Abstract: 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 demonstrate that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, coneshaped 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.

Abstract: During the first half of the viral life cycle, HIV-1 reverse transcribes its RNA genome and integrates the double-stranded DNA copy into a host cell chromosome. Despite progress in characterizing and inhibiting these processes, in situ mechanistic and structural studies remain challenging. This is because these operations are executed by individual viral preintegration complexes deep within cells. We therefore reconstituted and imaged the early stages of HIV-1 replication in a cell-free system. HIV-1 cores released from permeabilized virions supported efficient, capsid-dependent endogenous reverse transcription to produce double-stranded DNA genomes, which sometimes looped out from ruptured capsid walls. Concerted integration of both viral DNA ends into a target plasmid then proceeded in a cell extract-dependent reaction. This reconstituted system uncovers the role of the capsid in templating replication.

Abstract: Structures of flavivirus (dengue virus and Zika virus) particles are known to near-atomic resolution and show detailed structure and arrangement of their surface proteins (E and prM in immature virus or M in mature virus). By contrast, the arrangement of the capsid proteins:RNA complex, which forms the core of the particle, is poorly understood, likely due to inherent dynamics. Here, we stabilize immature Zika virus via an antibody that binds across the E and prM proteins, resulting in a subnanometer resolution structure of capsid proteins within the virus particle. Fitting of the capsid protein into densities shows the presence of a helix previously thought to be removed via proteolysis. This structure illuminates capsid protein quaternary organization, including its orientation relative to the lipid membrane and the genomic RNA, and its interactions with the transmembrane regions of the surface proteins. Results show the capsid protein plays a central role in the flavivirus assembly process. The structure of flavivirus surface proteins has been elucidated, but the conformation of capsid proteins within particles is less clear. Here, the authors provide a subnanometer resolution structure of Zika virus capsid protein within the virus particle, elucidating its quaternary organization and role in flavivirus packaging.

Abstract: Adeno-associated viruses (AAVs) are small, non-enveloped, and have a T=1 icosahedral capsid. They belong to the Parvoviridae, genus Dependoparvovirus. Interest in AAVs has grown over recent years as they have emerged as promising gene therapy vectors. The AAV capsid, encapsulating the transgene, consists, in total, of 60 subunits made up from three distinct viral proteins (VPs) originating from the same cap gene (VP1, VP2, and VP3), which vary only in their N-terminus. While all three VPs play a crucial and specific role in cell-entry and transduction, their exact stoichiometry and organization in AAV capsids has, despite the availability of several high-resolution structures remained elusive. Here we obtained a set of native mass spectra of intact AAV capsids (Mw ≈ 3.8 MDa) that display both highly resolved regions and regions wherein interferences occur. Through spectrum simulation we resolved and elucidated this spectral complexity, allowing us to directly assess the VP stoichiometries in a panel of serotypes from different production platforms. The data reveals an extremely heterogeneous population of capsids of variable composition. The relative abundance for each of the hundreds of co-occurring capsid compositions is accurately described by a model based upon stochastic assembly from a mixed pool of expressed VP1, VP2, and VP3. We show that even the single-most abundant VP stoichiometry represents only a few percent of the total AAV population. We estimate that virtually every AAV capsid in a particular preparation has a unique composition and arrangement, i.e. no particle is identical. The systematic scoring of the stochastic assembly model against experimental high-resolution native MS data offers a sensitive and accurate new method to characterize these exceptionally heterogeneous gene-delivery vectors.

Abstract: Gene delivery vectors based on adeno-associated virus (AAV) are highly promising due to several desirable features of this parent virus, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells and sustained maintenance of the viral genome. However, the conclusion from clinical data using these vectors is that there is a need to develop new AAVs with a higher transduction efficiency and specificity for relevant target tissues. To overcome these limitations, we chemically modified the surface of the capsid of AAV vectors. These modifications were achieved by chemical coupling of a ligand by the formation of a thiourea functionality between the amino group of the capsid proteins and the reactive isothiocyanate motif incorporated into the ligand. This strategy does not require genetic engineering of the capsid sequence. The proof of concept was first evidenced using a fluorophore (FITC). Next, we coupled the N-acetylgalactosamine ligand onto the surface of the AAV capsid for asialoglycoprotein receptor-mediated hepatocyte-targeted delivery. Chemically-modified capsids also showed reduced interactions with neutralizing antibodies. Taken together, our findings reveal the possibility of creating a specific engineered platform for targeting AAVs via chemical coupling.

Abstract: Capsid assembly is a critical step in the hepatitis B virus (HBV) life cycle, mediated by the core protein. Core is a potential target for new antiviral therapies, the capsid assembly modulators (CAMs). JNJ-56136379 (JNJ-6379) is a novel and potent CAM currently in phase II trials. We evaluated the mechanisms of action (MOAs) and antiviral properties of JNJ-6379 in vitro Size exclusion chromatography and electron microscopy studies demonstrated that JNJ-6379 induced the formation of morphologically intact viral capsids devoid of genomic material (primary MOA). JNJ-6379 accelerated the rate and extent of HBV capsid assembly in vitro JNJ-6379 specifically and potently inhibited HBV replication; its median 50% effective concentration (EC50) was 54 nM (HepG2.117 cells). In HBV-infected primary human hepatocytes (PHHs), JNJ-6379, when added with the viral inoculum, dose-dependently reduced extracellular HBV DNA levels (median EC50 of 93 nM) and prevented covalently closed circular DNA (cccDNA) formation, leading to a dose-dependent reduction of intracellular HBV RNA levels (median EC50 of 876 nM) and reduced antigen levels (secondary MOA). Adding JNJ-6379 to PHHs 4 or 5 days postinfection reduced extracellular HBV DNA and did not prevent cccDNA formation. Time-of-addition PHH studies revealed that JNJ-6379 most likely interfered with postentry processes. Collectively, these data demonstrate that JNJ-6379 has dual MOAs in the early and late steps of the HBV life cycle, which is different from the MOA of nucleos(t)ide analogues. JNJ-6379 is in development for chronic hepatitis B treatment and may translate into higher HBV functional cure rates.

Abstract: The tripartite-motif protein, TRIM5α, is an innate immune sensor that potently restricts retrovirus infection by binding to human immunodeficiency virus capsids. Higher-ordered oligomerization of this protein forms hexagonally patterned structures that wrap around the viral capsid, despite an anomalously low affinity for the capsid protein (CA). Several studies suggest TRIM5α oligomerizes into a lattice with a symmetry and spacing that matches the underlying capsid, to compensate for the weak affinity, yet little is known about how these lattices form. Using a combination of computational simulations and electron cryo-tomography imaging, we reveal the dynamical mechanisms by which these lattices self-assemble. Constrained diffusion allows the lattice to reorganize, whereas defects form on highly curved capsid surfaces to alleviate strain and lattice symmetry mismatches. Statistical analysis localizes the TRIM5α binding interface at or near the CypA binding loop of CA. These simulations elucidate the molecular-scale mechanisms of viral capsid cellular compartmentalization by TRIM5α.

Abstract: Adeno-associated viruses (AAVs) from clade E are often used as vectors in gene delivery applications. This clade includes rhesus isolate 10 (AAVrh.10) and 39 (AAVrh.39) which, unlike representative AAV8, are capable of crossing the blood-brain barrier (BBB), thereby enabling the delivery of therapeutic genes to the central nervous system. Here, the capsid structures of AAV8, AAVrh.10 and AAVrh.39 have been determined by cryo-electron microscopy and three-dimensional image reconstruction to 3.08-, 2.75-, and 3.39-A resolution, respectively, to enable a direct structural comparison. AAVrh.10 and AAVrh.39 are 98% identical in amino acid sequence but only ∼93.5% identical to AAV8. However, the capsid structures of all three viruses are similar, with only minor differences observed in the previously described surface variable regions, suggesting that specific residues S269 and N472, absent in AAV8, may confer the ability to cross the BBB in AAVrh.10 and AAVrh.39. Head-to-head comparison of empty and genome-containing particles showed DNA ordered in the previously described nucleotide-binding pocket, supporting the suggested role of this pocket in DNA packaging for the Dependoparvovirus The structural characterization of these viruses provides a platform for future vector engineering efforts toward improved gene delivery success with respect to specific tissue targeting, transduction efficiency, antigenicity, or receptor retargeting.IMPORTANCE Recombinant adeno-associated virus vectors (rAAVs), based on AAV8 and AAVrh.10, have been utilized in multiple clinical trials to treat different monogenetic diseases. The closely related AAVrh.39 has also shown promise in vivo As recently attained for other AAV biologics, e.g., Luxturna and Zolgensma, based on AAV2 and AAV9, respectively, the vectors in this study will likely gain U.S. Food and Drug Administration approval for commercialization in the near future. This study characterized the capsid structures of these clinical vectors at atomic resolution using cryo-electron microscopy and image reconstruction for comparative analysis. The analysis suggested two key residues, S269 and N472, as determinants of BBB crossing for AAVrh.10 and AAVrh.39, a feature utilized for central nervous system delivery of therapeutic genes. The structure information thus provides a platform for engineering to improve receptor retargeting or tissue specificity. These are important challenges in the field that need attention. Capsid structure information also provides knowledge potentially applicable for regulatory product approval.

Abstract: The HIV-1 capsid protein makes up the core of the virion and plays a critical role in early steps of HIV replication. Due to its exposure in the cytoplasm after entry, HIV capsid is a target for host cell factors that act directly to block infection such as TRIM5α and MxB. Several host proteins also play a role in facilitating infection, including in the protection of HIV-1 capsid from recognition by host cell restriction factors. Through an unbiased screening approach, called HIV-CRISPR, we show that the CPSF6-binding deficient, N74D HIV-1 capsid mutant is sensitive to restriction mediated by human TRIM34, a close paralog of the well-characterized HIV restriction factor TRIM5α. This restriction occurs at the step of reverse transcription, is independent of interferon stimulation, and limits HIV-1 infection in key target cells of HIV infection including CD4+ T cells and monocyte-derived dendritic cells. TRIM34 can also restrict some SIV capsids. TRIM34 restriction requires TRIM5α as knockout or knockdown of TRIM5α results in a loss of antiviral activity. Through immunofluorescence studies, we show that TRIM34 and TRIM5α colocalize to cytoplasmic bodies and are more frequently observed to be associated with infecting N74D capsids than with WT HIV-1 capsids. Our results identify TRIM34 as an HIV-1 CA-targeting restriction factor and highlight the potential role for heteromultimeric TRIM interactions in contributing to restriction of HIV-1 infection in human cells.

Abstract: Coronavirus disease 2019 (COVID-19) is a new emerging human infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, also previously known as 2019-nCoV), originated in Wuhan seafood and animal market, China. Since December 2019, more than 69,000 cases of COVID-19 have been confirmed in China and quickly spreads to other counties. Currently, researchers put their best efforts to identify effective drugs for COVID-19. The neutralizing antibody, which binds to viral capsid in a manner that inhibits cellular entry of virus and uncoating of the genome, is the specific defense against viral invaders. In this study, we investigate to identify neutralizing antibodies that can bind to SARS-CoV-2 Sipke (S) protein and interfere with the interaction between viral S protein and a host receptor by bioinformatic methods. The sequence analysis of S protein showed two major differences in the RBD region of the SARS-CoV-2 S protein compared to SARS-CoV and SARS-CoV related bat viruses (btSARS-CoV). The insertion regions were close to interacting residues with the human ACE2 receptor. Epitope analysis of neutralizing antibodies revealed that SARS-CoV neutralizing antibodies used conformational epitopes, whereas MERS-CoV neutralizing antibodies used a common linear epitope region, which contributes to form the β-sheet structure in MERS-CoV S protein and deleted in SARS-CoV-2 S protein. To identify effective neutralizing antibodies for SARS-CoV-2, the binding affinities of neutralizing antibodies with SARS-CoV-2 S protein were predicted and compared by antibody-antigen docking simulation. The result showed that CR3022 neutralizing antibody from human may have higher binding affinity with SARS-CoV-2 S protein than SARS-CoV S protein. We also found that F26G19 and D12 mouse antibodies could bind to SARS-CoV S protein with high affinity. Our findings provide crucial clues towards the development of antigen diagnosis, therapeutic antibody, and the vaccine against SARS-CoV-2.

Abstract: Enterovirus A71 (EV-A71), a positive-stranded RNA virus of the Picornaviridae family, may cause neurological complications or fatality in children. We examined specific factors responsible for this virulence using a chemical genetics approach. Known compounds from an anti-EV-A71 herbal medicine, Salvia miltiorrhiza (Danshen), were screened for anti-EV-A71. We identified a natural product, rosmarinic acid (RA), as a potential inhibitor of EV-A71 by cell-based antiviral assay and in vivo mouse model. Results also show that RA may affect the early stage of viral infection and may target viral particles directly, thereby interfering with virus-P-selectin glycoprotein ligand-1 (PSGL1) and virus-heparan sulfate interactions without abolishing the interaction between the virus and scavenger receptor B2 (SCARB2). Sequencing of the plaque-purified RA-resistant viruses revealed a N104K mutation in the five-fold axis of the structural protein VP1, which contains positively charged amino acids reportedly associated with virus-PSGL1 and virus-heparan sulfate interactions via electrostatic attraction. The plasmid-derived recombinant virus harbouring this mutation was confirmed to be refractory to RA inhibition. Receptor pull-down showed that this non-positively charged VP1-N104 is critical for virus binding to heparan sulfate. As the VP1-N104 residue is conserved among different EV-A71 strains, RA may be useful for inhibiting EV-A71 infection, even for emergent virus variants. Our study provides insight into the molecular mechanism of virus-host interactions and identifies a promising new class of inhibitors based on its antiviral activity and broad spectrum effects against a range of EV-A71.

Abstract: New hepatitis B virions released from infected hepatocytes are the result of an intricate maturation process that starts with the formation of the nucleocapsid providing a confined space where the viral DNA genome is synthesized via reverse transcription. Virion assembly is finalized by the enclosure of the icosahedral nucleocapsid within a heterogeneous envelope. The latter contains integral membrane proteins of three sizes, collectively known as hepatitis B surface antigen, and adopts multiple conformations in the course of the viral life cycle. The nucleocapsid conformation depends on the reverse transcription status of the genome, which in turn controls nucleocapsid interaction with the envelope proteins for virus exit. In addition, after secretion the virions undergo a distinct maturation step during which a topological switch of the large envelope protein confers infectivity. Here we review molecular determinants for envelopment and models that postulate molecular signals encoded in the capsid scaffold conducive or adverse to the recruitment of envelope proteins.

Abstract: The HIV-1 capsid protein performs multiple roles in virus replication both during assembly and particle release and during virus trafficking into the nucleus. In order to decipher the roles of capsid protein during early replication, a reliable method to follow its intracellular distribution is required. To complement existing approaches to track HIV-1 capsid during early infection, we developed an HIV-1 imaging strategy, relying on viruses incorporating enhanced green fluorescent protein (eGFP)-tagged capsid (CA-eGFP) protein and mCherry-tagged integrase (IN-mCherry). Wild-type infectivity and sensitivity to inhibition by PF74 point to the functionality of CA-eGFP-containing complexes. Low numbers of CA-eGFP molecules were located inside the viral core and imported into the nucleus without significant loss in intensity. Less than 5% of particles carrying both CA-eGFP and IN-mCherry retained both labelled proteins after nuclear entry, implying a major uncoating event at the nuclear envelope dissociating IN and CA. Still, 20% of all CA-eGFP-containing complexes were detected in the nucleus. Unlike for IN-mCherry complexes, addition of the integrase inhibitor raltegravir had no effect on CA-eGFP-containing complexes, suggesting that these may be not (yet) competent for integration. Our imaging strategy offers alternative visualization of viral capsid trafficking and helps clarify its potential role during integration.IMPORTANCE HIV-1 capsid protein (CA) builds a conical shell protecting viral genomic RNA inside the virus particles. Upon entry into host cells, this shell disassembles in a process of uncoating, which is coordinated with reverse transcription of viral RNA into DNA. After uncoating, a portion of CA remains associated with the viral DNA and mediates its nuclear import and, potentially, integration into host DNA. In this study, we tagged CA with eGFP to follow its trafficking in host cells and address potential CA roles in the nucleus. We found that while functional viruses import the tagged CA into the nucleus, this capsid protein is not part of integration-competent complexes. The roles of nuclear CA thus remain to be established.

Abstract: Flaviviruses are enveloped positive-sense single-stranded RNA arboviruses, infectious to humans and many other animals and are transmitted primarily via tick or mosquito vectors. Capsid is the primary structural protein to interact with viral genome within virus particles and is therefore necessary for efficient packaging. However, in cells, capsid interacts with many proteins and nucleic acids and we are only beginning to understand the broad range of functions of flaviviral capsids. It is known that capsid dimers interact with the membrane of lipid droplets, aiding in both viral packaging and storage of capsid prior to packaging. However, capsid dimers can bind a range of nucleic acid templates in vitro, and likely interact with a range of targets during the flavivirus lifecycle. Capsid may interact with host RNAs, resulting in altered RNA splicing and RNA transcription. Capsid may also bind short interfering-RNAs and has been proposed to sequester these species to protect flaviviruses from the invertebrate siRNA pathways. Capsid can also be found in the nucleolus, where it wreaks havoc on ribosome biogenesis. Here we review flavivirus capsid structure, nucleic acid interactions and how these give rise to multiple functions. We also discuss how these features might be exploited either in the design of effective antivirals or novel vaccine strategies.

Abstract: Use of the prototypical adeno-associated virus type 2 (AAV2) capsid delivered unexpectedly modest efficacy in an early liver-targeted gene therapy trial for hemophilia B. This result is consistent with subsequent data generated in chimeric mouse-human livers showing that the AAV2 capsid transduces primary human hepatocytes in vivo with low efficiency. In contrast, novel variants generated by directed evolution in the same model, such as AAV-NP59, transduce primary human hepatocytes with high efficiency. While these empirical data have immense translational implications, the mechanisms underpinning this enhanced AAV capsid transduction performance in primary human hepatocytes are yet to be fully elucidated. Remarkably, AAV-NP59 differs from the prototypical AAV2 capsid by only 11 aa and can serve as a tool to study the correlation between capsid sequence/structure and vector function. Using two orthogonal vectorological approaches, we have determined that just 2 of the 11 changes present in AAV-NP59 (T503A and N596D) account for the enhanced transduction performance of this capsid variant in primary human hepatocytes in vivo, an effect that we have associated with attenuation of heparan sulfate proteoglycan (HSPG) binding affinity. In support of this hypothesis, we have identified, using directed evolution, two additional single amino acid substitution AAV2 variants, N496D and N582S, which are highly functional in vivo. Both substitution mutations reduce AAV2's affinity for HSPG. Finally, we have modulated the ability of AAV8, a highly murine-hepatotropic serotype, to interact with HSPG. The results support our hypothesis that enhanced HSPG binding can negatively affect the in vivo function of otherwise strongly hepatotropic variants and that modulation of the interaction with HSPG is critical to ensure maximum efficiency in vivo. The insights gained through this study can have powerful implications for studies into AAV biology and capsid development for preclinical and clinical applications targeting liver and other organs.

Abstract: Human noroviruses are the most common viral agents of acute gastroenteritis. Recently, human intestinal enteroids were shown to be permissive for norovirus infection. We tested their suitability as a system to study norovirus neutralization. Hyperimmune sera raised against virus-like particles (VLPs) representing different genotypes showed highly-specific neutralization activity against GII.4 and GII.6 noroviruses. Carbohydrate blocking assays and neutralization exhibited similar patterns in antibody responses. Notably, sera produced against chimeric VLPs that presented swapped structural domains, shell and protruding (P), from different genotypes showed that neutralization is primarily mediated by antibodies mapping to the P domain of the norovirus capsid protein. This study provides empirical information on the antigenic differences among genotypes as measured by neutralization, which could guide vaccine design.

Abstract: Bacteriophage MS2 is a positive-sense, single-stranded RNA virus encapsulated in an asymmetric T = 3 pseudo-icosahedral capsid. It infects Escherichia coli through the F-pilus, in which it binds through a maturation protein incorporated into its capsid. Cryogenic electron microscopy has previously shown that its genome is highly ordered within virions, and that it regulates the assembly process of the capsid. In this study, we have assembled recombinant MS2 capsids with non-genomic RNA containing the capsid incorporation sequence, and investigated the structures formed, revealing that T = 3, T = 4 and mixed capsids between these two triangulation numbers are generated, and resolving structures of T = 3 and T = 4 capsids to 4 A and 6 A respectively. We conclude that the basic MS2 capsid can form a mix of T = 3 and T = 4 structures, supporting a role for the ordered genome in favouring the formation of functional T = 3 virions.

Abstract: Recently, we established an adeno-associated virus (AAV9) capsid-promoter interaction that directly determined cell-specific gene expression across two synthetic promoters, Cbh and CBA, in the rat striatum. These studies not only expand this capsid-promoter interaction to include another promoter in the rat striatum but also establish AAV capsid-promoter interactions in the nonhuman primate brain. When AAV serotype 9 (AAV9) vectors were injected into the rat striatum, the minimal synthetic promoter JetI drove green fluorescent protein (GFP) gene expression predominantly in oligodendrocytes. However, similar to our previous findings, the insertion of six alanines into VP1/VP2 of the AAV9 capsid (AAV9AU) significantly shifted JetI-driven GFP gene expression to neurons. In addition, previous retrograde tracing studies in the nonhuman primate brain also revealed the existence of a capsid-promoter interaction. When rAAV2-Retro vectors were infused into the frontal eye field (FEF) of rhesus macaques, local gene expression was prominent using either the hybrid chicken beta actin (CAG) or human synapsin (hSyn) promoters. However, only the CAG promoter, not the hSyn promoter, led to gene expression in the ipsilateral claustrum and contralateral FEF. Conversely, infusion of rAAV2-retro-hSyn vectors, but not rAAV2-retro-CAG, into the macaque superior colliculus led to differential and selective retrograde gene expression in cerebellotectal afferent cells. Clearly, this differential promoter/capsid expression profile could not be attributed to promoter inactivation from retrograde transport of the rAAV2-Retro vector. In summary, we document the potential for AAV capsid/promoter interactions to impact cell-specific gene expression across species, experimental manipulations, and engineered capsids, independent of capsid permissivity.

Abstract: A defining activity of retroviruses is reverse transcription, the process by which the viral genomic RNA is converted into the double-stranded DNA required for virus replication. Reverse transcriptase (RT), the viral enzyme responsible for this process, was identified in 1970 by assaying permeabilized retrovirus particles for DNA synthesis in vitro Such reactions are inefficient, with only a small fraction of viral genomes being converted to full-length double-stranded DNA molecules, possibly owing to disruption of the structure of the viral core. Here, we show that reverse transcription in purified HIV-1 cores is enhanced by the addition of the capsid-binding host cell metabolite inositol hexakisphosphate (IP6). IP6 potently enhanced full-length minus-strand synthesis, as did hexacarboxybenzene (HCB), which also stabilizes the HIV-1 capsid. Both IP6 and HCB stabilized the association of the viral CA and RT proteins with HIV-1 cores. In contrast to the wild type, cores isolated from mutant HIV-1 particles containing intrinsically hyperstable capsids exhibited relatively efficient reverse transcription in the absence of IP6, further indicating that the compound promotes reverse transcription by stabilizing the viral capsid. We also observed that the capsid-destabilizing antiviral compound PF74 inhibited endogenous reverse transcription with a potency that mirrors its ability to inhibit reverse transcription during infection. Our results show that the stabilization of the HIV-1 capsid permits efficient reverse transcription in HIV-1 cores, providing a sensitive experimental system for analyzing the functions of viral and host cell molecules and the role of capsid disassembly (uncoating) in the process.IMPORTANCE HIV-1 infection requires reverse transcription of the viral genome. While much is known about the biochemistry of reverse transcription from simplified biochemical reactions, reverse transcription during infection takes place within a viral core. However, endogenous reverse transcription reactions using permeabilized HIV-1 virions or purified viral cores have been inefficient. Using viral cores purified from infectious HIV-1 particles, we show that efficient reverse transcription is achieved in vitro by addition of the capsid-stabilizing metabolite inositol hexakisphosphate. The enhancement of reverse transcription was linked to the capsid-stabilizing effect of the compound, consistent with the known requirement for an intact or semi-intact viral capsid for HIV-1 infection. Our results establish a biologically relevant system for dissecting the function of the viral capsid and its disassembly during reverse transcription. The system should also prove useful for mechanistic studies of capsid-targeting antiviral drugs.

Abstract: From cellular deposition of the HIV-1 capsid to integration of the viral genome, the capsid constitutes a primary target of a variety of host proteins that work to either promote or inhibit HIV-1 infection. Successful progression of HIV-1 infection depends on interactions between the capsid and host factors involved in stability, cellular transport, nuclear import, and genome integration. The virus must also guard its reverse-transcribing genome inside the capsid from host restriction factors that bind the capsid and suppress infection. Understanding the structure and dynamics of the capsid protein (CA) component and the assembled capsid sheds light on the molecular underpinnings of overall capsid stability, architecture, and flexibility that govern HIV-1 capsid–host interactions. The vast majority of these interactions are mediated through recognition of higher order interfaces only present in the assembled capsid lattice. Patterns formed at these interfaces serve as signposts for capsid-binders. Here we provide a graphical summary of the intricate interactions between host factors and the HIV-1 capsid while highlighting recent research. Insights into how host proteins interact with the capsid is crucial for understanding the HIV-1 replication cycle and developing antiviral therapeutics to prevent viral genome integration.

Abstract: RNA interference (RNAi) is a conserved antiviral immune defense in eukaryotes, and numerous viruses have been found to encode viral suppressors of RNAi (VSRs) to counteract antiviral RNAi. Alphaviruses are a large group of positive-stranded RNA viruses that maintain their transmission and life cycles in both mosquitoes and mammals. However, there is little knowledge about how alphaviruses antagonize RNAi in both host organisms. In this study, we identified that Semliki Forest virus (SFV) capsid protein can efficiently suppress RNAi in both insect and mammalian cells by sequestrating double-stranded RNA and small interfering RNA. More importantly, when the VSR activity of SFV capsid was inactivated by reverse genetics, the resulting VSR-deficient SFV mutant showed severe replication defects in mammalian cells, which could be rescued by blocking the RNAi pathway. Besides, capsid protein of Sindbis virus also inhibited RNAi in cells. Together, our findings show that SFV uses capsid protein as VSR to antagonize RNAi in infected mammalian cells, and this mechanism is probably used by other alphaviruses, which shed new light on the knowledge of SFV and alphavirus.IMPORTANCE Alphaviruses are a genus of positive-stranded RNA viruses and include numerous important human pathogens, such as Chikungunya virus, Ross River virus, Western equine encephalitis virus, etc., which create the emerging and reemerging public health threat worldwide. RNA interference (RNAi) is one of the most important antiviral mechanisms in plants and insects. Accumulating evidence has provided strong support for the existence of antiviral RNAi in mammals. In response to antiviral RNAi, viruses have evolved to encode viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. It is unclear whether alphaviruses encode VSRs that can suppress antiviral RNAi during their infection in mammals. In this study, we first uncovered that capsid protein encoded by Semliki Forest virus (SFV), a prototypic alphavirus, had a potent VSR activity that can antagonize antiviral RNAi in the context of SFV infection in mammalian cells, and this mechanism is probably used by other alphaviruses.

Abstract: The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp’s top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly. Alphaviruses need to selectively package genomic viral RNA for transmission, but the packaging mechanism remains unclear. Here, Brown et al. combine PAR-CLIP with biotinylated capsid protein (Cp) retrieval and identify multiple Cp binding sites on genomic viral RNA that promote virion formation.

Abstract: During herpesvirus infection, egress of nascent viral capsids from the nucleus is mediated by the viral nuclear egress complex (NEC) NEC deforms the inner nuclear membrane (INM) around the capsid by forming a hexagonal array However, how the NEC coat interacts with the capsid and how curved coats are generated to enable budding is yet unclear Here, by structure-guided truncations, confocal microscopy, and cryoelectron tomography, we show that binding of the capsid protein UL25 promotes the formation of NEC pentagons rather than hexagons We hypothesize that during nuclear budding, binding of UL25 situated at the pentagonal capsid vertices to the NEC at the INM promotes formation of NEC pentagons that would anchor the NEC coat to the capsid Incorporation of NEC pentagons at the points of contact with the vertices would also promote assembly of the curved hexagonal NEC coat around the capsid, leading to productive egress of UL25-decorated capsids

Abstract: Hepatitis B virus (HBV) replicates its genomic DNA via viral DNA polymerase self-primed reverse transcription of a RNA pre-genome in the nucleocapsid assembled by 120 core protein (Cp) dimers. The arginine-rich carboxyl-terminal domain (CTD) of Cp plays an important role in the selective packaging of viral DNA polymerase-pregenomic (pg) RNA complex into nucleocapsid. Previous studies suggested that the CTD is initially phosphorylated at multiple sites to facilitate viral RNA packaging and subsequently dephosphorylated in association with viral DNA synthesis and secretion of DNA-containing virions. However, our recent studies suggested that Cp is hyper-phosphorylated as free dimers and its dephosphorylation is associated with pgRNA encapsidation. Herein, we provide further genetic and biochemical evidence supporting that extensive Cp dephosphorylation does take place during the assembly of pgRNA-containing nucleocapsids, but not empty capsids. Moreover, we found that cellular protein phosphatase 1 (PP1) is required for Cp dephosphorylation and pgRNA packaging. Interestingly, the PP1 catalytic subunits α and β were packaged into pgRNA-containing nucleocapsids, but not empty capsids, and treatment of HBV replicating cells with core protein allosteric modulators (CpAMs) promoted empty capsid assembly and abrogated the encapsidation of PP1 α and β. Our study thus identified PP1 as a host cellular factor that is co-packaged into HBV nucleocapsids, and plays an essential role in selective packaging of the viral DNA-polymerase-pgRNA complex through catalyzing Cp dephosphorylation.

Abstract: Generation and screening of libraries of adeno-associated virus (AAV) variants have emerged as a powerful method for identifying novel capsids for gene therapy applications. For the majority of libraries, vast population diversity requires multiplexed production, in which a library of inverted terminal repeat (ITR)-containing plasmid variants is transfected together into cells to generate the viral library. This process has the potential to be confounded by cross-packaging and mosaicism, in which particles are comprised of genomes and capsid monomers derived from different library members. Here, we investigate the prevalence of cross-packaging and mosaicism in simplified, minimal libraries using novel assays designed to assess capsid composition and packaging fidelity. We show that AAV library variants are prone to cross-packaging and capsid mosaic formation when produced at high plasmid levels, although to a lesser extent than in a recombinant context. We also provide experimental evidence that dilution of input library DNA significantly increases capsid monomer homogeneity and increases capsid:genome correlation in AAV libraries. Lastly, we determine that similar dilution methods yield higher-quality libraries when used for in vivo screens. Together, these findings quantitatively characterized the prevalence of cross-packaging and mosaicism in AAV libraries and established conditions that minimize related noise in subsequent screens.

Abstract: Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and has been shown to be closely associated with various malignancies. Here, we present a complete atomic model of EBV, including the icosahedral capsid, the dodecameric portal and the capsid-associated tegument complex (CATC). Our in situ portal from the tegumented capsid adopts a closed conformation with its channel valve holding the terminal viral DNA and with its crown region firmly engaged by three layers of ring-like dsDNA, which, together with the penton flexibility, effectively alleviates the capsid inner pressure placed on the portal cap. In contrast, the CATCs, through binding to the flexible penton vertices in a stoichiometric manner, accurately increase the inner capsid pressure to facilitate the pressure-driven genome delivery. Together, our results provide important insights into the mechanism by which the EBV capsid, portal, packaged genome and the CATCs coordinately achieve a pressure balance to simultaneously benefit both viral genome retention and ejection.