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Diarrhea, often severe, is a recognized and frequently early symptom during acute COVID-19 infection and may persist or develop for the first time in patients with long-COVID, with socioeconomic consequences. Diarrheal mechanisms in these cases are poorly understood. There is evidence for disruption of intestinal epithelial barrier function and also for changes in the gut microbiome, which is critical for gut immunity and metabolism. Whether the SARS-CoV-2 virus has adverse effects on intestinal transport proteins is unclear. However, the ability of the virus to inhibit expression and activity of an aldosterone-regulated epithelial sodium (Na+) channel (ENaC) present in human distal colon, which is responsible for Na+ and water salvage, points to possible disruption of other intestinal transport proteins during COVID-19 infection. In this Perspective, we develop this idea by highlighting possible intestinal transport protein targets for the SARS-CoV-2 virus and discussing how their interactions might be explored in the laboratory.

Following internalisation, viruses employ the changing environment of maturing endosomes as cues to promote endosomal escape, a process mediated by viral glycoproteins. Specifically, we previously showed that both high [K+] and low pH promote entry of Bunyamwera virus (BUNV), the prototypical bunyavirus. Here, we used sub-tomogram averaging combined with AlphaFold, to generate a pseudo-atomic model of the whole glycoprotein envelope of BUNV. This allowed us to unambiguously locate the Gc fusion domain and its chaperone Gn within the floor domain of the spike. We also confirmed that low pH and high [K+] alters the viral glycoproteins, resulting in an activated intermediate state functionally-distinct from the highly ordered ground state, and we localize these changes to the floor domain. Biochemical data suggests that in this intermediate state the viral fusion loops are partially exposed and selectively interact with host cell membranes. Taken together, we reveal new mechanistic understanding of the requirements for virus entry.

The Bunyavirales order of RNA viruses comprises emerging pathogens for which approved preventative or therapeutic measures for human use are not available. The genome of all Bunyavirales consists of negative-sense RNA segments wrapped by the virus-encoded nucleocapsid protein (NP) to form ribonucleoproteins (RNPs). RNPs represent the active template for RNA synthesis and the form in which the genome is packaged into virions, functions that require inherent flexibility. We present a pseudo-atomic model of a native RNP purified from Bunyamwera virus (BUNV), the prototypical Bunyavirales member, based on a cryo-electron microscopy (cryo-EM) average at 13 Å resolution with subsequent fitting of the BUNV NP crystal structure by molecular dynamics. We show the BUNV RNP possesses relaxed helical architecture, with successive helical turns separated by ∼18 Å. The model shows that adjacent NP monomers in the RNP chain interact laterally through flexible N- and C-terminal arms, with no helix-stabilizing interactions along the longitudinal axis. Instead, EM analysis of RNase-treated RNPs suggests their chain integrity is dependent on the encapsidated genomic RNA, thus providing the molecular basis for RNP flexibility. Overall, this work will assist in designing anti-viral compounds targeting the RNP and inform studies on bunyaviral RNP assembly, packaging and RNA replication.SignificanceBunyaviruses are emerging RNA viruses that cause significant disease and economic burden and for which vaccines or therapies approved for human use do not exist. The bunyavirus genome does not exist as naked RNA; instead it is wrapped up by the nucleoprotein (NP) to form a ribonucleoprotein (RNP). Using the prototypical bunyavirus, Bunyamwera virus, we determined the 3D structure of the native RNP, revealing a helical architecture with NP molecules linked by lateral contacts only, with no helix-stabilizing longitudinal contacts. Instead, the RNA genome itself plays a role in maintaining the helical architecture, allowing a high degree of flexibility that is critical for several stages of the virus replication cycle, such as segment circularization and genome packaging into virions.

Ribosomes have long been thought of as homogeneous macromolecular machines, but recent evidence suggests they are heterogeneous and could be specialised to regulate translation. Here, we have characterised ribosomal protein heterogeneity across 4 tissues of Drosophila melanogaster. We find that testes and ovaries contain the most heterogeneous ribosome populations, which occurs through a combination of paralog-enrichment and paralog-switching. We have solved structures of ribosomes purified from in vivo tissues by cryo-EM, revealing differences in precise ribosomal arrangement for testis and ovary 80S ribosomes. Differences in the amino acid composition of paralog pairs and their localisation on the ribosome exterior indicate paralog-switching could alter the ribosome surface, enabling different proteins to regulate translation. One testis-specific paralog-switching pair is also found in humans, suggesting this is a conserved site of ribosome heterogeneity. Overall, this work allows us to propose that mRNA translation might be regulated in the gonads through ribosome heterogeneity, providing a potential means of ribosome specialisation.

The Bunyavirales order of segmented negative sense RNA viruses includes over 500 isolates that infect insects, animals, and plants, and are often associated with severe and fatal disease in humans. To multiply and cause disease, bunyaviruses must transport their genomes from outside the cell into the cytosol, achieved by transit through the endocytic network. We have previously shown that the model bunyaviruses Bunyamwera virus (BUNV) and Hazara virus (HAZV) exploit the changing potassium concentration ([K+]) of maturing endosomes to release their genomes at the appropriate endosomal location. K+ was identified as a biochemical cue to activate the viral fusion machinery, promoting fusion between viral and cellular membranes, consequently permitting genome release. In this study, we further define the biochemical prerequisites for BUNV and HAZV entry and their K+ dependence. We report four major findings: (1) BUNV and HAZV require cellular cholesterol during virus infection; (2) cholesterol is required during BUNV endosomal escape; (3) cholesterol depletion from host cells impairs their ability to accumulate K+ in maturing endosomes, revealing new insights into endosomal K+ homeostasis; (4) ‘priming’ BUNV virions with K+ prior to infection alleviates BUNV cholesterol requirement, revealing the mechanism of cholesterol dependence. Taken together, we provide a new model in which cholesterol abundance influences K+ endosomal homeostasis and consequently the efficiency of bunyavirus infection. The ability to inhibit bunyaviruses with existing cholesterol lowering drugs offers new options for future anti-bunyavirus interventions for pathogenic family members.

Ion channels play key roles in almost all facets of cellular physiology and have emerged as key host cell factors for a multitude of viral infections. A catalogue of ion channel-blocking drugs have been shown to possess antiviral activity, some of which are in widespread human usage for ion channel-related diseases, highlighting new potential for drug repurposing. The emergence of ion channel–virus interactions has also revealed the intriguing possibility that channelopathies may explain some commonly observed virus induced pathologies. This field is rapidly evolving and an up-to-date summary of new discoveries can inform future perspectives. We herein discuss the role of ion channels during viral lifecycles, describe the recently identified ion channel drugs that can inhibit viral infections, and highlight the potential contribution of ion channels to virus-mediated disease.

The family Hantaviridae within the Bunyavirales order comprises tri-segmented negative sense RNA viruses, many of which are rodent-borne emerging pathogens associated with fatal human disease. In contrast, hantavirus infection of corresponding rodent hosts results in inapparent or latent infections, which can be recapitulated in cultured cells that become persistently infected. In this study, we used Tula virus (TULV) to investigate the location of hantavirus replication during early, peak and persistent phases of infection, over a 30-day time course. Using immunofluorescent (IF) microscopy, we showed that the TULV nucleocapsid protein (NP) is distributed within both punctate and filamentous structures, with the latter increasing in size as the infection progresses. Transmission electron microscopy of TULV-infected cell sections revealed these filamentous structures comprised aligned clusters of filament bundles. The filamentous NP-associated structures increasingly co-localized with the Golgi and with the stress granule marker TIA-1 over the infection time course, suggesting a redistribution of these cellular organelles. The analysis of the intracellular distribution of TULV RNAs using fluorescent in-situ hybridization revealed that both genomic and mRNAs co-localized with Golgi-associated filamentous compartments that were positive for TIA. These results show that TULV induces a dramatic reorganization of the intracellular environment, including the establishment of TULV RNA synthesis factories in re-modelled Golgi compartments.

Influenza virus is the causative agent of the ‘flu’. According to the World Health Organisation, Influenza causes up to 5 million cases of severe flu and 500 000 deaths annually. To release its genome inside the cell and start an infection, Influenza virus must fuse its envelope with the endosomal membrane of the host, making this process an excellent drug target. In order to attempt to control, or better yet, eradicate this pathogen, a greater understanding of its entry mechanism, a fundamental aspect of the viral life cycle, is required. It is well established that Influenza A virus (IAV) fusion is driven by the viral glycoprotein hemagglutinin (HA), which when exposed to low pH transitions from a meta-stable pre-fusion to post-fusion state. However, it is becoming increasingly apparent that the ionic balance of the endosomes also has a significant role in the entry of enveloped viruses. Specifically, our preliminary studies on Influenza virus suggest that K+ concentrations within the endocytic pathway play a significant role in Influenza A fusion events. Through the use of broad-spectrum K+ channel inhibitors, a dose-dependant reduction in Influenza infectivity in tissue culture can be observed. Further to these studies, we have isolated Affimers (novel antibody-like proteins that can be produced in large quantities in E. coli) that recognise HAs from different Influenza subtypes (H3N2 and H1N1). Using these Affimers, we aim to develop an early detection method to distinguish between bacterial respiratory infections and ‘the flu’ to alleviate mounting pressures on an already diminishing antibiotic treatment system.

Herpes Simplex Virus (HSV) afflicts ∼90 % of the population, causing a range of diseases from oral cold sores to encephalitis. Despite the widespread effects of HSV, current drugs cannot cure infection and there are no preventative vaccines. HSV infection requires entry into the host cell, which is mediated by the fusion protein, gB. Despite various structures being available for post-fusion gB, a high-resolution model for the pre-fusion state, required to understand the fundamental mechanism of HSV fusion, is still lacking. To elucidate this structure, full length gB was previously expressed on lipid vesicles and was shown via cryo-electron microscopy techniques to adopt the elusive pre-fusion state. Strikingly, two recent studies, one characterising such vesicles, generated two different pre-fusion gB models. The discrepancy between the models can be summarised by the position of the fusion loops: are they positioned facing away from the viral membrane or towards it in the pre-fusion state? Using the gB studded vesicle production method combined with a state-of-the-art Titan Krios cryo-electron microscope equipped with a Volta Phase Plate, a direct electron detector camera and an energy filter, all to increase contrast and signal-to-noise ratio, we aim to settle the dispute as to the structure of pre-fusion gB. Revealing the pre-fusion structure could lead to possible therapeutics to a disease that can have serious complications in newborns and the immunocompromised, a growing cohort in the modern world.

The Bunyavirales order are the largest group of negative stranded RNA viruses, infecting humans as well as a bewildering array of animals and plants, in which select members cause severe or fatal disease. To enter host cells, bunyaviruses undergo endosomal transport to specific cellular destinations and exploit the changing environment of maturing endocytic vesicles to mediate genome release. Several virus-endosome fusion triggers have previously been identified, including endosomal potassium (K+) recently identified by our group. Specifically, we demonstrated a role for K+ channels and endosomal K+ concentration ([K+]) in the ‘priming’ of virions for fusion and uncoating events. Interestingly for Bunyamwera virus (BUNV), both a reduced pH and elevated [K+] were required to permit endosomal escape of the virus. To understand the molecular basis for this requirement we have used cryo-electron tomography to study the changes in virion structure upon K+ and pH treatment. These studies reveal why endosomal [K+] and K+ channels are required for bunyavirus entry, highlighting the potential of K+channels as druggable anti-viral targets.

The herpesvirus family contains eight human pathogens that act as the causative agent of several human maladies. The herpesvirus family can be further subdivided alphaherpesviruses, betaherpesviruses, and the oncogenic gammaherpesviruses. Herpesvirus replication and capsid assembly occurs within the nucleus, from there capsids migrate into the cytoplasm. Following nuclear egress, viral capsids travel to the cellular membrane, where morphogenesis takes place. Recent analysis has demonstrated that the gammaherpesvirus Kaposi’s sarcoma associated herpesvirus (KSHV) upregulates the expression of a host miR-365 to target the cellular protein, DOCK5, to enhance KSHV egress. This hypothesis is supported by the observation that inhibition of miR-365 or overexpression of DOCK5 leads to the prevention of KSHV egress and accumulation of capsids at the plasma membrane. As herpesviruses share several mechanistic features, we are now employing electron microscopy to visualise the cytoplasmic capsid accumulation and to determine how DOCK5 is involved in herpesvirus egress. This will be achieved through advanced microscopy techniques such as correlative light microscopy, cryo-electron microscopy, and a novel nanobiopsy approach known as nanopipetting. Currently we have been establishing the optimal time of capsid egress in both KSHV and Herpes Simplex Virus-1 which will aid in visualising the process through transmission electron microscopy. A better understanding of herpesvirus egress will potentially help identify novel antiviral targets against herpesviruses.

Capsid (CA) plays multiple roles in the HIV-1 replication cycle. CA-CA domain interactions are responsible for multimerization of the Gag polyprotein at virus assembly sites, and in the mature virion, CA monomers assemble into a conical core that encapsidates the viral RNA genome. Multiple CA regions that contribute to the assembly and release of HIV-1 particles have been mapped and investigated. Here, we identified and characterized a Pro-rich loop in CA that is important for the formation of the immature Gag lattice. Changes in this region disrupt viral production and abrogate the formation of infectious, mature virions. Propagation of the mutants in culture led to the selection of second-site compensatory mutations within CA. These results expand our knowledge of the assembly and maturation steps in the viral replication cycle and may be relevant for development of antiviral drugs targeting CA., Late in the HIV-1 replication cycle, the viral structural protein Gag is targeted to virus assembly sites at the plasma membrane of infected cells. The capsid (CA) domain of Gag plays a critical role in the formation of the hexameric Gag lattice in the immature virion, and, during particle release, CA is cleaved from the Gag precursor by the viral protease and forms the conical core of the mature virion. A highly conserved Pro-Pro-Ile-Pro (PPIP) motif (CA residues 122 to 125) [PPIP(122–125)] in a loop connecting CA helices 6 and 7 resides at a 3-fold axis formed by neighboring hexamers in the immature Gag lattice. In this study, we characterized the role of this PPIP(122–125) loop in HIV-1 assembly and maturation. While mutations P123A and P125A were relatively well tolerated, mutation of P122 and I124 significantly impaired virus release, caused Gag processing defects, and abolished infectivity. X-ray crystallography indicated that the P122A and I124A mutations induce subtle changes in the structure of the mature CA lattice which were permissive for in vitro assembly of CA tubes. Transmission electron microscopy and cryo-electron tomography demonstrated that the P122A and I124A mutations induce severe structural defects in the immature Gag lattice and abrogate conical core formation. Propagation of the P122A and I124A mutants in T-cell lines led to the selection of compensatory mutations within CA. Our findings demonstrate that the CA PPIP(122–125) loop comprises a structural element critical for the formation of the immature Gag lattice.

Infectious bursal disease virus (IBDV), a nonenveloped, double-stranded RNA (dsRNA) virus with a T=13 icosahedral capsid, has a virion assembly strategy that initiates with a precursor particle based on an internal scaffold shell similar to that of tailed double-stranded DNA (dsDNA) viruses. In IBDV-infected cells, the assembly pathway results mainly in mature virions that package four dsRNA segments, although minor viral populations ranging from zero to three dsRNA segments also form. We used cryo-electron microscopy (cryo-EM), cryo-electron tomography, and atomic force microscopy to characterize these IBDV populations. The VP3 protein was found to act as a scaffold protein by building an irregular, ∼40-Å-thick internal shell without icosahedral symmetry, which facilitates formation of a precursor particle, the procapsid. Analysis of IBDV procapsid mechanical properties indicated a VP3 layer beneath the icosahedral shell, which increased the effective capsid thickness. Whereas scaffolding proteins are discharged in tailed dsDNA viruses, VP3 is a multifunctional protein. In mature virions, VP3 is bound to the dsRNA genome, which is organized as ribonucleoprotein complexes. IBDV is an amalgam of dsRNA viral ancestors and traits from dsDNA and single-stranded RNA (ssRNA) viruses. IMPORTANCE Structural analyses highlight the constraint of virus evolution to a limited number of capsid protein folds and assembly strategies that result in a functional virion. We report the cryo-EM and cryo-electron tomography structures and the results of atomic force microscopy studies of the infectious bursal disease virus (IBDV), a double-stranded RNA virus with an icosahedral capsid. We found evidence of a new inner shell that might act as an internal scaffold during IBDV assembly. The use of an internal scaffold is reminiscent of tailed dsDNA viruses, which constitute the most successful self-replicating system on Earth. The IBDV scaffold protein is multifunctional and, after capsid maturation, is genome bound to form ribonucleoprotein complexes. IBDV encompasses numerous functional and structural characteristics of RNA and DNA viruses; we suggest that IBDV is a modern descendant of ancestral viruses and comprises different features of current viral lineages.

Many enveloped viruses enter cells through the endocytic network, from which they must subsequently escape through fusion of viral and endosomal membranes. This membrane fusion is mediated by virus-encoded spikes that respond to the dynamic endosomal environment, which triggers conformational changes in the spikes that initiate the fusion process. Several fusion triggers have been identified and include pH, membrane composition, and endosome-resident proteins, and these cues dictate when and where viral fusion occurs. We recently reported that infection with an enveloped bunyavirus requires elevated potassium ion concentrations [K+], controlled by cellular K+ channels, that are encountered during viral transit through maturing endosomes. Here we reveal the molecular basis for the K+ requirement of bunyaviruses through the first direct visualization of a member of the Nairoviridae family, namely Hazara virus (HAZV), using cryo-EM. Using cryo-electron tomography, we observed HAZV spike glycoproteins within infectious HAZV particles exposed to both high and low [K+], which showed that exposure to K+ alone results in dramatic changes to the ultrastructural architecture of the virion surface. In low [K+], the spikes adopted a compact conformation arranged in locally ordered arrays, whereas, following exposure to high [K+], the spikes became extended, and spike–membrane interactions were observed. Viruses exposed to high [K+] also displayed enhanced infectivity, thus identifying K+ as a newly defined trigger that helps promote viral infection. Finally, we confirmed that K+ channel blockers are inhibitory to HAZV infection, highlighting the potential of K+ channels as anti-bunyavirus targets.

In order to multiply and cause disease a virus must transport its genome from outside the cell into the cytosol, most commonly achieved through the endocytic network. Endosomes transport virus particles to specific cellular destinations and viruses exploit the changing environment of maturing endocytic vesicles as triggers to mediate genome release. Previously we demonstrated that several bunyaviruses, which comprise the largest family of negative sense RNA viruses, require the activity of cellular potassium (K+) channels to cause productive infection. Specifically, we demonstrated a surprising role for K+ channels during virus endosomal trafficking. In this study, we have used the prototype bunyavirus, Bunyamwera virus (BUNV), as a tool to understand why K+ channels are required for progression of these viruses through the endocytic network. We report three major findings: First, the production of a dual fluorescently labelled bunyavirus to visualize virus trafficking in live cells. Second, we show that BUNV traffics through endosomes containing high [K+] and that these K+ ions influence the infectivity of virions. Third, we show that K+ channel inhibition can alter the distribution of K+ across the endosomal system and arrest virus trafficking in endosomes. These data suggest high endosomal [K+] is a critical cue that is required for virus infection, and is controlled by cellular K+ channels resident within the endosome network. This highlights cellular K+ channels as druggable targets to impede virus entry, infection and disease., Author summary To penetrate cells and cause disease most viruses traverse the cellular endocytic network and exploit its environment to trigger the release of their genetic material into the cytosol. Endosomal pH, virus-receptor interactions, proteolytic cleavage, and lipid composition of membranes have all been previously reported to act as endosomal triggers. Here, we reveal the identity of an additional critical endosomal trigger for virus entry, namely a requirement for exposure to specific K+ ion concentrations, which we demonstrate is regulated by cellular K+ channels. We show that by blocking K+ channels, we can disrupt the K+ concentration across the endosomal system, impeding virus trafficking. Here, we reveal cellular K+ channels as a new druggable target to inhibit virus entry, and thus disease.

Influenza virus, the causative agent of flu, enters the host cell by endocytosis. The low pH encountered inside endosomes triggers conformational changes in the viral glycoprotein hemagglutinin (HA), that mediate fusion of the viral and cellular membranes. This releases the viral genome into the cytoplasm of the infected cell, establishing the onset of the replication cycle. To investigate the structural basis of HA-mediated membrane fusion, a number of techniques have been employed. These include X-ray crystallography, which has provided atomic models of the HA ectodomain in its initial (pre-fusion) state and of part of HA in its final (post-fusion) state. However, this left an information deficit concerning many other aspects of the fusion process. Electron microscopy (EM) approaches are helping to fill this void. For example, influenza virions at neutral pH have been imaged by cryo-EM and cryo-electron tomography (cryo-ET); thin section EM has shown that influenza viruses enter the cell by endocytosis; the large-scale structural changes in HA when virions are exposed to low pH (pre-fusion to post-fusion states) have been visualized by negative staining and cryo-EM; acidification also induces structural changes in the M1 matrix layer and its separation from the viral envelope; intermediate HA conformations between its pre- and post-fusion states have been detected by cryo-ET supplemented with subtomogram averaging; and fusion of influenza virions with liposomes has been visualized by cryo-ET. In this review, we survey EM-based contributions towards the characterization of influenza virus-mediated membrane fusion and anticipate the potential for future developments.

Recombinant hemagglutinin from influenza viruses with pandemic potential can be produced rapidly in various cell substrates. In this study, we compared the functionality and immunogenicity of bacterially produced oligomeric or monomeric HA1 proteins from H5N1 (A/Vietnam/1203/04) with those of the egg-based licensed subunit H5N1 (SU-H5N1) vaccine in ferrets challenged with homologous or heterologous H5N1 highly pathogenic influenza strains. Ferrets were vaccinated twice with the oligomeric or monomeric rHA1 or with SU-H5N1 (Sanofi Pasteur) emulsified with Titermax adjuvant and were challenged with wild-type homologous (A/Vietnam/1203/04; clade 1) or heterologous (A/Whooperswan/Mongolia/244/2005; clade 2.2) virus. Only the oligomeric rHA1 (not the monomeric rHA1) immunogen and the SU-H5N1 vaccine provided protection against the lethality and morbidity of homologous and heterologous highly pathogenic H5N1. Oligomeric rHA1 generated more cross-neutralizing antibodies and higher levels of serum antibody binding to HA1, with stronger avidity and a better IgG/IgM ratio, than monomeric HA1 and SU-H5N1 vaccines, as determined by surface plasmon resonance (SPR). Importantly, viral loads after heterologous H5N1 challenge were more efficiently controlled in ferrets vaccinated with the oligomeric rHA1 immunogen than in SU-H5N1-vaccinated ferrets. The reduction of viral loads in the nasal washes correlated strongly with higher-avidity antibodies to oligomeric rHA1 derived from H5N1 clade 1 and clade 2.2 viruses, as measured by SPR. This is the first study to show the role of antibody avidity for the HA1 globular head domain in reduction of viral loads in the upper respiratory tract, which could significantly reduce viral transmission.

Summary Viral factories are novel structures built by viruses in infected cells. During their construction organelles are recruited and build a large scaffold for viral replication and morphogenesis. We have studied how a bunyavirus uses the Golgi to build the factory. With the help of confocal and 3D ultrastructural imaging together with molecular mapping in situ and in vitro we have characterized a tubular structure that harbours the viral replication complexes in a globular domain. Numerous ribonucleoproteins were released from purified tubes disrupted in vitro. Actin and myosin I were identified by peptide mass fingerprinting in isolated tubes while actin and the viral NSm non‐structural protein were detected in the tubes' internal proteinaceous scaffold by immunogold labelling. Studies with NSm deletion mutants and drugs affecting actin showed that both NSm and actin are key factors for tube and virus assembly in Golgi. Three‐dimensional reconstructions based on oriented serial sections of infected cells showed that tubes anchor cell organelles to Golgi stacks and make contacts with intracellular viruses. We propose that this new structure, unique among enveloped viruses, assembles in association with the most stable component of Golgi stacks, the actin‐containing matrix scaffold, connecting viral replication and morphogenesis inside viral factories.

The herpes simplex virus 1 (HSV-1) capsid is a massive particle (~200 MDa; 1,250-Å diameter) with T=16 icosahedral symmetry. It initially assembles as a procapsid with ~4,000 protein subunits of 11 different kinds. The procapsid undergoes major changes in structure and composition as it matures, a process driven by proteolysis and expulsion of the internal scaffolding protein. Assembly also relies on an external scaffolding protein, the triplex, an α2β heterotrimer that coordinates neighboring capsomers in the procapsid and becomes a stabilizing clamp in the mature capsid. To investigate the mechanisms that regulate its assembly, we developed a novel isolation procedure for the metastable procapsid and collected a large set of cryo-electron microscopy data. In addition to procapsids, these preparations contain maturation intermediates, which were distinguished by classifying the images and calculating a three-dimensional reconstruction for each class. Appraisal of the procapsid structure led to a new model for assembly; in it, the protomer (assembly unit) consists of one triplex, surrounded by three major capsid protein (MCP) subunits. The model exploits the triplexes’ departure from 3-fold symmetry to explain the highly skewed MCP hexamers, the triplex orientations at each 3-fold site, and the T=16 architecture. These observations also yielded new insights into maturation., IMPORTANCE This paper addresses the molecular mechanisms that govern the self-assembly of large, structurally complex, macromolecular particles, such as the capsids of double-stranded DNA viruses. Although they may consist of thousands of protein subunits of many different kinds, their assembly is precise, ranking them among the largest entities in the biosphere whose structures are uniquely defined to the atomic level. Assembly proceeds in two stages: formation of a precursor particle (procapsid) and maturation, during which major changes in structure and composition take place. Our analysis of the HSV procapsid by cryo-electron microscopy suggests a hierarchical pathway in which multisubunit “protomers” are the building blocks of the procapsid but their subunits are redistributed into different subcomplexes upon being incorporated into a nascent procapsid and are redistributed again in maturation. Assembly is a highly virus-specific process, making it a potential target for antiviral intervention.

Upon release of HIV-1 particles from the infected cell, the viral protease cleaves the Gag polyprotein at specific sites, triggering maturation. During this process, which is essential for infectivity, the capsid protein (CA) reassembles into a conical core. Maturation inhibitors (MIs) block HIV-1 maturation by interfering with protease-mediated CA-spacer peptide 1 (CA-SP1) processing, concomitantly stabilizing the immature CA-SP1 lattice; virions from MI-treated cells retain an immature-like CA-SP1 lattice, whereas mutational abolition of cleavage at the CA-SP1 site results in virions in which the CA-SP1 lattice converts to a mature-like form. We previously reported that propagation of HIV-1 in the presence of MI PF-46396 selected for assembly-defective, compound-dependent mutants with amino acid substitutions in the major homology region (MHR) of CA. Propagation of these mutants in the absence of PF-46396 resulted in the acquisition of second-site compensatory mutations. These included a Thr-to-Ile substitution at SP1 residue 8 (T8I), which results in impaired CA-SP1 processing. Thus, the T8I mutation phenocopies PF-46396 treatment in terms of its ability to rescue the replication defect imposed by the MHR mutations and to impede CA-SP1 processing. Here, we use cryo-electron tomography to show that, like MIs, the T8I mutation stabilizes the immature-like CA-SP1 lattice. These results have important implications for the mechanism of action of HIV-1 MIs; they also suggest that T8I may provide a valuable tool for structural definition of the CA-SP1 boundary region, which has thus far been refractory to high-resolution analysis, apparently because of conformational flexibility in this region of Gag. IMPORTANCE HIV-1 maturation involves dissection of the Gag polyprotein by the viral protease and assembly of a conical capsid enclosing the viral ribonucleoprotein. Maturation inhibitors (MIs) prevent the final cleavage step at the site between the capsid protein (CA) and spacer peptide 1 (SP1), apparently by binding at this site and denying the protease access. Additionally, MIs stabilize the immature-like CA-SP1 lattice, preventing release of CA into the soluble pool. We previously found that T8I, a mutation in SP1, rescues a PF-46396-dependent CA mutant and blocks CA-SP1 cleavage. In this study, we imaged T8I virions by cryo-electron tomography and showed that T8I mutants, like MI-treated virions, contain an immature CA-SP1 lattice. These results lay the groundwork needed to understand the structure of the CA-SP1 interface region and further illuminate the mechanism of action of MIs.

Summary Rubella virus (RUB) assembles its replication complexes (RCs) in modified organelles of endo-lysosomal origin, known as cytopathic vacuoles (CPVs). These peculiar structures are key elements of RUB factories, where rough endoplasmic reticulum, mitochondria, and Golgi are recruited. Bicistronic RUB replicons expressing an antibiotic resistance gene either in the presence or the absence of the RUB capsid (C) gene were used to study the structure of RCs in transfected cells. Confocal microscopy showed that the RUB replicase components P90 and P150 localized to CPVs, as did double-stranded RNA (dsRNA), a marker for RNA synthesis. Electron microscopy (EM) showed that replicons generated CPVs containing small vesicles and large vacuoles, similar to CPVs from RUB-infected cells and that the replicase proteins were sufficient for organelle recruitment. Some of these CPVs contained straight membranes. When cross-sectioned, these rigid membranes appeared to be sheets of closely packed proteins. Immuno-EM revealed that these sheets, apparently in contact with the cytosol, contained both P150 and P90, as well as dsRNA, and thus could be two-dimensional arrays of functional viral replicases. Labelling of dsRNA after streptolysin-O permeabilization showed that replication of viral genome takes place on the cytoplasmic side of CPVs. When present, C accumulated around CPVs. Mitochondrial protein P32 was detected within modified CPVs, the first demonstration of involvement of this protein, which interacts with C, with RCs.

Genome replication and assembly of viruses often takes place in specific intracellular compartments where viral components concentrate, thereby increasing the efficiency of the processes. For a number of viruses the formation of ‘factories’ has been described, which consist of perinuclear or cytoplasmic foci that mostly exclude host proteins and organelles but recruit specific cell organelles, building a unique structure. The formation of the viral factory involves a number of complex interactions and signalling events between viral and cell factors. Mitochondria, cytoplasmic membranes and cytoskeletal components frequently participate in the formation of viral factories, supplying basic and common needs for key steps in the viral replication cycle.

Living cells compartmentalize materials and enzymatic reactions to increase metabolic efficiency. While eukaryotes use membrane-bound organelles, bacteria and archaea rely primarily on protein-bound nanocompartments. Encapsulins constitute a class of nanocompartments widespread in bacteria and archaea whose functions have hitherto been unclear. Here, we characterize the encapsulin nanocompartment from Myxococcus xanthus, which consists of a shell protein (EncA, 32.5 kDa) and three internal proteins (EncB, 17 kDa; EncC, 13 kDa; EncD, 11 kDa). Using cryo-electron microscopy, we determined that EncA self-assembles into an icosahedral shell 32 nm in diameter (26 nm internal diameter), built from 180 subunits with the fold first observed in bacteriophage HK97 capsid. The internal proteins, of which EncB and EncC have ferritin-like domains, attach to its inner surface. Native nanocompartments have dense iron-rich cores. Functionally, they resemble ferritins, cage-like iron storage proteins, but with a massively greater capacity (~30,000 iron atoms versus ~3,000 in ferritin). Physiological data reveal that few nanocompartments are assembled during vegetative growth, but they increase fivefold upon starvation, protecting cells from oxidative stress through iron sequestration.

The M1 matrix protein of influenza A virus, which plays multiple roles in virion assembly and infection, underlies the viral envelope. However, previous studies have given differing accounts of the number of layers in the M1-envelope complex and their thicknesses and compositions. To resolve this issue, we performed cryo-electron microscopy and cryo-electron tomography on the self-same specimens. At neutral pH, there were two kinds of complexes, corresponding to a lipid bilayer with embedded glycoproteins, with and without a closely associated, 4-nm-thick sheet of M1 protein. The reported discrepancies arose from differences in imaging conditions, i.e., in defocus and in whole-particle projections versus thin tomographic slices. Exposure of virions to low pH (as in the endosome) promotes membrane fusion, and previous work has shown that the M2 ion channel causes the virion interior to acidify also. We found that after 5 min at pH 4.9, the proportion of virions lacking an M1 layer increased from 10% to 50%. In virions retaining an M1 layer under these conditions, the M1-envelope complex exhibited two states, viz, the original, neutral-pH state and one in which the M1 layer appeared thinner and/or closer to the membrane. These observations extend previous indications that acidic pH causes the M1 layer to dissociate, leaving the envelope more pliable and, consequently, fusion compatible, and they show that dissociation is preceded by a conformational change in M1.

HIV-1 maturation involves proteolysis of the precursor polyproteins, Gag and Gag-Pol, and assembly of a conical core containing the viral ribonucleoprotein (vRNP), comprised of the vRNA genome and associated proteins, notably nucleocapsid (NC). Allosteric integrase inhibitors (ALLINIs) are antiviral drugs initially thought to impede the viral integrase from inserting the DNA version of the genome into a host chromosome. However, recent studies indicate that their primary effects are on maturation. To investigate these effects, we imaged virions produced in the presence of ALLINIs by cryo-electron tomography (cryo-ET) and “tomo-bubblegram” imaging, a novel labeling technique that exploits the fact that vitrified protein subjected to relatively high levels of electron irradiation generates bubbles of hydrogen gas, which are readily visible. First, a regular cryo-tomogram is calculated. Then, after priming the specimen with additional exposures, a second tilt series is recorded, visualizing the bubbles. With HIV, we exploited the serendipitously discovered susceptibility of NC to bubbling.Most wild-type virions have conical cores with internal density, putatively the vRNP. In contrast, most ALLINI-treated virions have non-conical or conical cores with sparse internal contents but contain “eccentric condensates”, outside the capsid. Tomo-bubblegrams showed that the bubbles specifically label the contents of filled cores and eccentric condensates. In immature virions, in which the domains of Gag and Gag-Pol are radially ordered in a spherical shell, the bubbles appear in the NC layer of that shell. Immature virions in which NC is replaced by a leucine zipper (another RNA binder) yielded no bubbles, confirming NC as the bubbling component. Taken together, these observations provide strong evidence that ALLINIs sabotage capsid assembly and vRNP encapsidation. They also illustrate the potential of bubblegram imaging for mapping components of large macromolecular complexes.

Retrovirus infection starts with the binding of envelope glycoproteins to host cell receptors. Subsequently, conformational changes in the glycoproteins trigger fusion of the viral and cellular membranes. Some retroviruses, such as avian sarcoma/leukosis virus (ASLV), employ a two-step mechanism in which receptor binding precedes low-pH activation and fusion. We used cryo-electron tomography to study virion/receptor/liposome complexes that simulate the interactions of ASLV virions with cells. Binding the soluble receptor at neutral pH resulted in virions capable of binding liposomes tightly enough to alter their curvature. At virion-liposome interfaces, the glycoproteins are ∼3-fold more concentrated than elsewhere in the viral envelope, indicating specific recruitment to these sites. Subtomogram averaging showed that the oblate globular domain in the prehairpin intermediate (presumably the receptor-binding domain) is connected to both the target and the viral membrane by 2.5-nm-long stalks and is partially disordered, compared with its native conformation. Upon lowering the pH, fusion took place. Fusion is a stochastic process that, once initiated, must be rapid, as only final (postfusion) products were observed. These fusion products showed glycoprotein spikes on their surface, with their interiors occupied by patches of dense material but without capsids, implying their disassembly. In addition, some of the products presented a density layer underlying and resolved from the viral membrane, which may represent detachment of the matrix protein to facilitate the fusion process.

Vaccine production and initiation of mass vaccination is a key factor in rapid response to new influenza pandemic. During the 2009–2010 H1N1 pandemic, several bottlenecks were identified, including the delayed availability of vaccine potency reagents. Currently, antisera for the single-radial immunodiffusion (SRID) potency assay are generated in sheep immunized repeatedly with HA released and purified after bromelain-treatment of influenza virus grown in eggs. This approach was a major bottleneck for pandemic H1N1 (H1N1pdm09) potency reagent development in 2009. Alternative approaches are needed to make HA immunogens for generation of SRID reagents in the shortest possible time. In this study, we found that properly folded recombinant HA1 globular domain (rHA1) from several type A viruses including H1N1pdm09 and two H5N1 viruses could be produced efficiently by using a bacterial expression system and subsequent purification. The rHA1 proteins were shown to form functional oligomers of trimers, similar to virus derived HA, and elicited high titer of neutralizing antibodies in rabbits and sheep. Importantly, the immune sera formed precipitation rings with reference antigens in the SRID assay in a dose-dependent manner. The HA contents in multiple H1N1 vaccine products from different manufacturers (and in several lots) as determined with the rHA1-generated sheep sera were similar to the values obtained with a traditionally generated sheep serum from NIBSC. We conclude that bacterially-expressed recombinant HA1 proteins can be produced rapidly and used to generate SRID potency reagents shortly after new influenza strains with pandemic potential are identified.

Viral factories are complex structures in the infected cell where viruses compartmentalize their life cycle. Rubella virus (RUBV) assembles factories by recruitment of rough endoplasmic reticulum (RER), mitochondria and Golgi around modified lysosomes known as cytopathic vacuoles or CPVs. These organelles contain active replication complexes that transfer replicated RNA to assembly sites in Golgi membranes. We have studied the structure of RUBV factory in three dimensions by electron tomography and freeze-fracture. CPVs contain stacked membranes, rigid sheets, small vesicles and large vacuoles. These membranes are interconnected and in communication with the endocytic pathway since they incorporate endocytosed BSA-gold. RER and CPVs are coupled through protein bridges and closely apposed membranes. Golgi vesicles attach to the CPVs but no tight contacts with mitochondria were detected. Immunogold labelling confirmed that the mitochondrial protein p32 is an abundant component around and inside CPVs where it could play important roles in factory activities.

Electron tomography allows the structures such as individual macromolecules, viruses and parts and even whole cells, to be reconstructed in their near-native state in three dimensions. In electron tomography, the transmission electron microscope is used to obtain the projection images of a sample from multiple angles, and those images are back-projected to reconstruct the original object in three dimensions.

12 pages, 9 figures.-- PMID: 19114107 [PubMed].-- Supplementary information (Suppl. movies S1-S3) available at: http://dx.doi.org/10.1016/j.jsb.2008.11.009, Available online Dec 10, 2008 [Epub ahead of print]., Identification of proteins in 3D maps of cells is a main challenge in structural cell biology. For light microscopy (LM) clonable reagents such as green fluorescent protein represented a real revolution and equivalent reagents for transmission electron microscopy (TEM) have been pursued for a long time. To test the viability of the metal-binding protein metallothionein (MT) as a tag for TEM in cells we have studied three MT-fusion proteins in Escherichia coli: AmiC, a component of the division ring, RecA, a DNA-binding protein, and a truncated cytoplasmic form of maltose-binding protein (MBP). Proteins fused to MT were expressed in E. coli. live cells treated with gold salts were processed by fast-freezing and freeze-substitution. Small electron-dense particles were detected in sections of bacteria expressing the MT-fusion proteins and immunogold labelling confirmed that these particles were associated to the fusion proteins. The distribution of the particles correlated with the functional locations of these proteins: MBP–MT3 concentrated in the cytoplasm, AmiC-MT1 in the bacterial division ring and RecAMT1 in the nucleoid. The electron-dense tag was easily visualized by electron tomography and in frozen-hydrated cells., Authors want to thank the support from the UPMCIFR83 electron microscopy service and Rocío Arranz (CNB-CSIC) for assistance with cryoEM studies. This work has been funded by Grants PIF06-004/200620F0024 from the Consejo Superior de Investigaciones Científicas (CSIC), BFU2006-04584/BMC from the Ministerio de Educación y Ciencia of Spain (C.R.), 3DEM European network (LSHG-CT-2004-502828) and ANR (PCV06 142771) (S.M.). E.D. is funded by the JAE-CSIC program of Spain and J.F. by the Comunidad de Madrid.

During virion maturation, HIV-1 capsid protein assembles into a conical core containing the viral ribonucleoprotein (vRNP) complex, thought to be composed mainly of the viral RNA and nucleocapsid protein (NC). After infection, the viral RNA is reverse transcribed into double-stranded DNA, which is then incorporated into host chromosomes by integrase (IN) catalysis. Certain IN mutations (class II) and antiviral drugs (allosteric IN inhibitors [ALLINIs]) adversely affect maturation, resulting in virions that contain “eccentric condensates,” electron-dense aggregates located outside seemingly empty capsids. Here we demonstrate that in addition to this mislocalization of electron density, a class II IN mutation and ALLINIs each increase the fraction of virions with malformed capsids (from ∼12% to ∼53%). Eccentric condensates have a high NC content, as demonstrated by “tomo-bubblegram” imaging, a novel labeling technique that exploits the susceptibility of NC to radiation damage. Tomo-bubblegrams also localized NC inside wild-type cores and lining the spherical Gag shell in immature virions. We conclude that eccentric condensates represent nonpackaged vRNPs and that either genetic or pharmacological inhibition of IN can impair vRNP incorporation into mature cores. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with internal electron density and the infectivity of a class II IN deletion mutant virus. Moreover, the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose a role for IN in initiating core morphogenesis and vRNP incorporation into the mature core during HIV-1 maturation. IMPORTANCE Maturation, a process essential for HIV-1 infectivity, involves core assembly, whereby the viral ribonucleoprotein (vRNP, composed of vRNA and nucleocapsid protein [NC]) is packaged into a conical capsid. Allosteric integrase inhibitors (ALLINIs) affect multiple viral processes. We have characterized ALLINIs and integrase mutants that have the same phenotype. First, by comparing the effects of ALLINIs on several steps of the viral cycle, we show that inhibition of maturation accounts for compound potency. Second, by using cryoelectron tomography, we find that ALLINIs impair conical capsid assembly. Third, by developing tomo-bubblegram imaging, which specifically labels NC protein, we find that ALLINIs block vRNP packaging; instead, vRNPs form “eccentric condensates” outside the core. Fourth, malformed cores, typical of integrase-deleted virus, are partially replaced by conical cores when integrase is supplied in trans . Fifth, vRNA is necessary for ALLINI-induced eccentric condensate formation. These observations suggest that integrase is involved in capsid morphogenesis and vRNP packaging.