suggesting that cell-to-cell transfer promoted replication of the mutants. We conclude that interactions at the three-fold interface are adaptable, key mediators of capsid stability in target cells and are able to antagonize even severe capsid instability to promote infection.

Viruses exploit host cytoskeletal elements and motor proteins for trafficking through the dense cytoplasm. Yet the molecular mechanism that describes how viruses connect to the motor machinery is unknown. Here, we demonstrate the first example of viral microtubule trafficking from purified components: HIV-1 hijacking microtubule transport machinery. We discover that HIV-1 directly binds to the retrograde microtubule-associated motor, dynein, and not via a cargo adaptor, as previously suggested. Moreover, we show that HIV-1 motility is supported by multiple, diverse dynein cargo adaptors as HIV-1 binds to dynein light and intermediate chains on dynein's tail. Further, we demonstrate that multiple dynein motors tethered to rigid cargoes, like HIV-1 capsids, display reduced motility, distinct from the behavior of multiple motors on membranous cargoes. Our results introduce a new model of viral trafficking wherein a pathogen opportunistically 'hijacks' the microtubule transport machinery for motility, enabling multiple transport pathways through the host cytoplasm.