Biology subjects

Dick, R. A.

Publications and source records attributed to Dick, R. A..

11 recordsLinked to original sources

bioRxiv2026

Retroviral CA subdomains encode their immature assembly properties independently of strict subdomain co-pairing

Retroviral capsid (CA) proteins display a conserved two-domain architecture consisting of independently folded N-terminal (CA-NTD) and a C-terminal (CA-CTD) subdomains. Despite a strong CA protein fold conservation, different retrovirus species are employing remarkably variable geometrical solutions to stabilize the CA lattices that form the immature virus particle. For instance, while HTLV-1 has been reported to use the CA-NTD to stabilize its immature lattice, HIV-1 relies on a contiguous structural unit formed of the CA-CTD together with the short spacer peptide 1 (SP1). This raises the question whether immature assembly phenotypes are modular features encoded independently into individual CA subdomains, or if they are emergent features requiring strict co-pairing or co-evolution between CA subdomains of a given species. To test for these two options, we have designed chimeric proteins pairing the CA subdomains of HTLV-1 and HIV-1. Our results show that the CA subdomains of HIV-1 and HTLV-1 can be clearly distinguished into assembly-passive and assembly-driving categories. Moreover, pairing assembly-driving subdomains from the two retrovirus species still allows them to independently form their native immature contacts, while at the same time trying to impose their preferred curvature on each other. Mutations of either subdomain within the chimeric context do not disrupt assembly, but rather shift the phenotype balance while relieving curvature strains, demonstrating that all immature assembly properties can be encoded into a single subdomain. Our results therefore highlight that the two-domain architecture of retroviral capsid is a result of a division of labor approach built on structural modularity, where, at least for immature assembly, one of the subdomains is the key driver for assembly competence while the other could serve more regulatory functions.

microbiology↗

bioRxiv2026

Cryo-electron tomography of Nipah virus structural protein complexes in virus-like particles

Nipah virus (NiV) is a BSL-4 zoonotic paramyxovirus with [~]75% human mortality. The matrix protein (M) of NiV and other paramyxoviruses binds the inner leaflet of the cellular plasma membrane, orchestrating virion assembly by bringing together transmembrane glycoproteins (F/G) and ribonucleoprotein complexes (N). However, the interactions of these full-length proteins within membrane complexes remain elusive. Using cryo-electron tomography and subtomogram averaging of virus like particles (VLPs), we interrogated the protein:protein interactions of the main NiV structural proteins M/N/F/G. The M lattice structure determined to 7[A] revealed a novel M-dimer arrangement that yielded two distinct repeating holes. Notably, F-trimers were arranged above only one of the two holes, dependent on Fs cytoplasmic tail. G was enriched in regions of higher M-VLP curvature, while N dramatically increased M-VLP pleomorphism. This work provides novel insights into paramyxoviral protein complexes, structures, and morphology.

microbiology↗

bioRxiv2026

Structural Basis of Polypurine Track Strand Displacement by HIV-1 Reverse Transcriptase

To complete reverse transcription, HIV-1 reverse transcriptase (RT) must displace the RNase H-resistant polypurine tract (PPT) primers. This enables synthesis of the long terminal repeats and formation of the central cDNA flap. However, the molecular mechanism of this PPT strand displacement (SD) has remained unknown, and no structural data exist on how a retroviral polymerase execute these reactions. We report the first cryo-EM structures of HIV-1 RT bound to nucleic acid substrates containing either a PPTRNA or PPTDNA displacement strand, with incoming dATP positioned at the polymerase catalytic site. These structures reveal key features of the PPT displacement mechanism by RT. Specifically, we observed a binding mode where the template nucleotide (T1) base-paired to the first displacement nucleotide (D1) undergoes a 90{degrees} rotation relative to the preceding template base (T0). This sharp template flip positions D1 [~]30 [A] away from the primers 3-end and is coordinated by RTp66 residues at the SD interface: F61 and R78 contact T1/T0 to drive template translocation, while W24 engages both T1 and D1 to stabilize the displacement strand. Biochemical and virological mutagenesis experiments confirm that interactions with F61 and R78 are essential for both canonical cDNA polymerization and SD, whereas the W24-nucleotide interactions are required exclusively for SD but are dispensable for standard cDNA synthesis. These results contribute to the structural and functional understanding of PPT strand displacement by HIV-1 RT and reveal a distinct mechanistic vulnerability for the design of next-generation antiretrovirals.

biochemistry↗

bioRxiv2026

Damaging the conical morphology of HIV-1 capsid by targeting the FG-binding pocket and disfavoring pentameric subunits needed for core closure

The HIV-1 capsid is an essential viral component, targeted by the long-acting antiretroviral Lenacapavir (LEN). LEN binds to the HIV-1 capsid protein (CA) at the phenylalanine-glycine (FG) binding pocket (FGBP), a site for multiple host-factor and antiviral interactions in CA hexamers (CAHEX). Previously, we generated a chemical library to investigate the FGBP; ZW-1261, a lead compound, exhibits potent antiviral activity and strong inter-subunit interactions within CAHEX. Here, we report the molecular mechanism by which ZW-1261 affects the morphology and integrity of capsid lattice. ZW-1261 alone rapidly induces tubular CA assemblies; simultaneous addition of ZW-1261 with the assembly cofactor inositol hexaphosphate (IP6) forms morphologically distinct tubes. In mature virions, IP6 is required for the assembly of both CAHEX and CA pentamers (CAPENT). Cryogenic-electron microscopy analysis of in vitro assembled capsid-like particles (CLPs) with IP6 suggests that ZW-1261 leads to the absence of CAPENT and damages the pre-formed conical lattice. To elucidate how this FGBP-targeting antiviral impacts CAPENT, we further solved structures of CAPENT-only icosahedral assemblies (T = 1), formed by reported mutations, that were treated with ZW-1261. We find that ZW-1261 binding in these constrained T = 1 assemblies converts CAPENT to a CAHEX-like conformation. Collectively, this suggests a mechanism by which addition of FGBP-binding inhibitor to native cores leads to the absence of CAPENT, impacting capsid closure and core integrity.

molecular biology↗

bioRxiv2026

Lenacapavir prevents production of infectious HIV-1 by abrogating immature virus assembly.

The HIV-1 capsid effector Lenacapavir (LEN) acts by disrupting the early (reverse transcription and nuclear entry) and late (assembly and maturation) stages of the viral lifecycle. The early stage requires an intact Capsid consisting of a lattice of capsid protein (CA) hexamers and pentamers. Phenylalanine-Glycine (FG) containing nuclear pore proteins interact with Capsid hexamers at their FG pockets, facilitating nuclear entry. Disruption of Capsid lattice stability, or competitive binding to the FG pocket, by LEN blocks infection. Here, we provide insight into the effects of LEN on the late stage. Using a combination of cryo-EM structure determination, in vitro assembly, and in situ viral assays, we determined that treatment of producer cells with LEN abrogates the production of infectious virus via multiple mechanisms. Previous studies have shown that HIV-1 produced from LEN treated cells have improperly formed Capsids. However, how LEN interacts with the CA domain of the Gag polyprotein during assembly, prior to maturation, is unclear. Using a viral protease (PR) defective HIV-1 clone, which traps the Gag lattice in an immature state, we found that LEN dramatically remodulates the CA domain. The resulting CA layer was mature-like despite the absence of PR cleavage. We dubbed this unnatural state as "premature" lattice. Proper immature and mature lattice formation requires inositol hexakisphosphate (IP6), but our cryo-EM work revealed a lack of IP6 in the premature lattice. These findings provide insight into the mechanisms by which LEN prevents infectious HIV-1 production.

molecular biology↗

bioRxiv2025

Translocation of HIV capsid core through the Nuclear Pore Complex by affinity gradient

The HIV capsid core encapsulates the viral genome for subsequent integration into host cellular DNA. Prior to nuclear entry, the core must translocate through the Nuclear Pore Complex (NPC). This transit involves interactions between the capsid core and phenylalanine-glycine (FG) repeats found in nucleoporins within the NPC. Despite this critical role in the viral replication cycle, the molecular mechanism of capsid core translocation remains unclear. FG repeats consist of three classes of canonical motifs: FG, GLFG, and FxFG motifs. These are segregated within the NPC to define distinct zones of the gating machinery. FG- and FxFG-type motifs are enriched in the cytoplasmic and nuclear ("nuclear basket") peripheries of the NPC while GLFG motifs are enriched in regions adjacent to the core of the NPC. To investigate the capsid core translocation, we use biochemical, biophysical, and structural approaches to study FG-capsid interactions in a quantitative manner. We show that the capsid (CA) interacts with a range of diverse FG repeats with varying affinities. GLFG motifs of core NUP98 exhibit increased affinity to CA proteins compared to other conventional FG/FxFG. However, the non-canonical FxFG motif of NUP153 at the "nuclear basket" significantly increases binding affinity to CA compared to canonical FxFG, therefore called FG super-motif. In addition, C-terminal motif of NUP153 consists of a stretch of basic residues, which enhances the affinity of this non-canonical FG super-motif to capsid core at the NPC nuclear periphery. We identified other binding enhancers of the NPC core FG-NUPs, NUP58 and POM121. The relationship between the binding strength of FG/FxFG binding enhancers of NUP58, POM121, NUP153 and their position within the NPC also shows capsid core binding affinity increases with increasing proximity to the "nuclear basket." Based on our data, the difference in binding affinities between the canonical FxFG motif and the enhancer-coupled FG super-motif of NUP153 to capsid cores at the "nuclear basket" is approximately 1,000-fold. Therefore, the diverse FG motifs and binding enhancers, which are naturally distributed within the NPC into distinct zones, create an avidity gradient--with changes in both concentration and binding affinity--along the cytoplasmic-nuclear axis. We suggest that HIV capsid translocation into the nucleus is potentiated by this gradient in a unidirectional manner (outside- to-inside) within the NPC.

microbiology↗

bioRxiv2024

The conserved HIV-1 spacer peptide 2 triggers matrix lattice maturation

HIV-1 particles are released in an immature, non-infectious form. Proteolytic cleavage of the main structural polyprotein Gag into functional domains induces rearrangement into mature, infectious virions. In immature virus particles, the Gag membrane binding domain, MA, forms a hexameric protein lattice that undergoes structural transition upon cleavage into a distinct, mature MA lattice. The mechanism of MA lattice maturation is unknown. Here we show that released spacer peptide 2 (SP2), a conserved peptide of unknown function situated [~]300 residues downstream of MA, binds MA to induce structural maturation. By high-resolution in-virus structure determination of MA, we show that MA does not bind lipid into a side pocket as previously thought, but instead binds SP2 as an integral part of the protein-protein interfaces that stabilise the mature lattice. Analysis of Gag cleavage site mutants showed that SP2 release is required for MA maturation, and we demonstrate that SP2 is sufficient to induce maturation of purified MA on lipid layers in vitro. SP2-triggered MA maturation correlated with faster fusion of virus with target cells. Our results reveal a new, unexpected interaction between two HIV-1 components, provide a high-resolution structure of mature MA, establish the trigger of MA structural maturation, and assign function to the SP2 peptide.

microbiology↗

bioRxiv2022

Structural insights into HIV-1 polyanion-dependent capsid lattice formation revealed by singleparticle cryo-EM

The HIV-1 capsid houses the viral genome and interacts extensively with host cell proteins throughout the viral life cycle. It is composed of capsid protein (CA), which assembles into a conical fullerene lattice composed of roughly 200 CA hexamers and 12 CA pentamers. Previous structural analyses of individual CA hexamers and pentamers have provided valuable insight into capsid structure and function, but high-resolution information about these assemblies in the broader context of the capsid lattice is lacking. In this study, we combined cryo-electron tomography and single particle analysis cryo-electron microscopy to determine high-resolution structures of continuous regions of the capsid lattice containing both hexamers and pentamers. We also developed a new method of in vitro lattice assembly that enabled us to directly study the lattice under a wider range of conditions than has previously been possible. Using this approach, we identified a critical role for inositol hexakisphosphate (IP6) in pentamer formation and determined the structure of the CA lattice bound to the capsid-targeting antiretroviral drug GS-6207 (Lenacapvir). Our work reveals new structural details of the mature HIV-1 CA lattice and establishes the combination of lattice templating and single particle analysis as a robust strategy for studying retroviral capsid structure and capsid interactions with host proteins and antiviral compounds. Significance statementThe mature HIV-1 capsid is composed of the capsid (CA) protein arranged in a conical lattice of hexamers and pentamers. Numerous structures of individual CA hexamers and pentamers alone have been published, but the molecular details of these assemblies in a more global, lattice-wide context are lacking. Here, we present high-resolution cryo-electron microscopy structures of continuous regions of the capsid lattice containing both hexamers and pentamers. We also describe key differences in the assembly and structures of these oligomers that have important implications for understanding retroviral maturation and for ongoing efforts to pharmacologically target the HIV-1 capsid.

biochemistry↗

bioRxiv2022

Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs

The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) image processing has been well documented for single particle analysis (SPA). Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4-6 [A] resolution range. In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 [A]. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.

molecular biology↗

bioRxiv2020

Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer

Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 was [~]100-fold more potent at promoting RSV mature CA assembly than observed for HIV-1 and removal of IP6 in vivo reduced infectivity by 100-fold. By cryo-electron tomography and subtomogram averaging, mature virus-like particles (VLPs) showed an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 had opposing effects on CA in vitro assembly, inducing formation of T=1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles revealed that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.

molecular biology↗

bioRxiv2020

Inositol hexakisphosphate (IP6) and inositol pentakisphosphate (IP5) are required for viral particle release of retroviruses belonging to the primate lentivirus genus

Inositol hexakisphosphate (IP6) potently stimulates HIV-1 particle assembly in vitro and infectious particle production in vivo. However, knockout cells lacking the enzyme inositol-pentakisphosphate 2-kinase (IPPK-KO), which adds the final phosphate to inositol pentakisphosphate (IP5) to produce IP6, were still able to produce infectious HIV-1 particles at a greatly reduced rate. HIV-1 in vitro assembly can also be stimulated to a lesser extent with IP5, but it was not known if IP5 could also function in promoting assembly in vivo. IPPK-KO cells expressed no detectable IP6 but elevated IP5 levels and displayed a 20-100-fold reduction in infectious particle production, correlating with lost virus release. Transient transfection of an IPPK expression vector stimulated infectious particle production and release in IPPK-KOs but not in wildtype cells. Several attempts to make an IP6 and IP5 deficient stable cell line were not successful, but transient expression of multiple inositol polyphosphate phosphatase-1 (MINPP1) into IPPK-KOs resulted in the near ablation of IP6 and IP5. Under these conditions, HIV-1 infectious particle production and virus release were essentially abolished (1000-fold reduction). However, other retroviruses including a Gammaretrovirus, a Betaretrovirus, and two non-primate Lentiviruses displayed only a modest (3-fold) reduction in infectious particle production from IPPK-KOs and were not significantly altered by expression of IPPK or MINPP1. The only other retrovirus found that showed a clear IP6/IP5 dependence was the primate (macaque) Lentivirus Simian Immunodeficiency Virus (SIV-mac), which displayed similar sensitivity to IP6/IP5 levels as HIV-1. Finally, we found that loss of IP6/IP5 in viral target cells had no effect on permissiveness to HIV-1 infection. However, because it was not possible to generate viral particles devoid of IP6 and IP5, we were not able to determine if IP6 or IP5 derived from the virus producer cells is required at additional steps beyond assembly. Author SummaryInositol hexakisphosphate (IP6) is a co-factor required for efficient production of infectious HIV-1 particles. The HIV-1 structural protein Gag forms a hexagonal lattice structure. The negatively charged IP6 sits in the middle of the hexamer and stabilizes a ring of positively charged lysines. Previously described results show that depletion of IP6 reduces, but does not eliminate, infectious virus production. This depletion was achieved through knock-out of inositol-pentakisphosphate 2-kinase (IPPK-KO), the enzyme responsible for adding the sixth and final phosphate to the molecule. Whether IP6 is required, another inositol phosphate can substitute, or IP6 is simply acting as an enhancer for virus production was unknown. Here, we show that loss of IP6 and inositol pentakisphosphate (IP5) abolishes infectious HIV-1 production from cells. We do this through a cell-based system using transiently expressed proteins to restore or deplete IP6 and IP5 in the IPPK-KO cell line. We further show that the IP6 and IP5 requirement is a feature of primate lentiviruses, but not all retroviruses, and that IP6 and IP5 is required in the producer but not the target cell for HIV-1 infection.

microbiology↗