Determination of the structure of the recombinant T = 1 capsid of Sesbania mosaic virus

The recombinant coat protein (CP) of Sesbania mosaic virus lacking segments of different lengths from the N-terminus expressed in E. coli was shown to selfassemble into a variety of distinct capsids encapsidating 23S rRNA from the host and CP mRNA in vivo. Particles with 60 copies (T = 1) of protein subunits were observed when protein lacking 65 amino acids from the N-terminus was expressed. This recombinant protein possesses the sequence corresponding to the S-domain of the native, T = 3 icosahedral particles but lacks the β-annulus, the βA strand (residues 67–70) and the arginine-rich ARM motif (residues 28–36). Purified T = 1 particles crystallized in the monoclinic space group P21 with cell parameters of a = 188.4 Å, b = 194.6Å, c = 272.1Å and β=92.6°. The structure of the T = 1 particles was determined by X-ray diffraction at 3.0 Å resolution. As expected, the poly-peptide fold of the subunit closely resembles that of the S-domain of the native virus. The recombinant particles bind calcium ions in a manner indistinguishable from that of the native capsids. The structure reveals the major differences in the quaternary organization responsible for the formation of T = 1 against T = 3 particles

Drosophila S2 Cells Are Non-Permissive for Vaccinia Virus DNA Replication Following Entry via Low pH-Dependent Endocytosis and Early Transcription

Vaccinia virus (VACV), a member of the chordopox subfamily of the Poxviridae, abortively infects insect cells. We have investigated VACV infection of Drosophila S2 cells, which are useful for protein expression and genome-wide RNAi screening. Biochemical and electron microscopic analyses indicated that VACV entry into Drosophila S2 cells depended on the VACV multiprotein entry-fusion complex but appeared to occur exclusively by a low pH-dependent endocytic mechanism, in contrast to both neutral and low pH entry pathways used in mammalian cells. Deep RNA sequencing revealed that the entire VACV early transcriptome, comprising 118 open reading frames, was robustly expressed but neither intermediate nor late mRNAs were made. Nor was viral late protein synthesis or inhibition of host protein synthesis detected by pulse-labeling with radioactive amino acids. Some reduction in viral early proteins was noted by Western blotting. Nevertheless, synthesis of the multitude of early proteins needed for intermediate gene expression was demonstrated by transfection of a plasmid containing a reporter gene regulated by an intermediate promoter. In addition, expression of a reporter gene with a late promoter was achieved by cotransfection of intermediate genes encoding the late transcription factors. The requirement for transfection of DNA templates for intermediate and late gene expression indicated a defect in viral genome replication in VACV-infected S2 cells, which was confirmed by direct analysis. Furthermore, VACV-infected S2 cells did not support the replication of a transfected plasmid, which occurs in mammalian cells and is dependent on all known viral replication proteins, indicating a primary restriction of DNA synthesis

Seroprevalence and Risk Factors Possibly Associated with Emerging Zoonotic Vaccinia Virus in a Farming Community, Colombia

In 2014, vaccinia virus (VACV) infections were identified among farmworkers in Caquetá Department, Colombia; additional cases were identified in Cundinamarca Department in 2015. VACV, an orthopoxvirus (OPXV) used in the smallpox vaccine, has caused sporadic bovine and human outbreaks in countries such as Brazil and India. In response to the emergence of this disease in Colombia, we surveyed and collected blood from 134 farmworkers and household members from 56 farms in Cundinamarca Department. We tested serum samples for OPXV antibodies and correlated risk factors with seropositivity by using multivariate analyses. Fifty-two percent of farmworkers had OPXV antibodies; this percentage decreased to 31% when we excluded persons who would have been eligible for smallpox vaccination. The major risk factors for seropositivity were municipality, age, smallpox vaccination scar, duration of time working on a farm, and animals having vaccinia-like lesions. This investigation provides evidence for possible emergence of VACV as a zoonosis in South America.https://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0000318507https://scholar.google.com.co/citations?user=cU2KyT4AAAAJ&hl=enhttps://scienti.minciencias.gov.co/gruplac/jsp/visualiza/visualizagr.jsp?nro=00000000008981https://orcid.org/0000-0002-8093-054

The Membrane Fusion Step of Vaccinia Virus Entry Is Cooperatively Mediated by Multiple Viral Proteins and Host Cell Components

For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV) enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditional-lethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry

Characterization of a Newly Identified 35-Amino-Acid Component of the Vaccinia Virus Entry/Fusion Complex Conserved in All Chordopoxviruses▿

The original annotation of the vaccinia virus (VACV) genome was limited to open reading frames (ORFs) of at least 65 amino acids. Here, we characterized a 35-amino-acid ORF (O3L) located between ORFs O2L and I1L. ORFs similar in length to O3L were found at the same genetic locus in all vertebrate poxviruses. Although amino acid identities were low, the presence of a characteristic N-terminal hydrophobic domain strongly suggested that the other poxvirus genes were orthologs. Further studies demonstrated that the O3 protein was expressed at late times after infection and incorporated into the membrane of the mature virion. An O3L deletion mutant was barely viable, producing tiny plaques and a 3-log reduction in infectious progeny. A mutant VACV with a regulated O3L gene had a similar phenotype in the absence of inducer. There was no apparent defect in virus morphogenesis, though O3-deficient virus had low infectivity. The impairment was shown to be at the stage of virus entry, as cores were not detected in the cytoplasm after virus adsorption. Furthermore, O3-deficient virus did not induce fusion of infected cells when triggered by low pH. These characteristics are hallmarks of a group of proteins that form the entry/fusion complex (EFC). Affinity purification experiments demonstrated an association of O3 with EFC proteins. In addition, the assembly or stability of the EFC was impaired when expression of O3 was repressed. Thus, O3 is the newest recognized component of the EFC and the smallest VACV protein shown to have a function

Vaccinia Virus H7 Protein Contributes to the Formation of Crescent Membrane Precursors of Immature Virions ▿

Crescent membranes are the first viral structures that can be discerned during poxvirus morphogenesis. The crescents consist of a lipoprotein membrane and an outer lattice scaffold, which provides uniform curvature. Relatively little is known regarding the composition of the crescent membrane or its mode of formation. Here, we show that the H7 protein, which is conserved in all vertebrate poxviruses but has no discernible functional motifs or nonpoxvirus homologs, contributes to the formation of crescents and immature virions. Synthesis of the 17-kDa H7 protein was dependent on DNA replication and occurred late during vaccinia virus infection. Unlike many late proteins, however, H7 was not incorporated into mature virions or localized in cellular organelles. To gain insight into the role of H7, an inducible mutant was constructed and shown to have a conditional lethal phenotype: H7 expression and infectious virus formation were dependent on isopropyl-beta-d-thiogalactopyranoside. In the absence of inducer, viral late proteins were made, but membrane and core proteins were not processed by the I7 protease. A block in morphogenesis was demonstrated by transmission electron microscopy: neither typical crescents nor immature virions were detected in the absence of inducer. Instead, factory areas of the cytoplasm contained large, electron-dense inclusions, some of which had partially coated membrane segments at their surfaces. Separate, lower-density inclusions containing the D13 scaffold protein and endoplasmic reticulum membranes were also present. These features are most similar to those previously seen when expression of A11, another conserved nonvirion protein, is repressed

Spot the Diagnosis

A 73-year-old lady was admitted with abdominal pain, vomiting and significant weight loss. Her initial biochemical profiles were normal. Plain radiographs of the chest and abdomen were normal. An abdominal ultrasound scan revealed stones in the gallbladder as well as in the common bile duct. She underwent endoscopic retrograde cholangiopancreaticography (ERCP) with removal of duct stones. Three days later, she developed severe abdominal pain associated with abdominal distension and obstipation. The plain radiograph done at this stage is depicted as Figure 1. What is the Diagnosis

Inhibition of the Ubiquitin-Proteasome System Prevents Vaccinia Virus DNA Replication and Expression of Intermediate and Late Genes▿

The ubiquitin-proteasome system has a central role in the degradation of intracellular proteins and regulates a variety of functions. Viruses belonging to several different families utilize or modulate the system for their advantage. Here we showed that the proteasome inhibitors MG132 and epoxomicin blocked a postentry step in vaccinia virus (VACV) replication. When proteasome inhibitors were added after virus attachment, early gene expression was prolonged and the expression of intermediate and late genes was almost undetectable. By varying the time of the removal and addition of MG132, the adverse effect of the proteasome inhibitors was narrowly focused on events occurring 2 to 4 h after infection, the time of the onset of viral DNA synthesis. Further analyses confirmed that genome replication was inhibited by both MG132 and epoxomicin, which would account for the effect on intermediate and late gene expression. The virus-induced replication of a transfected plasmid was also inhibited, indicating that the block was not at the step of viral DNA uncoating. UBEI-41, an inhibitor of the ubiquitin-activating enzyme E1, also prevented late gene expression, supporting the role of the ubiquitin-proteasome system in VACV replication. Neither the overexpression of ubiquitin nor the addition of an autophagy inhibitor was able to counter the inhibitory effects of MG132. Further studies of the role of the ubiquitin-proteasome system for VACV replication may provide new insights into virus-host interactions and suggest potential antipoxviral drugs

Crystal structure of the serine protease domain of Sesbania mosaic virus polyprotein and mutational analysis of residues forming the S1-binding pocket

Sesbania mosaic virus (SeMV) polyprotein is processed by its N-terminal serine protease domain. The crystal structure of the protease domain was determined to a resolution of 2.4 Å using multiple isomorphous replacement and anomalous scattering. The SeMV protease domain exhibited the characteristic trypsin fold and was found to be closer to cellular serine proteases than to other viral proteases. The residues of the S1-binding pocket, H298, T279 and N308 were mutated to alanine in the ΔN70-Protease-VPg polyprotein, and the cis-cleavage activity was examined. The H298A and T279A mutants were inactive, while the N308A mutant was partially active, suggesting that the interactions of H298 and T279 with P1-glutamate are crucial for the E-T/S cleavage. A region of exposed aromatic amino acids, probably essential for interaction with VPg, was identified on the protease domain, and this interaction could play a major role in modulating the function of the protease