Enhanced Zika virus-like particle development using Baculovirus spp. constructs.

Zika virus (ZIKV) is one of several examples of an unprecedented pandemic spread and against which there is currently no suitable vaccine or treatment. Here, we constructed and characterized recombinant baculovirus-derived ZIKV-like particles (Zika VLPs) to study ZIKV-antibody interactions. These VLPs, uniquely consisted of the full-length ZIKV capsid (C), pre-membrane (prM), and envelope (E) proteins with either: a) the viral nonstructural NS2B and NS3 protease unit under one or two different promoters or b) an alternative host-cell furin protease encoding cleavage sequence inserted between the C and prM genes, together with lobster tropomyosin leader and honeybee signal sequences with one promoter for increased extracellular secretion. All these Zika VLPs displayed typical virion morphology in transmission electron microscopic analysis when expressed in both insect (Sf9) and mammalian (HEK293T) cells and no uncleaved prM glycoprotein was detected, as are present on immature virions. The importance of glycosylation of the E glycoprotein was shown by the effects on both polyclonal and monoclonal antibody reactions after these N-linked carbohydrate residues were disrupted by oxidation or enzymatic cleavage. Importantly, the construct which contained the host-cell furin protease cleavage sequence together with a lobster tropomyosin leader and honeybee signal sequences under one promoter produced higher Zika VLP titers and protein concentrations and which can now be tested as a superior construct in multifunctional diagnostic (ELISA and neutralization/antibody-dependent enhancement) assays and immunogenic assessments possibly leading to vaccine trials.

Membrane-assisted assembly and selective secretory autophagy of enteroviruses.

Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. It is unknown how virions assemble around these newly synthesized genomes and how they are then loaded into autophagic membranes for release through secretory autophagy. Here, we use cryo-electron tomography of infected cells to show that poliovirus assembles directly on replication membranes. Pharmacological untethering of capsids from membranes abrogates RNA encapsidation. Our data directly visualize a membrane-bound half-capsid as a prominent virion assembly intermediate. Assembly progression past this intermediate depends on the class III phosphatidylinositol 3-kinase VPS34, a key host-cell autophagy factor. On the other hand, the canonical autophagy initiator ULK1 is shown to restrict virion production since its inhibition leads to increased accumulation of virions in vast intracellular arrays, followed by an increased vesicular release at later time points. Finally, we identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from other types of autophagosome contents. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle.

A New Infectious Unit: Extracellular Vesicles Carrying Virus Populations.

Viral egress and transmission have long been described to take place through single free virus particles. However, viruses can also shed into the environment and transmit as populations clustered inside extracellular vesicles (EVs), a process we had first called vesicle-mediated en bloc transmission. These membrane-cloaked virus clusters can originate from a variety of cellular organelles including autophagosomes, plasma membrane, and multivesicular bodies. Their viral cargo can be multiples of nonenveloped or enveloped virus particles or even naked infectious genomes, but egress is always nonlytic, with the cell remaining intact. Here we put forth the thesis that EV-cloaked viral clusters are a distinct form of infectious unit as compared to free single viruses (nonenveloped or enveloped) or even free virus aggregates. We discuss how efficient and prevalent these infectious EVs are in the context of virus-associated diseases and highlight the importance of their proper detection and disinfection for public health.

Entry-competent-replication-abortive African horse sickness virus strains elicit robust immunity in ponies against all serotypes.

African horse sickness virus (AHSV) is an Orbivirus within the Reoviridae family, spread by Culicoides species of midges, which infects equids with high mortality, particularly in horses and has a considerable impact on the equine industry. In order to control the disease, we previously described Entry Competent Replication Abortive (ECRA) virus strains for each of the nine distinct AHSV serotypes and demonstrated their potential as vaccines, first in type I interferon receptor (IFNAR-/-) knockout mice, and then in ponies. In this report we have investigated whether or not a combination ECRA vaccine comprising nine vaccine strains as two different cocktails is as efficient in ponies and the duration of the immunity triggered by ECRA vaccines. In one study, a group of ponies were vaccinated with a cocktail of 4 vaccine strains, followed by a vaccination of the remaining 5 vaccine strains, mimicking the current live attenuated vaccine regimen. In the second study, ponies were vaccinated with a single ECRA-AHSV strain and monitored for 6 months. The first group of ponies developed neutralising antibody responses against all 9 serotypes, indicating that no cross-serotype interference occurred, while the second group developed robust neutralising antibody responses against the single serotype that were sustained at the same level throughout a 6-month study. The results support our previous data and further validate ECRA vaccines as a safe and efficacious replacement of current live vaccines.

ß-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway.

ß-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that ß-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of ß-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. ß-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

A Calcium Sensor Discovered in Bluetongue Virus Nonstructural Protein 2 Is Critical for Virus Replication.

Many viruses use specific viral proteins to bind calcium ions (Ca2+) for stability or to modify host cell pathways; however, to date, no Ca2+ binding protein has been reported in bluetongue virus (BTV), the causative agent of bluetongue disease in livestock. Here, using a comprehensive bioinformatics screening, we identified a putative EF-hand-like Ca2+ binding motif in the carboxyl terminal region of BTV nonstructural phosphoprotein 2 (NS2). Subsequently, using a recombinant NS2, we demonstrated that NS2 binds Ca2+ efficiently and that Ca2+ binding was perturbed when the Asp and Glu residues in the motif were substituted by alanine. Using circular dichroism analysis, we found that Ca2+ binding by NS2 triggered a helix-to-coil secondary structure transition. Further, cryo-electron microscopy in the presence of Ca2+ revealed that NS2 forms helical oligomers which, when aligned with the N-terminal domain crystal structure, suggest an N-terminal domain that wraps around the C-terminal domain in the oligomer. Further, an in vitro kinase assay demonstrated that Ca2+ enhanced the phosphorylation of NS2 significantly. Importantly, mutations introduced at the Ca2+ binding site in the viral genome by reverse genetics failed to allow recovery of viable virus, and the NS2 phosphorylation level and assembly of viral inclusion bodies (VIBs) were reduced. Together, our data suggest that NS2 is a dedicated Ca2+ binding protein and that calcium sensing acts as a trigger for VIB assembly, which in turn facilitates virus replication and assembly.IMPORTANCE After entering the host cells, viruses use cellular host factors to ensure a successful virus replication process. For replication in infected cells, members of the Reoviridae family form inclusion body-like structures known as viral inclusion bodies (VIB) or viral factories. Bluetongue virus (BTV) forms VIBs in infected cells through nonstructural protein 2 (NS2), a phosphoprotein. An important regulatory factor critical for VIB formation is phosphorylation of NS2. In our study, we discovered a characteristic calcium-binding EF-hand-like motif in NS2 and found that the calcium binding preferentially affects phosphorylation level of the NS2 and has a role in regulating VIB assembly.

Differential Localization of Structural and Non-Structural Proteins during the Bluetongue Virus Replication Cycle.

Members of the Reoviridae family assemble virus factories within the cytoplasm of infected cells to replicate and assemble virus particles. Bluetongue virus (BTV) forms virus inclusion bodies (VIBs) that are aggregates of viral RNA, certain viral proteins, and host factors, and have been shown to be sites of the initial assembly of transcriptionally active virus-like particles. This study sought to characterize the formation, composition, and ultrastructure of VIBs, particularly in relation to virus replication. In this study we have utilized various microscopic techniques, including structured illumination microscopy, and virological assays to show for the first time that the outer capsid protein VP5, which is essential for virus maturation, is also associated with VIBs. The addition of VP5 to assembled virus cores exiting VIBs is required to arrest transcriptionally active core particles, facilitating virus maturation. Furthermore, we observed a time-dependent association of the glycosylated non-structural protein 3 (NS3) with VIBs, and report on the importance of the two polybasic motifs within NS3 that facilitate virus trafficking and egress from infected cells at the plasma membrane. Thus, the presence of VP5 and the dynamic nature of NS3 association with VIBs that we report here provide novel insight into these previously less well-characterized processes.

Atomic structure of the translation regulatory protein NS1 of bluetongue virus.

Bluetongue virus (BTV) non-structural protein 1 (NS1) regulates viral protein synthesis and exists as tubular and non-tubular forms in infected cells, but how tubules assemble and how protein synthesis is regulated are unknown. Here, we report near-atomic resolution structures of two NS1 tubular forms determined by cryo-electron microscopy. The two tubular forms are different helical assemblies of the same NS1 monomer, consisting of an amino-terminal foot, a head and body domains connected to an extended carboxy-terminal arm, which wraps atop the head domain of another NS1 subunit through hydrophobic interactions. Deletion of the C terminus prevents tubule formation but not viral replication, suggesting an active non-tubular form. Two zinc-finger-like motifs are present in each NS1 monomer, and tubules are disrupted by divalent cation chelation and restored by cation addition, including Zn2+, suggesting a regulatory role of divalent cations in tubule formation. In vitro luciferase assays show that the NS1 non-tubular form upregulates BTV mRNA translation, whereas zinc-finger disruption decreases viral mRNA translation, tubule formation and virus replication, confirming a functional role for the zinc-fingers. Thus, the non-tubular form of NS1 is sufficient for viral protein synthesis and infectious virus replication, and the regulatory mechanism involved operates through divalent cation-dependent conversion between the non-tubular and tubular forms.

Mapping the pH Sensors Critical for Host Cell Entry by a Complex Nonenveloped Virus.

Bluetongue virus (BTV), in the family Reoviridae, is an insect-borne, double-capsid virus causing hemorrhagic disease in livestock around the world. Here, we elucidate how outer capsid proteins VP2 and VP5 coordinate cell entry of BTV. To identify key functional residues, we used atomic-level structural data to guide mutagenesis of VP2 and VP5 and a series of biological and biochemical approaches, including site-directed mutagenesis, reverse genetics-based virus recovery, expression and characterization of individual recombinant mutant proteins, and various in vitro and in vivo assays. We demonstrate the dynamic nature of the conformational change process, revealing that a unique zinc finger (CCCH) in VP2 acts as the major low pH sensor, coordinating VP2 detachment, subsequently allowing VP5 to sense low pH via specific histidine residues at key positions. We show that single substitution of only certain histidine residues has a lethal effect, indicating that the location of histidine in VP5 is critical to inducing changes in VP5 conformation that facilitates membrane penetration. Further, we show that the VP5 anchoring domain alone recapitulates sensing of low pH. Our data reveal a novel, multiconformational process that overcomes entry barriers faced by this multicapsid nonenveloped virus.IMPORTANCE Virus entry into a susceptible cell is the first step of infection and a significant point at which infection can be prevented. To enter effectively, viruses must sense the cellular environment and, when appropriate, initiate a series of changes that eventually jettison the protective shell and deposit virus genes into the cytoplasm. Many viruses sense pH, but how this happens and the events that follow are often poorly understood. Here, we address this question for a large multilayered bluetongue virus. We show key residues in outer capsid proteins, a pH-sensing histidine of a zinc finger within the receptor-binding VP2 protein, and certain histidine residues in the membrane-penetrating VP5 protein that detect cellular pH, leading to irreversible changes and propel the virus through the cell membrane. Our data reveal a novel mechanism of cell entry for a nonenveloped virus and highlight mechanisms which may also be used by other viruses.

The Interaction of Bluetongue Virus VP6 and Genomic RNA Is Essential for Genome Packaging.

The genomes of the Reoviridae, including the animal pathogen bluetongue virus (BTV), are multisegmented double-stranded RNA (dsRNA). During replication, single-stranded (ss) positive-sense RNA segments are packaged into the assembling virus capsid, triggering genomic dsRNA synthesis. However, exactly how this packaging event occurs is not clear. A minor capsid protein, VP6, unique for the orbiviruses, has been proposed to be involved in the RNA-packaging process. In this study, we sought to characterize the RNA binding activity of VP6 and its functional relevance. A novel proteomic approach was utilized to map the ssRNA/dsRNA binding sites of a purified recombinant protein and the genomic dsRNA binding sites of the capsid-associated VP6. The data revealed that each VP6 protein has multiple distinct RNA-binding regions and that only one region is shared between recombinant and capsid-associated VP6. A combination of targeted mutagenesis and reverse genetics identified the RNA-binding region that is essential for virus replication. Using an in vitro RNA-binding competition assay, a unique cell-free assembly assay, and an in vivo single-cycle replication assay, it was possible to identify a motif within the shared binding region that binds BTV ssRNA preferentially in a manner consistent with specific RNA recruitment during capsid assembly. These data highlight the critical roles that this unique protein plays in orbivirus genome packaging and replication.IMPORTANCE Genome packaging is a critical stage during virus replication. For viruses with segmented genomes, the genome segments need to be correctly packaged into a newly formed capsid. However, the detailed mechanism of this packaging is unclear. Here we focus on VP6, a minor viral protein of bluetongue virus, which is critical for genome packaging. We used multiple approaches, including a robust RNA-protein fingerprinting assay, to map the ssRNA binding sites of recombinant VP6 and the genomic dsRNA binding sites of capsid-associated VP6. By these means, together with virological and biochemical methods, we identify the viral RNA-packaging motif of a segmented dsRNA virus for the first time.

Protective efficacy of multivalent replication-abortive vaccine strains in horses against African horse sickness virus challenge.

African horse sickness virus (AHSV) is an orbivirus, a member of the Reoviridae family. Nine different serotypes have been described so far. AHSV is vectored by Culicoides spp. to equids, causing high mortality, particularly in horses, with considerable economic impacts. For development of a safe attenuated vaccine, we previously established an efficient reverse genetics (RG) system to generate Entry Competent Replication-Abortive (ECRA) virus strains, for all nine serotypes and demonstrated the vaccine potential of these strains in type I interferon receptor (IFNAR)-knockout mice. Here, we evaluated the protective efficacies of these ECRA viruses in AHSV natural hosts. One monoserotype (ECRA.A4) vaccine and one multivalent cocktail (ECRA.A1/4/6/8) vaccine were tested in ponies and subsequently challenged with a virulent AHSV4. In contrast to control animals, all vaccinated ponies were protected and did not develop severe clinical symptoms of AHS. Furthermore, the multivalent cocktail vaccinated ponies produced neutralizing antibodies against all serotypes present in the cocktail, and a foal born during the trial was healthy and had no viremia. These results validate the suitability of these ECRA strains as a new generation of vaccines for AHSV.

Involvement of an Arginine Triplet in M1 Matrix Protein Interaction with Membranes and in M1 Recruitment into Virus-Like Particles of the Influenza A(H1N1)pdm09 Virus.

The influenza A(H1N1)pdm09 virus caused the first influenza pandemic of the 21st century. In this study, we wanted to decipher the role of conserved basic residues of the viral M1 matrix protein in virus assembly and release. M1 plays many roles in the influenza virus replication cycle. Specifically, it participates in viral particle assembly, can associate with the viral ribonucleoprotein complexes and can bind to the cell plasma membrane and/or the cytoplasmic tail of viral transmembrane proteins. M1 contains an N-terminal domain of 164 amino acids with two basic domains: the nuclear localization signal on helix 6 and an arginine triplet (R76/77/78) on helix 5. To investigate the role of these two M1 basic domains in influenza A(H1N1)pdm09 virus molecular assembly, we analyzed M1 attachment to membranes, virus-like particle (VLP) production and virus infectivity. In vitro, M1 binding to large unilamellar vesicles (LUVs), which contain negatively charged lipids, decreased significantly when the M1 R76/77/78 motif was mutated. In cells, M1 alone was mainly observed in the nucleus (47%) and in the cytosol (42%). Conversely, when co-expressed with the viral proteins NS1/NEP and M2, M1 was relocated to the cell membranes (55%), as shown by subcellular fractionation experiments. This minimal system allowed the production of M1 containing-VLPs. However, M1 with mutations in the arginine triplet accumulated in intracellular clusters and its incorporation in VLPs was strongly diminished. M2 over-expression was essential for M1 membrane localization and VLP production, whereas the viral trans-membrane proteins HA and NA seemed dispensable. These results suggest that the M1 arginine triplet participates in M1 interaction with membranes. This R76/77/78 motif is essential for M1 incorporation in virus particles and the importance of this motif was confirmed by reverse genetic demonstrating that its mutation is lethal for the virus. These results highlight the molecular mechanism of M1-membrane interaction during the formation of influenza A(H1N1)pdm09 virus particles which is essential for infectivity.