Biophysical and structural characterization of a multifunctional viral genome packaging motor.

The large dsDNA viruses replicate their DNA as concatemers consisting of multiple covalently linked genomes. Genome packaging is catalyzed by a terminase enzyme that excises individual genomes from concatemers and packages them into preassembled procapsids. These disparate tasks are catalyzed by terminase alternating between two distinct states-a stable nuclease that excises individual genomes and a dynamic motor that translocates DNA into the procapsid. It was proposed that bacteriophage λ terminase assembles as an anti-parallel dimer-of-dimers nuclease complex at the packaging initiation site. In contrast, all characterized packaging motors are composed of five terminase subunits bound to the procapsid in a parallel orientation. Here, we describe biophysical and structural characterization of the λ holoenzyme complex assembled in solution. Analytical ultracentrifugation, small angle X-ray scattering, and native mass spectrometry indicate that 5 subunits assemble a cone-shaped terminase complex. Classification of cryoEM images reveals starfish-like rings with skewed pentameric symmetry and one special subunit. We propose a model wherein nuclease domains of two subunits alternate between a dimeric head-to-head arrangement for genome maturation and a fully parallel arrangement during genome packaging. Given that genome packaging is strongly conserved in both prokaryotic and eukaryotic viruses, the results have broad biological implications.

Polγ coordinates DNA synthesis and proofreading to ensure mitochondrial genome integrity.

Accurate replication of mitochondrial DNA (mtDNA) by DNA polymerase γ (Polγ) is essential for maintaining cellular energy supplies, metabolism, and cell cycle control. To illustrate the structural mechanism for Polγ coordinating polymerase (pol) and exonuclease (exo) activities to ensure rapid and accurate DNA synthesis, we determined four cryo-EM structures of Polγ captured after accurate or erroneous incorporation to a resolution of 2.4-3.0 Å. The structures show that Polγ employs a dual-checkpoint mechanism to sense nucleotide misincorporation and initiate proofreading. The transition from replication to error editing is accompanied by increased dynamics in both DNA and enzyme, in which the polymerase relaxes its processivity and the primer-template DNA unwinds, rotates, and backtracks to shuttle the mismatch-containing primer terminus 32 Å to the exo site for editing. Our structural and functional studies also provide a foundation for analyses of Polγ mutation-induced human diseases and aging.

Lipid-coated mesoporous silica nanoparticles for anti-viral applications via delivery of CRISPR-Cas9 ribonucleoproteins.

Emerging and re-emerging viral pathogens present a unique challenge for anti-viral therapeutic development. Anti-viral approaches with high flexibility and rapid production times are essential for combating these high-pandemic risk viruses. CRISPR-Cas technologies have been extensively repurposed to treat a variety of diseases, with recent work expanding into potential applications against viral infections. However, delivery still presents a major challenge for these technologies. Lipid-coated mesoporous silica nanoparticles (LCMSNs) offer an attractive delivery vehicle for a variety of cargos due to their high biocompatibility, tractable synthesis, and amenability to chemical functionalization. Here, we report the use of LCMSNs to deliver CRISPR-Cas9 ribonucleoproteins (RNPs) that target the Niemann-Pick disease type C1 gene, an essential host factor required for entry of the high-pandemic risk pathogen Ebola virus, demonstrating an efficient reduction in viral infection. We further highlight successful in vivo delivery of the RNP-LCMSN platform to the mouse liver via systemic administration.

Suppression of amyloid-ß fibril growth by drug-engineered polymorph transformation.

Fibrillization of the protein amyloid ß is assumed to trigger Alzheimer's pathology. Approaches that target amyloid plaques, however, have garnered limited clinical success, and their failures may relate to the scarce understanding of the impact of potential drugs on the intertwined stages of fibrillization. Here, we demonstrate that bexarotene, a T-cell lymphoma medication with known antiamyloid activity both in vitro and in vivo, suppresses amyloid fibrillization by promoting an alternative fibril structure. We employ time-resolved in situ atomic force microscopy to quantify the kinetics of growth of individual fibrils and supplement it with structure characterization by cryo-EM. We show that fibrils with structure engineered by the drug nucleate and grow substantially slower than "normal" fibrils; remarkably, growth remains stunted even in drug-free solutions. We find that the suppression of fibril growth by bexarotene is not because of the drug binding to the fibril tips or to the peptides in the solution. Kinetic analyses attribute the slow growth of drug-enforced fibril polymorph to the distinctive dynamics of peptide chain association to their tips. As an additional benefit, the bexarotene fibrils kill primary rat hippocampal neurons less efficiently than normal fibrils. In conclusion, the suggested drug-driven polymorph transformation presents a mode of action to irreversibly suppress toxic aggregates not only in Alzheimer's but also potentially in myriad diverse pathologies that originate with protein condensation.

Environmentally-triggered contraction of the norovirus virion determines diarrheagenic potential.

Noroviruses are the leading cause of severe childhood diarrhea and foodborne disease worldwide. While they are a major cause of disease in all age groups, infections in the very young can be quite severe with annual estimates of 50,000-200,000 fatalities in children under 5 years old. In spite of the remarkable disease burden associated with norovirus infections in people, very little is known about the pathogenic mechanisms underlying norovirus diarrhea, principally because of the lack of tractable small animal models. We recently demonstrated that wild-type neonatal mice are susceptible to murine norovirus (MNV)-induced acute self-resolving diarrhea in a time course mirroring human norovirus disease. Using this robust pathogenesis model system, we demonstrate that virulence is regulated by the responsiveness of the viral capsid to environmental cues that trigger contraction of the VP1 protruding (P) domain onto the particle shell, thus enhancing receptor binding and infectivity. The capacity of a given MNV strain to undergo this contraction positively correlates with infection of cells expressing low abundance of the virus receptor CD300lf, supporting a model whereby virion contraction triggers infection of CD300lflo cell types that are responsible for diarrhea induction. These findings directly link environmentally-influenced biophysical features with norovirus disease severity.

The transcription factor Tbx5 regulates direction-selective retinal ganglion cell development and image stabilization.

The diversity of visual input processed by the mammalian visual system requires the generation of many distinct retinal ganglion cell (RGC) types, each tuned to a particular feature. The molecular code needed to generate this cell-type diversity is poorly understood. Here, we focus on the molecules needed to specify one type of retinal cell: the upward-preferring ON direction-selective ganglion cell (up-oDSGC) of the mouse visual system. Single-cell transcriptomic profiling of up- and down-oDSGCs shows that the transcription factor Tbx5 is selectively expressed in up-oDSGCs. The loss of Tbx5 in up-oDSGCs results in a selective defect in the formation of up-oDSGCs and a corresponding inability to detect vertical motion. A downstream effector of Tbx5, Sfrp1, is also critical for vertical motion detection but not up-oDSGC formation. These results advance our understanding of the molecular mechanisms that specify a rare retinal cell type and show how disrupting this specification leads to a corresponding defect in neural circuitry and behavior.

Compact IF2 allows initiator tRNA accommodation into the P site and gates the ribosome to elongation.

During translation initiation, initiation factor 2 (IF2) holds initiator transfer RNA (fMet-tRNAifMet) in a specific orientation in the peptidyl (P) site of the ribosome. Upon subunit joining IF2 hydrolyzes GTP and, concomitant with inorganic phosphate (Pi) release, changes conformation facilitating fMet-tRNAifMet accommodation into the P site and transition of the 70 S ribosome initiation complex (70S-IC) to an elongation-competent ribosome. The mechanism by which IF2 separates from initiator tRNA at the end of translation initiation remains elusive. Here, we report cryo-electron microscopy (cryo-EM) structures of the 70S-IC from Pseudomonas aeruginosa bound to compact IF2-GDP and initiator tRNA. Relative to GTP-bound IF2, rotation of the switch 2 α-helix in the G-domain bound to GDP unlocks a cascade of large-domain movements in IF2 that propagate to the distal tRNA-binding domain C2. The C2-domain relocates 35 angstroms away from tRNA, explaining how IF2 makes way for fMet-tRNAifMet accommodation into the P site. Our findings provide the basis by which IF2 gates the ribosome to the elongation phase.

Structural Studies on the Shapeshifting Murine Norovirus.

Noroviruses are responsible for almost a fifth of all cases of gastroenteritis worldwide. The calicivirus capsid is composed of 180 copies of VP1 with a molecular weight of ~58 kDa. This coat protein is divided into the N-terminus (N), the shell (S) and C-terminal protruding (P) domains. The S domain forms a shell around the viral RNA genome, while the P domains dimerize to form protrusions on the capsid surface. The P domain is subdivided into P1 and P2 subdomains, with the latter containing the binding sites for cellular receptors and neutralizing antibodies. Reviewed here are studies on murine norovirus (MNV) showing that the capsid responds to several physiologically relevant cues; bile, pH, Mg2+, and Ca2+. In the initial site of infection, the intestinal tract, high bile and metal concentrations and low pH cause two significant conformational changes: (1) the P domain contracts onto the shell domain and (2) several conformational changes within the P domain lead to enhanced receptor binding while blocking antibody neutralization. In contrast, the pH is neutral, and the concentrations of bile and metals are low in the serum. Under these conditions, the loops at the tip of the P domain are in the open conformation with the P domain floating on a linker or tether above the shell. This conformational state favors antibody binding but reduces interactions with the receptor. In this way, MNV uses metabolites and environmental cues in the intestine to optimize cellular attachment and escape antibody binding but presents a wholly different structure to the immune system in the serum. To our knowledge, this is the first example of a virus shapeshifting in this manner to escape the immune response.

Multiple Signals in the Gut Contract the Mouse Norovirus Capsid To Block Antibody Binding While Enhancing Receptor Affinity.

Human norovirus is the leading cause of gastroenteritis worldwide, with no approved vaccine or antiviral treatment to mitigate infection. These plus-strand RNA viruses have T = 3 icosahedral protein capsids with 90 pronounced protruding (P) domain dimers, to which antibodies and cellular receptors bind. We previously demonstrated that bile binding to the capsid of mouse norovirus (MNV) causes several major conformational changes; the entire P domain rotates by ∼90° and contracts onto the shell, the P domain dimers rotate about each other, and the structural equilibrium of the epitopes at the top of the P domain shifts toward the closed conformation, which favors receptor binding while blocking antibody binding. Here, we demonstrate that MNV undergoes reversible conformational changes at pH 5.0 that are nearly identical to those observed when bile binds. Notably, at low pH or when metals bind, a cluster of acidic resides in the G'-H' loop interact and distort the G'-H' loop, and this may drive C'-D' loop movement toward the closed conformation. Enzyme-linked immunosorbent assays with infectious virus particles at low pH or in the presence of metals demonstrated that all tested antibodies do not bind to this contracted form, akin to what was observed with the MNV-bile complex. Therefore, low pH, cationic metals, and bile salts are physiological triggers in the gut for P domain contraction and structural rearrangement, which synergistically prime the virus for receptor binding while blocking antibody binding. IMPORTANCE The protruding domains on the calicivirus capsids are recognized by cell receptors and antibodies. We demonstrated that MNV P domains are highly mobile, and bile causes contraction onto the shell surface while allosterically blocking antibody binding. We present the near-atomic cryo-electron microscopy structures of infectious MNV at pH 5.0 and pH 7.5. Surprisingly, low pH is sufficient to cause the same conformational changes as when bile binds. A cluster of acidic residues on the G'-H' loop were most likely involved in the pH effects. These residues also bound divalent cations and had the same conformation as observed here at pH 5. Binding assays demonstrated that low pH and metals block antibody binding, and thus the G'-H' loop might be driving the conformational changes. Therefore, low pH, cationic metals, and bile salts in the gut synergistically prime the virus for receptor binding while blocking antibody binding.

A Norovirus Uses Bile Salts To Escape Antibody Recognition While Enhancing Receptor Binding.

Noroviruses, members of the Caliciviridae family, are the major cause of epidemic gastroenteritis in humans, causing ∼20 million cases annually. These plus-strand RNA viruses have T=3 icosahedral protein capsids with 90 pronounced protruding (P) domain dimers to which antibodies and cellular receptors bind. In the case of mouse norovirus (MNV), bile salts have been shown to enhance receptor (CD300lf) binding to the P domain. We demonstrated previously that the P domains of several genotypes are markedly flexible and "float" over the shell, but the role of this flexibility was unclear. Recently, we demonstrated that bile causes a 90° rotation and collapse of the P domain onto the shell surface. Since bile binds distally to the P-shell interface, it was not at all clear how it could cause such dramatic changes. Here, we present the near-atomic resolution cryo-electron microscopy (cryo-EM) structure of the MNV protruding domain complexed with a neutralizing Fab. On the basis of previous results, we show here that bile salts cause allosteric conformational changes in the P domain that block antibody recognition of the top of the P domain. In addition, bile causes a major rearrangement of the P domain dimers that is likely responsible for the bile-induced collapse of the P domain onto the shell. In the contracted shell conformation, antibodies to the P1 and shell domains are not expected to bind. Therefore, at the site of infection in the gut, the host's own bile allows the virus to escape antibody-mediated neutralization while enhancing cell attachment. IMPORTANCE The major feature of calicivirus capsids is the 90 protruding domains (P domains) that are the site of cell receptor attachment and antibody epitopes. We demonstrated previously that these P domains are highly mobile and that bile causes these "floating" P domains in mouse norovirus (MNV) to contract onto the shell surface. Here, we present the near-atomic cryo-EM structure of the isolated MNV P domain complexed with a neutralizing Fab fragment. Our data show that bile causes two sets of changes. First, bile causes allosteric conformational changes in the epitopes at the top of the P domain that block antibody binding. Second, bile causes the P domain dimer subunits to rotate relative to each other, causing a contraction of the P domain that buries epitopes at the base of the P and shell domains. Taken together, the results show that MNV uses the host's own metabolites to enhance cell receptor binding while simultaneously blocking antibody recognition.

Mesoscopic protein-rich clusters host the nucleation of mutant p53 amyloid fibrils.

The protein p53 is a crucial tumor suppressor, often called "the guardian of the genome"; however, mutations transform p53 into a powerful cancer promoter. The oncogenic capacity of mutant p53 has been ascribed to enhanced propensity to fibrillize and recruit other cancer fighting proteins in the fibrils, yet the pathways of fibril nucleation and growth remain obscure. Here, we combine immunofluorescence three-dimensional confocal microscopy of human breast cancer cells with light scattering and transmission electron microscopy of solutions of the purified protein and molecular simulations to illuminate the mechanisms of phase transformations across multiple length scales, from cellular to molecular. We report that the p53 mutant R248Q (R, arginine; Q, glutamine) forms, both in cancer cells and in solutions, a condensate with unique properties, mesoscopic protein-rich clusters. The clusters dramatically diverge from other protein condensates. The cluster sizes are decoupled from the total cluster population volume and independent of the p53 concentration and the solution concentration at equilibrium with the clusters varies. We demonstrate that the clusters carry out a crucial biological function: they host and facilitate the nucleation of amyloid fibrils. We demonstrate that the p53 clusters are driven by structural destabilization of the core domain and not by interactions of its extensive unstructured region, in contradistinction to the dense liquids typical of disordered and partially disordered proteins. Two-step nucleation of mutant p53 amyloids suggests means to control fibrillization and the associated pathologies through modifying the cluster characteristics. Our findings exemplify interactions between distinct protein phases that activate complex physicochemical mechanisms operating in biological systems.

Discovery of Exosomes From Tick Saliva and Salivary Glands Reveals Therapeutic Roles for CXCL12 and IL-8 in Wound Healing at the Tick-Human Skin Interface.

Ticks secrete various anti-coagulatory, anti-vasoconstrictory, anti-inflammatory, and anti-platelet aggregation factors in their saliva at the bite site during feeding to evade host immunological surveillance and responses. For the first time, we report successful isolation of exosomes (small membrane-bound extracellular signaling vesicles) from saliva and salivary glands of partially fed or unfed ixodid ticks. Our data showed a novel role of these in vivo exosomes in the inhibition of wound healing via downregulation of C-X-C motif chemokine ligand 12 (CXCL12) and upregulation of interleukin-8 (IL-8). Cryo-electron microscopy (cryo-EM) analysis revealed that tick saliva and salivary glands are composed of heterogeneous populations of in vivo exosomes with sizes ranging from 30 to 200 nm. Enriched amounts of tick CD63 ortholog protein and heat shock protein 70 (HSP70) were evident in these exosomes. Treatment of human skin keratinocytes (HaCaT cells) with exosomes derived from tick saliva/salivary glands or ISE6 cells dramatically delayed cell migration, wound healing, and repair process. Wound healing is a highly dynamic process with several individualized processes including secretion of cytokines. Cytokine array profiling followed by immunoblotting and quantitative-PCR analysis revealed that HaCaT cells treated with exosomes derived from tick saliva/salivary glands or ISE6 cells showed enhanced IL-8 levels and reduced CXCL12 loads. Inhibition of IL-8 or CXCL12 further delayed exosome-mediated cell migration, wound healing, and repair process, suggesting a skin barrier protection role for these chemokines at the tick bite site. In contrast, exogenous treatment of CXCL12 protein completely restored this delay and enhanced the repair process. Taken together, our study provides novel insights on how tick salivary exosomes secreted in saliva can delay wound healing at the bite site to facilitate successful blood feeding.

The Dynamic Life of Virus Capsids.

Protein-shelled viruses have been thought as "tin cans" that merely carry the genomic cargo from cell to cell. However, through the years, it has become clear that viruses such as rhinoviruses and caliciviruses are active and dynamic structures waiting for the right environmental cues to deliver their genomic payload to the host cell. In the case of human rhinoviruses, the capsid has empty cavities that decrease the energy required to cause conformational changes, resulting in the capsids "breathing", waiting for the moment when the receptor binds for it to release its genome. Most strikingly, the buried N-termini of VP1 and VP4 are transiently exposed during this process. A more recent example of a "living" protein capsid is mouse norovirus (MNV). This family of viruses have a large protruding (P) domain that is loosely attached to the shell via a single-polypeptide tether. Small molecules found in the gut, such as bile salts, cause the P domains to rotate and collapse onto the shell surface. Concomitantly, bile alters the conformation of the P domain itself from one that binds antibodies to one that recognizes receptors. In this way, MNV appears to use capsid flexibility to present one face to the immune system and a completely different one to attack the host tissue. Therefore, it appears that even protein-shelled viruses have developed an impressive array of tricks to dodge our immune system and efficiently attack the host.

Near-Atomic-Resolution Cryo-Electron Microscopy Structures of Cucumber Leaf Spot Virus and Red Clover Necrotic Mosaic Virus: Evolutionary Divergence at the Icosahedral Three-Fold Axes.

Members of the Tombusviridae family have highly similar structures, and yet there are important differences among them in host, transmission, and capsid stabilities. Viruses in the Tombusviridae family have single-stranded RNA (ssRNA) genomes with T=3 icosahedral protein shells with a maximum diameter of ∼340 Å. Each capsid protein is comprised of three domains: R (RNA binding), S (shell), and P (protruding). Between the R domain and S domain is the "arm" region that studies have shown to play a critical role in assembly. To better understand how the details of structural differences and similarities influence the Tombusviridae viral life cycles, the structures of cucumber leaf spot virus (CLSV; genus Aureusvirus) and red clover necrotic mosaic virus (RCNMV; genus Dianthovirus) were determined to resolutions of 3.2 Å and 2.9 Å, respectively, with cryo-electron microscopy and image reconstruction methods. While the shell domains had homologous structures, the stabilizing interactions at the icosahedral 3-fold axes and the R domains differed greatly. The heterogeneity in the R domains among the members of the Tombusviridae family is likely correlated with differences in the sizes and characteristics of the corresponding genomes. We propose that the changes in the R domain/RNA interactions evolved different arm domain interactions at the ß-annuli. For example, RCNMV has the largest genome and it appears to have created the necessary space in the capsid by evolving the shortest R domain. The resulting loss in RNA/R domain interactions may have been compensated for by increased intersubunit ß-strand interactions at the icosahedral 3-fold axes. Therefore, the R and arm domains may have coevolved to package different genomes within the conserved and rigid shell.IMPORTANCE Members of the Tombusviridae family have nearly identical shells, and yet they package genomes that range from 4.6 kb (monopartite) to 5.3 kb (bipartite) in size. To understand how this genome flexibility occurs within a rigidly conserved shell, we determined the high-resolution cryo-electron microscopy (cryo-EM) structures of cucumber leaf spot virus and red clover necrotic mosaic virus. In response to genomic size differences, it appears that the ssRNA binding (R) domain of the capsid diverged evolutionarily in order to recognize the different genomes. The next region, the "arm," seems to have also coevolved with the R domain to allow particle assembly via interactions at the icosahedral 3-fold axes. In addition, there are differences at the icosahedral 3-fold axes with regard to metal binding that are likely important for transmission and the viral life cycle.

Bile Salts Alter the Mouse Norovirus Capsid Conformation: Possible Implications for Cell Attachment and Immune Evasion.

Caliciviruses are single-stranded RNA viruses with 180 copies of capsid protein comprising the T=3 icosahedral capsids. The main capsid feature is a pronounced protruding (P) domain dimer formed by adjacent subunits on the icosahedral surface while the shell domain forms a tight icosahedral sphere around the genome. While the P domain in the crystal structure of human Norwalk virus (genotype I.1) was tightly associated with the shell surface, the cryo-electron microscopy (cryo-EM) structures of several members of the Caliciviridae family (mouse norovirus [MNV], rabbit hemorrhagic disease virus, and human norovirus genotype II.10) revealed a "floating" P domain that hovers above the shell by nearly 10 to 15 Å in physiological buffers. Since this unusual feature is shared among, and unique to, the Caliciviridae, it suggests an important biological role. Recently, we demonstrated that bile salts enhance cell attachment to the target cell and increase the intrinsic affinity between the P domain and receptor. Presented here are the cryo-EM structures of MNV-1 in the presence of bile salts (∼3 Å) and the receptor CD300lf (∼8 Å). Surprisingly, bile salts cause the rotation and contraction of the P domain onto the shell surface. This both stabilizes the P domain and appears to allow for a higher degree of saturation of receptor onto the virus. Together, these results suggest that, as the virus moves into the gut and the associated high concentrations of bile, the entire capsid face undergoes a conformational change to optimize receptor avidity while the P domain itself undergoes smaller conformational changes to improve receptor affinity.IMPORTANCE Mouse norovirus and several other members of the Caliciviridae have been shown to have a highly unusual structure with the receptor binding protruding (P) domain only loosely tethered to the main capsid shell. Recent studies demonstrated that bile salts enhance the intrinsic P domain/receptor affinity and is necessary for cell attachment. Presented here are the high-resolution cryo-EM structures of apo MNV, MNV/bile salt, and MNV/bile salt/receptor. Bile salts cause a 90° rotation and collapse of the P domain onto the shell surface that may increase the number of available receptor binding sites. Therefore, bile salts appear to be having several effects on MNV. Bile salts shift the structural equilibrium of the P domain toward a form that binds the receptor and away from one that binds antibody. They may also cause the entire P domain to optimize receptor binding while burying a number of potential epitopes.

Exosomes mediate Zika virus transmission through SMPD3 neutral Sphingomyelinase in cortical neurons.

The harmful effects of ZIKA virus (ZIKV) infection are reflected by severe neurological manifestations such as microcephaly in neonates and other complications associated with Guillain-Barré syndrome in adults. The transmission dynamics of ZIKV in or between neurons, or within the developing brains of the foetuses are not fully understood. Using primary cultures of murine cortical neurons, we show that ZIKV uses exosomes as mediators of viral transmission between neurons. Cryo-electron microscopy showed heterogeneous population of neuronal exosomes with a size range of 30-200 nm. Increased production of exosomes from neuronal cells was noted upon ZIKV infection. Neuronal exosomes contained both ZIKV viral RNA and protein(s) that were highly infectious to naïve cells. RNaseA and neutralizing antibodies treatment studies suggest the presence of viral RNA/proteins inside exosomes. Exosomes derived from time- and dose-dependent incubations showed increasing viral loads suggesting higher packaging and delivery of ZIKV RNA and proteins. Furthermore, we noted that ZIKV induced both activity and gene expression of neutral Sphingomyelinase (nSMase)-2/SMPD3, an important molecule that regulates production and release of exosomes. Silencing of SMPD3 in neurons resulted in reduced viral burden and transmission through exosomes. Treatment with SMPD3 specific inhibitor GW4869, significantly reduced ZIKV loads in both cortical neurons and in exosomes derived from these neuronal cells. Taken together, our results suggest that ZIKV modulates SMPD3 activity in cortical neurons for its infection and transmission through exosomes perhaps leading to severe neuronal death that may result in neurological manifestations such as microcephaly in the developing embryonic brains.

Explorative Study on Isolation and Characterization of a Microviridae G4 Bacteriophage, EMCL318, against Multi-Drug-resistant Escherichia coli 15-318.

Bacteriophages screened and isolated from sewage water samples exhibited antibacterial activities against multi-drug-resistant Escherichia coli strains. Five different water samples from Canadian habitats such as Kamloops Wastewater Treatment Center, Domtar, the Pacific Ocean, Bisaro Anima Cave, and alkali ponds, were used in this study. Four Enterobacteriaceae strains including one non-resistant and three clinical multi-drug Escherichia coli strains (E. coli 15-102, E. coli 15-124, and E. coli 15-318) were selected as target bacteria to screen for the bacteriophages from these collected water samples. Seeded agar assay technique was implemented for the screening. It was found that only sewage water sample exhibited a significant number of plaques count with the E. coli 15-318 (1.82 × 10² plaques/plate) cells in comparison to E. coli non-resistant strain K12 (8 plaques/plate). The phage did not produce plaques in the E. coli 15-124 and E. coli 15-102 strains. The bacteriophage, designated EMCL318, was isolated, purified, characterized, and identified to belong to the G4 species of the Family Microviridae, GenBank accession number MG563770. This is an explorative study conducted in order to reveal the viruses as alternative potentials to fight against emerging and existing multi-drug-resistant infectious diseases.

Lipid-Coated Mesoporous Silica Nanoparticles for the Delivery of the ML336 Antiviral to Inhibit Encephalitic Alphavirus Infection.

Venezuelan equine encephalitis virus (VEEV) poses a major public health risk due to its amenability for use as a bioterrorism agent and its severe health consequences in humans. ML336 is a recently developed chemical inhibitor of VEEV, shown to effectively reduce VEEV infection in vitro and in vivo. However, its limited solubility and stability could hinder its clinical translation. To overcome these limitations, lipid-coated mesoporous silica nanoparticles (LC-MSNs) were employed. The large surface area of the MSN core promotes hydrophobic drug loading while the liposome coating retains the drug and enables enhanced circulation time and biocompatibility, providing an ideal ML336 delivery platform. LC-MSNs loaded 20 ± 3.4 µg ML336/mg LC-MSN and released 6.6 ± 1.3 µg/mg ML336 over 24 hours. ML336-loaded LC-MSNs significantly inhibited VEEV in vitro in a dose-dependent manner as compared to unloaded LC-MSNs controls. Moreover, cell-based studies suggested that additional release of ML336 occurs after endocytosis. In vivo safety studies were conducted in mice, and LC-MSNs were not toxic when dosed at 0.11 g LC-MSNs/kg/day for four days. ML336-loaded LC-MSNs showed significant reduction of brain viral titer in VEEV infected mice compared to PBS controls. Overall, these results highlight the utility of LC-MSNs as drug delivery vehicles to treat VEEV.

An attenuated replication-competent chikungunya virus with a fluorescently tagged envelope.

BACKGROUND: Chikungunya virus (CHIKV) is the most common alphavirus infecting humans worldwide, causing acute and chronically debilitating arthralgia at a great economic expense. METHODOLOGY/PRINCIPAL FINDINGS: To facilitate our study of CHIKV, we generated a mCherry tagged replication-competent chimeric virus, CHIKV 37997-mCherry. Single particle cryoEM demonstrated icosahedral organization of the chimeric virus and the display of mCherry proteins on virus surface. CHIKV 37997-mCherry is attenuated in both IFNαR knockout and wild-type mice. Strong anti-CHIKV and anti-mCherry antibody responses were induced in CHIKV 37997-mCherry infected mice. CONCLUSIONS/SIGNIFICANCE: Our work suggests that chimeric alphaviruses displaying foreign antigen can serve as vaccines against both aphaviruses and other pathogens and diseases.

Neutralizing Antibodies Inhibit Chikungunya Virus Budding at the Plasma Membrane.

Neutralizing antibodies (NAbs) are traditionally thought to inhibit virus infection by preventing virion entry into target cells. In addition, antibodies can engage Fc receptors (FcRs) on immune cells to activate antiviral responses. We describe a mechanism by which NAbs inhibit chikungunya virus (CHIKV), the most common alphavirus infecting humans, by preventing virus budding from infected human cells and activating IgG-specific Fcγ receptors. NAbs bind to CHIKV glycoproteins on the infected cell surface and induce glycoprotein coalescence, preventing budding of nascent virions and leaving structurally heterogeneous nucleocapsids arrested in the cytosol. Furthermore, NAbs induce clustering of CHIKV replication spherules at sites of budding blockage. Functionally, these densely packed glycoprotein-NAb complexes on infected cells activate Fcγ receptors, inducing a strong, antibody-dependent, cell-mediated cytotoxicity response from immune effector cells. Our findings describe a triply functional antiviral pathway for NAbs that might be broadly applicable across virus-host systems, suggesting avenues for therapeutic innovation through antibody design.