Insights into the structure of RNPs from segmented negative-sense RNA viruses.

The genome of segmented negative-sense single-stranded RNA viruses, such as influenza virus and bunyaviruses, is coated by viral nucleoproteins (NPs), forming a ribonucleoprotein (RNP). In this issue of Structure, Dick et al.1 expand our knowledge on the RNPs of these viruses by solving the structures of Thogoto virus NP and RNP.

Exploiting the Affimer platform against influenza A virus.

Influenza A virus (IAV) is well known for its pandemic potential. While current surveillance and vaccination strategies are highly effective, therapeutic approaches are often short-lived due to the high mutation rates of IAV. Recently, monoclonal antibodies (mAbs) have emerged as a promising therapeutic approach, both against current strains and future IAV pandemics. In addition to mAbs, several antibody-like alternatives exist, which aim to improve upon mAbs. Among these, Affimers stand out for their short development time, high expression levels in Escherichia coli, and animal-free production. In this study, we utilized the Affimer platform to isolate and produce specific and potent inhibitors of IAV. Using a monomeric version of the IAV trimeric hemagglutinin (HA) fusion protein, we isolated 12 Affimers that inhibit IAV infection in vitro. Two of these Affimers were characterized in detail and exhibited nanomolar-binding affinities to the target H3 HA protein, specifically binding to the HA1 head domain. Cryo-electron microscopy (cryo-EM), employing a novel spray approach to prepare cryo-grids, allowed us to image HA-Affimer complexes. Combined with functional assays, we determined that these Affimers inhibit IAV by blocking the interaction of HA with the host-cell receptor, sialic acid. Furthermore, these Affimers inhibited IAV strains closely related to the one used for their isolation. Overall, our results support the use of Affimers as a viable alternative to existing targeted therapies for IAV and highlight their potential as diagnostic reagents. IMPORTANCE: Influenza A virus is one of the few viruses that can cause devastating pandemics. Due to the high mutation rates of this virus, annual vaccination is required, and antivirals are short-lived. Monoclonal antibodies present a promising approach to tackle influenza virus infections but are associated with some limitations. To improve on this strategy, we explored the Affimer platform, which are antibody-like proteins made in bacteria. By performing phage-display against a monomeric version of influenza virus fusion protein, an established viral target, we were able to isolate Affimers that inhibit influenza virus infection in vitro. We characterized the mechanism of inhibition of the Affimers by using assays targeting different stages of the viral replication cycle. We additionally characterized HA-Affimer complex structure, using a novel approach to prepare samples for cryo-electron microscopy. Overall, these results show that Affimers are a promising tool against influenza virus infection.

Cellular endosomal potassium ion flux regulates arenavirus uncoating during virus entry.

Lymphocytic choriomeningitis virus (LCMV) is an enveloped and segmented negative-sense RNA virus classified within the Arenaviridae family of the Bunyavirales order. LCMV is associated with fatal disease in immunocompromised populations and, as the prototypical arenavirus member, acts as a model for the many highly pathogenic members of the Arenaviridae family, such as Junín, Lassa, and Lujo viruses, all of which are associated with devastating hemorrhagic fevers. To enter cells, the LCMV envelope fuses with late endosomal membranes, for which two established requirements are low pH and interaction between the LCMV glycoprotein (GP) spike and secondary receptor CD164. LCMV subsequently uncoats, where the RNA genome-associated nucleoprotein (NP) separates from the Z protein matrix layer, releasing the viral genome into the cytosol. To further examine LCMV endosome escape, we performed an siRNA screen which identified host cell potassium ion (K+) channels as important for LCMV infection, with pharmacological inhibition confirming K+ channel involvement during the LCMV entry phase completely abrogating productive infection. To better understand the K+-mediated block in infection, we tracked incoming virions along their entry pathway under physiological conditions, where uncoating was signified by separation of NP and Z proteins. In contrast, K+ channel blockade prevented uncoating, trapping virions within Rab7 and CD164-positive endosomes, identifying K+ as a third LCMV entry requirement. K+ did not increase GP-CD164 binding or alter GP-CD164-dependent fusion. Thus, we propose that K+ mediates uncoating by modulating NP-Z interactions within the virion interior. These results suggest K+ channels represent a potential anti-arenaviral target.IMPORTANCEArenaviruses can cause fatal human disease for which approved preventative or therapeutic options are not available. Here, using the prototypical LCMV, we identified K+ channels as critical for arenavirus infection, playing a vital role during the entry phase of the infection cycle. We showed that blocking K+ channel function resulted in entrapment of LCMV particles within late endosomal compartments, thus preventing productive replication. Our data suggest K+ is required for LCMV uncoating and genome release by modulating interactions between the viral nucleoprotein and the matrix protein layer inside the virus particle.

Convalescent human plasma candidate reference materials protect against Crimean-Congo haemorrhagic fever virus (CCHFV) challenge in an A129 mouse model.

Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is spread by infected ticks or direct contact with blood, tissues and fluids from infected patients or livestock. Infection with CCHFV causes severe haemorrhagic fever in humans which is fatal in up to 83 % of cases. CCHFV is listed as a priority pathogen by the World Health Organization (WHO) and there are currently no widely-approved vaccines. Defining a serological correlate of protection against CCHFV infection would support the development of vaccines by providing a 'target threshold' for pre-clinical and clinical immunogenicity studies to achieve in subjects and potentially obviate the need for in vivo protection studies. We therefore sought to establish titratable protection against CCHFV using pooled human convalescent plasma, in a mouse model. Convalescent plasma collected from seven individuals with a known previous CCHFV virus infection were characterised using binding antibody and neutralisation assays. All plasma recognised nucleoprotein and the Gc glycoprotein, but some had a lower Gn glycoprotein response by ELISA. Pooled plasma and two individual donations from convalescent donors were administered intraperitoneally to A129 mice 24 h prior to intradermal challenge with CCHFV (strain IbAr10200). A partial protective effect was observed with all three convalescent plasmas characterised by longer survival post-challenge and reduced clinical score. These protective responses were titratable. Further characterisation of the serological reactivities within these samples will establish their value as reference materials to support assay harmonisation and accelerate vaccine development for CCHFV.

Lymphocytic choriomeningitis arenavirus requires cellular COPI and AP-4 complexes for efficient virion production.

Lymphocytic choriomeningitis virus (LCMV) is a bisegmented negative-sense RNA virus classified within the Arenaviridae family of the Bunyavirales order. LCMV is associated with fatal disease in immunocompromized populations, and as the prototypical arenavirus, acts as a model for the many serious human pathogens within this group. Here, we examined the dependence of LCMV multiplication on cellular trafficking components using a recombinant LCMV expressing enhanced green fluorescent protein in conjunction with a curated siRNA library. The screen revealed a requirement for subunits of both the coat protein 1 (COPI) coatomer and adapter protein 4 (AP-4) complexes. By rescuing a recombinant LCMV harboring a FLAG-tagged glycoprotein (GP-1) envelope spike (rLCMV-GP1-FLAG), we showed infection resulted in marked co-localization of individual COPI and AP-4 components with both LCMV nucleoprotein (NP) and GP-1, consistent with their involvement in viral processes. To further investigate the role of both COPI and AP-4 complexes during LCMV infection, we utilized the ARF-I inhibitor brefeldin A (BFA) that prevents complex formation. Within a single 12-h cycle of virus multiplication, BFA pre-treatment caused no significant change in LCMV-specific RNA synthesis, alongside no significant change in LCMV NP expression, as measured by BFA time-of-addition experiments. In contrast, BFA addition resulted in a significant drop in released virus titers, approaching 50-fold over the same 12-h period, rising to over 600-fold over 24 h. Taken together, these findings suggest COPI and AP-4 complexes are important host cell factors required for the formation and release of infectious LCMV. IMPORTANCE: Arenaviruses are rodent-borne, segmented, negative-sense RNA viruses, with several members responsible for fatal human disease, with the prototypic member lymphocytic choriomeningitis virus (LCMV) being under-recognised as a pathogen capable of inflicting neurological infections with fatal outcome. A detailed understanding of how arenaviruses subvert host cell processes to complete their multiplication cycle is incomplete. Here, using a combination of gene ablation and pharmacological inhibition techniques, we showed that host cellular COPI and AP-4 complexes, with native roles in cellular vesicular transport, were required for efficient LCMV growth. We further showed these complexes acted on late stages of the multiplication cycle, post-gene expression, with a significant impact on infectious virus egress. Collectively, our findings improve the understanding of arenaviruses host-pathogen interactions and reveal critical cellular trafficking pathways required during infection.

Organisation of the orthobunyavirus tripodal spike and the structural changes induced by low pH and K+ during entry.

Following endocytosis, enveloped viruses employ the changing environment of maturing endosomes as cues to promote endosomal escape, a process often mediated by viral glycoproteins. We previously showed that both high [K+] and low pH promote entry of Bunyamwera virus (BUNV), the prototypical bunyavirus. Here, we use sub-tomogram averaging and AlphaFold, to generate a pseudo-atomic model of the whole BUNV glycoprotein envelope. We unambiguously locate the Gc fusion domain and its chaperone Gn within the floor domain of the spike. Furthermore, viral incubation at low pH and high [K+], reminiscent of endocytic conditions, results in a dramatic rearrangement of the BUNV envelope. Structural and biochemical assays indicate that pH 6.3/K+ in the absence of a target membrane elicits a fusion-capable triggered intermediate state of BUNV GPs; but the same conditions induce fusion when target membranes are present. Taken together, we provide mechanistic understanding of the requirements for bunyavirus entry.

Is intestinal transport dysfunctional in COVID-19-related diarrhea?

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.

The Native Orthobunyavirus Ribonucleoprotein Possesses a Helical Architecture.

The Bunyavirales order is the largest group of negative-sense RNA viruses, containing many lethal human pathogens for which approved anti-infective measures are not available. The bunyavirus genome consists of multiple negative-sense RNA segments enwrapped by the virus-encoded nucleocapsid protein (NP), which together with the viral polymerase form ribonucleoproteins (RNPs). RNPs represent substrates for RNA synthesis and virion assembly, which require inherent flexibility, consistent with the appearance of RNPs spilled from virions. These observations have resulted in conflicting models describing the overall RNP architecture. Here, we purified RNPs from Bunyamwera virus (BUNV), the prototypical orthobunyavirus. The lengths of purified RNPs imaged by negative staining resulted in 3 populations of RNPs, suggesting that RNPs possess a consistent method of condensation. Employing microscopy approaches, we conclusively show that the NP portion of BUNV RNPs is helical. Furthermore, we present a pseudo-atomic model for this portion based on a cryo-electron microscopy average at 13 Å resolution, which allowed us to fit the BUNV NP crystal structure by molecular dynamics. This model was confirmed by NP mutagenesis using a mini-genome system. The model shows that adjacent NP monomers in the RNP chain interact laterally through flexible N- and C-terminal arms only, with no longitudinal helix-stabilizing interactions, thus providing a potential model for the molecular basis for RNP flexibility. Excessive RNase treatment disrupts native RNPs, suggesting that RNA was key in maintaining the RNP structure. Overall, this work will inform studies on bunyaviral RNP assembly, packaging, and RNA replication, and aid in future antiviral strategies. IMPORTANCE Bunyaviruses are emerging RNA viruses that cause significant disease and economic burden and for which vaccines or therapies approved for humans are not available. The bunyavirus genome is wrapped up by the nucleoprotein (NP) and interacts with the viral polymerase, forming a ribonucleoprotein (RNP). This is the only form of the genome active for viral replication and assembly. However, until now how NPs are organized within an RNP was not known for any orthobunyavirus. Here, we purified RNPs from the prototypical orthobunyavirus, Bunyamwera virus, and employed microscopy approaches to show that the NP portion of the RNP was helical. We then combined our helical average with the known structure of an NP monomer, generating a pseudo-atomic model of this region. This arrangement allowed the RNPs to be highly flexible, which was critical for several stages of the viral replication cycle, such as segment circularization.

Ribosome heterogeneity in Drosophila melanogaster gonads through paralog-switching.

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.

Reproducibility of Dietary Intake Measurement From Diet Diaries, Photographic Food Records, and a Novel Sensor Method.

Objective: No data currently exist on the reproducibility of photographic food records compared to diet diaries, two commonly used methods to measure dietary intake. Our aim was to examine the reproducibility of diet diaries, photographic food records, and a novel electronic sensor, consisting of counts of chews and swallows using wearable sensors and video analysis, for estimating energy intake. Method: This was a retrospective analysis of data from a previous study, in which 30 participants (15 female), aged 29 ± 12 y and having a BMI of 27.9 ± 5.5, consumed three identical meals on different days. Four different methods were used to estimate total mass and energy intake on each day: (1) weighed food record; (2) photographic food record; (3) diet diary; and (4) novel mathematical model based on counts of chews and swallows (CCS models) obtained via the use of electronic sensors and video monitoring system. The study staff conducted weighed food records for all meals, took pre- and post-meal photographs, and ensured that diet diaries were completed by participants at the end of each meal. All methods were compared against the weighed food record, which was used as the reference method. Results: Reproducibility was significantly different between the diet diary and photographic food record for total energy intake (p = 0.004). The photographic record had greater reproducibility vs. the diet diary for all parameters measured. For total energy intake, the novel sensor method exhibited good reproducibility (repeatability coefficient (RC) of 59.9 (45.9, 70.4), which was better than that for the diet diary [RC = 79.6 (55.5, 103.3)] but not as repeatable as the photographic method [RC = 43.4 (32.1, 53.9)]. Conclusion: Photographic food records offer superior precision to the diet diary and, therefore, would be valuable for longitudinal studies with repeated measures of dietary intake. A novel electronic sensor also shows promise for the collection of longitudinal dietary intake data.

Ion Channels as Therapeutic Targets for Viral Infections: Further Discoveries and Future Perspectives.

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 RNA Replication Site of Tula Orthohantavirus Resides within a Remodelled Golgi Network.

: 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.

Cellular cholesterol abundance regulates potassium accumulation within endosomes and is an important determinant in bunyavirus entry.

The Bunyavirales order of segmented negative-sense RNA viruses includes more than 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 translocate 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. Using drug-mediated cholesterol extraction along with viral entry and K+ uptake assays, we report three major findings: BUNV and HAZV require cellular cholesterol during endosomal escape; cholesterol depletion from host cells impairs K+ accumulation in maturing endosomes, revealing new insights into endosomal K+ homeostasis; and "priming" BUNV and HAZV virions with K+ before infection alleviates their cholesterol requirement. Taken together, our findings suggest a model in which cholesterol abundance influences endosomal K+ levels and, consequently, the efficiency of bunyavirus infection. The ability to inhibit bunyaviruses with existing cholesterol-lowering drugs may offer new options for future antiviral interventions for pathogenic bunyaviruses.

Identification of a Structural Element in HIV-1 Gag Required for Virus Particle Assembly and Maturation.

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.IMPORTANCE 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.

The RNA-Binding Protein of a Double-Stranded RNA Virus Acts like a Scaffold Protein.

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.

Potassium is a trigger for conformational change in the fusion spike of an enveloped RNA virus.

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.

Bunyavirus requirement for endosomal K+ reveals new roles of cellular ion channels during infection.

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.

The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane.

All enveloped viruses, including herpesviruses, must fuse their envelope with the host membrane to deliver their genomes into target cells, making this essential step subject to interference by antibodies and drugs. Viral fusion is mediated by a viral surface protein that transits from an initial prefusion conformation to a final postfusion conformation. Strikingly, the prefusion conformation of the herpesvirus fusion protein, gB, is poorly understood. Herpes simplex virus (HSV), a model system for herpesviruses, causes diseases ranging from mild skin lesions to serious encephalitis and neonatal infections. Using cryo-electron tomography and subtomogram averaging, we have characterized the structure of the prefusion conformation and fusion intermediates of HSV-1 gB. To this end, we have set up a system that generates microvesicles displaying full-length gB on their envelope. We confirmed proper folding of gB by nondenaturing electrophoresis-Western blotting with a panel of monoclonal antibodies (MAbs) covering all gB domains. To elucidate the arrangement of gB domains, we labeled them by using (i) mutagenesis to insert fluorescent proteins at specific positions, (ii) coexpression of gB with Fabs for a neutralizing MAb with known binding sites, and (iii) incubation of gB with an antibody directed against the fusion loops. Our results show that gB starts in a compact prefusion conformation with the fusion loops pointing toward the viral membrane and suggest, for the first time, a model for gB's conformational rearrangements during fusion. These experiments further illustrate how neutralizing antibodies can interfere with the essential gB structural transitions that mediate viral entry and therefore infectivity.IMPORTANCE The herpesvirus family includes herpes simplex virus (HSV) and other human viruses that cause lifelong infections and a variety of diseases, like skin lesions, encephalitis, and cancers. As enveloped viruses, herpesviruses must fuse their envelope with the host membrane to start an infection. This process is mediated by a viral surface protein that transitions from an initial conformation (prefusion) to a final, more stable, conformation (postfusion). However, the prefusion conformation of the herpesvirus fusion protein (gB) is poorly understood. To elucidate the structure of the prefusion conformation of HSV type 1 gB, we have employed cryo-electron microscopy to study gB molecules expressed on the surface of vesicles. Using different approaches to label gB's domains allowed us to model the structures of the prefusion and intermediate conformations of gB. Overall, our findings enhance our understanding of HSV fusion and lay the groundwork for the development of new ways to prevent and block HSV infection.

The Primary Enveloped Virion of Herpes Simplex Virus 1: Its Role in Nuclear Egress.

Many viruses migrate between different cellular compartments for successive stages of assembly. The HSV-1 capsid assembles in the nucleus and then transfers into the cytoplasm. First, the capsid buds through the inner nuclear membrane, becoming coated with nuclear egress complex (NEC) protein. This yields a primary enveloped virion (PEV) whose envelope fuses with the outer nuclear membrane, releasing the capsid into the cytoplasm. We investigated the associated molecular mechanisms by isolating PEVs from US3-null-infected cells and imaging them by cryo-electron microscopy and tomography. (pUS3 is a viral protein kinase in whose absence PEVs accumulate in the perinuclear space.) Unlike mature extracellular virions, PEVs have very few glycoprotein spikes. PEVs are ~20% smaller than mature virions, and the little space available between the capsid and the NEC layer suggests that most tegument proteins are acquired later in the egress pathway. Previous studies have proposed that NEC is organized as hexamers in honeycomb arrays in PEVs, but we find arrays of heptameric rings in extracts from US3-null-infected cells. In a PEV, NEC contacts the capsid predominantly via the pUL17/pUL25 complexes which are located close to the capsid vertices. Finally, the NEC layer dissociates from the capsid as it leaves the nucleus, possibly in response to pUS3-mediated phosphorylation. Overall, nuclear egress emerges as a process driven by a program of multiple weak interactions.IMPORTANCE On its maturation pathway, the newly formed HSV-1 nucleocapsid must traverse the nuclear envelope, while respecting the integrity of that barrier. Nucleocapsids (125 nm in diameter) are too large to pass through the nuclear pore complexes that conduct most nucleocytoplasmic traffic. It is now widely accepted that the process involves envelopment/de-envelopment of a key intermediate-the primary enveloped virion. In wild-type infections, PEVs are short-lived, which has impeded study. Using a mutant that accumulates PEVs in the perinuclear space, we were able to isolate PEVs in sufficient quantity for structural analysis by cryo-electron microscopy and tomography. The findings not only elucidate the maturation pathway of an important human pathogen but also have implications for cellular processes that involve the trafficking of large macromolecular complexes.