Specific inter-domain interactions stabilize a compact HIV-1 Gag conformation.

HIV-1 Gag is a large multidomain poly-protein with flexible unstructured linkers connecting its globular subdomains. It is compact when in solution but assumes an extended conformation when assembled within the immature HIV-1 virion. Here, we use molecular dynamics (MD) simulations to quantitatively characterize the intra-domain interactions of HIV-1 Gag. We find that the matrix (MA) domain and the C-terminal subdomain CActd of the CA capsid domain can form a bound state. The bound state, which is held together primarily by interactions between complementary charged and polar residues, stabilizes the compact state of HIV-1 Gag. We calculate the depth of the attractive free energy potential between the MA/ CActd sites and find it to be about three times larger than the dimerization interaction between the CActd domains. Sequence analysis shows high conservation within the newly-found intra-Gag MA/CActd binding site, as well as its spatial proximity to other well known elements of Gag -such as CActd's SP1 helix region, its inositol hexaphosphate (IP6) binding site and major homology region (MHR), as well as the MA trimerization site. Our results point to a high, but yet undetermined, functional significance of the intra-Gag binding site. Recent biophysical experiments that address the binding specificity of Gag are interpreted in the context of the MA/CActd bound state, suggesting an important role in selective packaging of genomic RNA by Gag.

Assessment of Inhibitors of Pathogenic Crimean-Congo Hemorrhagic Fever Virus Strains Using Virus-Like Particles.

Crimean-Congo hemorrhagic fever (CCHF) is an often lethal, acute inflammatory illness that affects a large geographic area. The disease is caused by infection with CCHF virus (CCHFV), a nairovirus from the Bunyaviridae family. Basic research on CCHFV has been severely hampered by biosafety requirements and lack of available strains and molecular tools. We report the development of a CCHF transcription- and entry-competent virus-like particle (tecVLP) system that can be used to study cell entry and viral transcription/replication over a broad dynamic range (~4 orders of magnitude). The tecVLPs are morphologically similar to authentic CCHFV. Incubation of immortalized and primary human cells with tecVLPs results in a strong reporter signal that is sensitive to treatment with neutralizing monoclonal antibodies and by small molecule inhibitors of CCHFV. We used glycoproteins and minigenomes from divergent CCHFV strains to generate tecVLPs, and in doing so, we identified a monoclonal antibody that can prevent cell entry of tecVLPs containing glycoproteins from 3 pathogenic CCHFV strains. In addition, our data suggest that different glycoprotein moieties confer different cellular entry efficiencies, and that glycoproteins from the commonly used strain IbAr10200 have up to 100-fold lower ability to enter primary human cells compared to glycoproteins from pathogenic CCHFV strains.

Isolation and characterization of a bacteriophage with broad host range, displaying potential in preventing bovine diarrhoea.

Phage therapy has been previously tried for treatment of diarrhoea in calves, pigs and lambs but those trials were conducted without any detailed information of used phages. Here, we report isolation of a broad-spectrum phage which showed bactericidal activity against 47.3 % of calf diarrhoeal isolates of Escherichia coli, in vitro. The isolated phage resembled the characteristics of Myoviridae family and showed ~97 % similarity with earlier reported bacteriophages of sub family-Tevenvirinae, genus-T4-like virus, based on nucleotide sequence of major head protein-gp23 gene. The phage exhibits the potential to be used as drug substitute tool against E. coli causing diarrhoea in cattle in farm environments.

Phosphorylation of Beet black scorch virus coat protein by PKA is required for assembly and stability of virus particles.

Plant virus coat proteins (CPs) play a fundamental role in protection of genomic RNAs, virion assembly, and viral movement. Although phosphorylation of several CPs during virus infection have been reported, little information is available about CP phosphorylation of the spherical RNA plant viruses. Here, we demonstrate that the CP of Beet black scorch virus (BBSV), a member of the genus Necrovirus, can be phosphorylated at threonine-41 (T41) by cAMP-dependent protein kinase (PKA)-like kinase in vivo and in vitro. Mutant viruses containing a T41A non-phosphorylatable alanine substitution, and a T41E glutamic acid substitution to mimic threonine phosphorylation were able to replicate but were unable to move systemically in Nicotiana benthamiana. Interestingly, the T41A and T41E mutants generated unstable 17 nm virus-like particles that failed to package viral genomic (g) RNA, compared with wild-type BBSV with 30 nm virions during viral infection in N. benthamiana. Further analyses showed that the T41 mutations had little effect on the gRNA-binding activity of the CP. Therefore, we propose a model whereby CP phosphorylation plays an essential role in long-distance movement of BBSV that involves formation of stable virions.

Molecular characterization of a Chinese isolate of potato virus A (PVA) and evidence of a genome recombination event between PVA variants at the 3'-proximal end of the genome.

Potato plants that exhibited mosaic symptoms were collected in Xiangxi, Hunan province, China. Multiplex RT-PCR screening for common viruses revealed the presence of potato virus A (PVA) in these samples. ELISA with virus-specific antibodies confirmed infection by PVA in the plants. Rod-shaped virions of ~750 nm in length and ~13 nm in width were observed by transmission electron microscopy. One virus isolate (designated PVA-Hunan) was subjected to molecular characterization. The viral genome consisted of 9,567 nucleotides, excluding the poly(A) tail, and encoded a polyprotein of 3,059 amino acids. A second characteristic potyvirus open reading frame (ORF), pretty interesting Potyviridae ORF (pipo), was located at nucleotides 2,834-3,139. The isolate shared 84% to 98% and 93% to 99% sequence identity with other PVA isolates at the nucleotide and amino acid level, respectively. Phylogenetic analysis demonstrated that, within the PVA group, PVA-Hunan clustered most closely with the Finnish isolate Her, then with isolates 143, U, Ali, M and B11. The isolate TamMV stood alone at a separate branch. However, scanning of complete genome sequences using SimPlot revealed 99%-sequence identity between PVA-Hunan and TamMV in the 3'-proximal end of the genome (~nt 9,160 to the 3'end) and a 50%-94% (average~83%) identity upstream of nt 9,160. In contrast, 98% identity between PVA-Hunan and isolates M and B11 was detected for nucleotides 1 to ~9,160, but only ~94% for the 3'-proximal region, suggesting a genome recombination event (RE) at nt 9,133. The recombination breakpoint also was identified by the Recombination Detection Program (RDP). The RE was further confirmed by analysis of the CP gene, where the apparent RE was located.

Preclinical characterization of GLS4, an inhibitor of hepatitis B virus core particle assembly.

Hepatitis B virus (HBV)-associated chronic liver diseases are treated with nucleoside analogs that target the virus polymerase. While these analogs are potent, drugs are needed to target other virus-encoded gene products to better block the virus replication cycle and chronic liver disease. This work further characterized GLS4 and compared it to the related BAY 41-4109, both of which trigger aberrant HBV core particle assembly, where the virus replication cycle occurs. This was done in HepAD38 cells, which replicate HBV to high levels. In vitro, GLS4 was significantly less toxic for primary human hepatocytes (P < 0.01 up to 100 µM), inhibited virus accumulation in the supernantant of HepAD38 cells (P < 0.02 up to 100 nM), inhibited HBV replicative forms in the liver with a significantly lower 50% effective concentration (EC50) (P < 0.02), and more strongly inhibited core gene expression (P < 0.001 at 100 to 200 nM) compared to BAY 41-4109. In vivo characterization was performed in nude mice inoculated with HepAD38 cells, which grew out as tumors, resulting in viremia. Treatment of mice with GLS4 and BAY 41-4109 showed strong and sustained suppression of virus DNA to about the same extents both during and after treatment. Both drugs reduced the levels of intracellular core antigen in the tumors. Alanine aminotransferase levels were normal. Tumor and total body weights were not affected by treatment. Thus, GLS4 was as potent as the prototype, BAY 41-4109, and was superior to lamivudine, in that there was little virus relapse after the end of treatment and no indication of toxicity.

T4-like phage Bp7, a potential antimicrobial agent for controlling drug-resistant Escherichia coli in chickens.

Chicken-pathogenic Escherichia coli is severely endangering the poultry industry in China and worldwide, and antibiotic therapy is facing an increasing problem of antibiotic resistance. Bacteriophages can kill bacteria with no known activity in human or animal cells, making them an attractive alternative to antibiotics. In this study, we present the characteristics of a novel virulent bacteriophage, Bp7, specifically infecting pathogenic multidrug-resistant E. coli. Phage Bp7 was isolated from chicken feces. Bp7 belongs to the family Myoviridae, possessing an elongated icosahedral head and contractile sheathed tail. It has a 168-kb double-stranded DNA genome. For larger yields, its optimal multiplicity of infection (MOI) to infect E. coli was about 0.001. The latent period was 10 to 15 min, and the burst size was 90 PFU/infected cell. It was stable both at pH 5.0 to 10.0 and at 40°C or 50°C for at least 1 h. Bp7 could infect 46% of pathogenic clinical E. coli strains. Bp7 harbored 791 open reading frames (ORFs) and 263 possible genes. Among the 263 genes, 199 possessed amino acid sequence identities with ORFs of phage T4, 62 had identities with other T4-like phages, and only one lacked any database match. The genome of Bp7 manifested obvious division and rearrangement compared to phages T4, JS98, and IME08. Bp7 is a new member of the "T4-like" genus, family Myoviridae. Its wide host range, strong cell-killing activity, and high stability to pH make it an alternative to antimicrobials for controlling drug-resistant E. coli in chickens.

Therapeutic effects of bacteriophages against Salmonella gallinarum infection in chickens.

In this study the isolation and characterization of three bacteriophages (ST4, L13, and SG3) infecting Salmonella gallinarum were carried out. They were further tested for their in vivo efficacy in phage therapy. All three phages belong to the Siphoviridae family with isometric heads and non-contractile tails. They have a broad host range among serovars of Salmonella enterica. The burst sizes were observed to be 1670, 80, and 28 for ST4, L13, and SG3, respectively. The in vivo efficacy of the phages was tested in chickens. Layer chickens were challenged with S. gallinarum, whereas contact chickens were cohabited without direct challenge. Each bacteriophage was orally inoculated in the form of feed additives. Mortality was observed and S. gallinarum was periodically re-isolated from the livers, spleens, and cecums of the chickens. Bacterial re-isolation from the organs and mortality decreased significantly in both challenged and contact chickens treated with the bacteriophages compared with untreated chickens serving as the control. The three bacteriophages may be effective alternatives to antibiotics for the control of fowl typhoid disease in chickens.

A polyphenol-rich extract from Chaenomeles sinensis (Chinese quince) inhibits influenza A virus infection by preventing primary transcription in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruits of Chaenomeles sinensis Koehne (Chinese quince) are distributed throughout China and Japan. It has traditionally been known to have a therapeutic effect against respiratory symptoms caused by infectious diseases. AIM OF THE STUDY: The polyphenol-rich extract, CSD3, from Chaenomeles sinensis has previously been shown to neutralize influenza virus infectivity. The aim of this study was to clarify which step(s) in the replication cycle in vitro were inhibited. MATERIALS AND METHODS: We examined cell-binding, hemagglutination and hemolytic activities and infectivity of A/Udorn/72(H3N2) virus after pre-treatment with CSD3. We also investigated the time course of synthesis for viral mRNA, cRNA, and vRNA in Madin-Darby canine kidney epithelial cells (MDCK) cells infected with CSD3-treated virus. Finally, we studied the effect of CSD3-treatment on the ultrastructure of the influenza virion. RESULTS: Pre-treatment with CSD3 mildly reduced cell-binding, hemagglutination and hemolytic activities. These activities were reduced by 70% to be equivalent to 30% of the control at 1µg/ml. CSD3 severely reduced infectivity to 1% of the control at 1µg/ml. Primary transcription in MDCK cells infected with CSD3 (1µg/ml)-treated virus was decreased to about 1% of that in cells infected with mock-treated virus. Synthesis of viral cRNA, vRNA and secondary mRNA was also severely decreased. Electron microscopy revealed that the integrity of the virus envelope was damaged by CSD3 and was permeable to uranyl acetate. CONCLUSIONS: The main target step(s) of CSD3 in the replication cycle is after cell-binding but before or at primary transcription. Involvement of the increased permeability of virus envelope as the inhibition mechanism was proposed. CSD3 could be useful in preventing influenza virus infection, and be employed as a lozenge or mouthwash for daily use.

wksl3, a New biocontrol agent for Salmonella enterica serovars enteritidis and typhimurium in foods: characterization, application, sequence analysis, and oral acute toxicity study.

Of the Salmonella enterica serovars, S. Enteritidis and S. Typhimurium are responsible for most of the Salmonella outbreaks implicated in the consumption of contaminated foods in the Republic of Korea. Because of the widespread occurrence of antimicrobial-resistant Salmonella in foods and food processing environments, bacteriophages have recently surfaced as an alternative biocontrol tool. In this study, we isolated a virulent bacteriophage (wksl3) that could specifically infect S. Enteritidis, S. Typhimurium, and several additional serovars. Transmission electron microscopy revealed that phage wksl3 belongs to the family Siphoviridae. Complete genome sequence analysis and bioinformatic analysis revealed that the DNA of phage wksl3 is composed of 42,766 bp with 64 open reading frames. Since it does not encode any phage lysogeny factors, toxins, pathogen-related genes, or food-borne allergens, phage wksl3 may be considered a virulent phage with no side effects. Analysis of genetic similarities between phage wksl3 and four of its relatives (SS3e, vB_SenS-Ent1, SE2, and SETP3) allowed wksl3 to be categorized as a SETP3-like phage. A single-dose test of oral toxicity with BALB/c mice resulted in no abnormal clinical observations. Moreover, phage application to chicken skin at 8°C resulted in an about 2.5-log reduction in the number of Salmonella bacteria during the test period. The strong, stable lytic activity, the significant reduction of the number of S. Enteritidis bacteria after application to food, and the lack of clinical symptoms of this phage suggest that wksl3 may be a useful agent for the protection of foods against S. Enteritidis and S. Typhimurium contamination.

Poxvirus membrane biogenesis: rupture not disruption.

Enveloped viruses acquire their membrane from the host by budding at, or wrapping by, cellular membranes. Transmission electron microscopy (TEM) images, however, suggested that the prototype member of the poxviridae, vaccinia virus (VACV), may create its membrane 'de novo' with free open ends exposed in the cytosol. Within the frame of the German-wide priority programme we re-addressed the biogenesis and origin of the VACV membrane using electron tomography (ET), cryo-EM and lipid analysis of purified VACV using mass spectrometry (MS). This review discussed how our data led to a model of unconventional membrane biogenesis involving membrane rupture and the generation of a single open membrane from open membrane intermediates. Lipid analyses of purified virus by MS suggest an ER origin with a relatively low cholesterol content compared with whole cells, confirming published data. Unlike previous reports using thin-layer chromatography, no depletion of phosphatidylethanolamine was detected. We did detect, however, an enrichment for phosphatidic acid, diacylglycerol and phosphatidylinositol in the virion. Our data are discussed in the light of other pathogens that may requirecellular membrane rupture during their intracellular life cycle.

Distribution of Potato virus Y in potato plant organs, tissues, and cells.

The distribution of Potato virus Y (PVY) in the systemically infected potato (Solanum tuberosum) plants of the highly susceptible cultivar Igor was investigated. Virus presence and accumulation was analyzed in different plant organs and tissues using real-time polymerase chain reaction and transmission electron microscopy (TEM) negative staining methods. To get a complete insight into the location of viral RNA within the tissue, in situ hybridization was developed and optimized for the detection of PVY RNA at the cellular level. PVY was shown to accumulate in all studied leaf and stem tissues, in shoot tips, roots, and tubers; however, the level of virus accumulation was specific for each organ or tissue. The highest amounts of viral RNA and viral particles were found in symptomatic leaves and stem. By observing cell ultrastructure with TEM, viral cytoplasmic inclusion bodies were localized in close vicinity to the epidermis and in trichomes. Our results show that viral RNA, viral particles, and cytoplasmic inclusion bodies colocalize within the same type of cells or in close vicinity.

Molecular variability and genetic structure of the population of Onion yellow dwarf virus infecting garlic in Iran.

Onion yellow dwarf virus (OYDV) is one of the most important viral diseases of garlic crops worldwide. This study surveyed the occurrence of OYDV in 26 garlic ecotypes collected from different regions in Iran during 2008-2009. Using an electron microscope, we detected filamentous particles with about 700-800 nm in length and 12 nm in width in five samples. These features are typical of the genus Potyvirus. The coat protein (CP) gene from 26 samples was PCR amplified, cloned, sequenced, and compared with the sequences available in GenBank. Phylogenetic analysis using 235 deduced amino acids of the CP gene showed that virus isolates fell into two groups, group A and group B. Members of group A were divided into two subgroups: A-I and A-II. The subgroup A-I appears to be a new subgroup comprising 17 Iranian isolates. The identity levels among the amino acid of 26 Iranian isolates ranged between 90 and 100%. The results indicated that the genetic diversity found in Iran is due to local OYDV populations rather than introduction from other geographical regions. This study is the first report about the molecular structure and geographically diverse range of OYDV populations in this country.

Atomic force microscopy investigation of the giant mimivirus.

Mimivirus was investigated by atomic force microscopy in its native state following serial degradation by lysozyme and bromelain. The 750-nm diameter virus is coated with a forest of glycosylated protein fibers of lengths about 140 nm with diameters 1.4 nm. Fibers are capped with distinctive ellipsoidal protein heads of estimated Mr=25 kDa. The surface fibers are attached to the particle through a layer of protein covering the capsid, which is in turn composed of the major capsid protein (MCP). The latter is organized as an open network of hexagonal rings with central depressions separated by 14 nm. The virion exhibits an elaborate apparatus at a unique vertex, visible as a star shaped depression on native particles, but on defibered virions as five arms of 50 nm width and 250 nm length rising above the capsid by 20 nm. The apparatus is integrated into the capsid and not applied atop the icosahedral lattice. Prior to DNA release, the arms of the star disengage from the virion and it opens by folding back five adjacent triangular faces. A membrane sac containing the DNA emerges from the capsid in preparation for fusion with a membrane of the host cell. Also observed from disrupted virions were masses of distinctive fibers of diameter about 1 nm, and having a 7-nm periodicity. These are probably contained within the capsid along with the DNA bearing sac. The fibers were occasionally observed associated with toroidal protein clusters interpreted as processive enzymes modifying the fibers.

Morphology and general characteristics of bacteriophages infectious to Robinia pseudoacacia mesorhizobia.

Four phages infectious to Mesorhizobium strains were identified in soil samples taken from local Robinia pseudoacacia stands. Based on their polyhedral heads and short noncontractile tails, three of the phages, Mlo30, Mam12, and Mam20, were assigned to group C of Bradley's classification, the Podoviridae family, while phage Mlo1, with its elongated hexagonal head and a long flexible tail represented subgroup B2 bacteriophages, the Siphoviridae family. The phages were homogeneous in respect of their virulence, as they only lysed Mesorhizobium strains, but did not affect strains of Rhizobium or Bradyrhizobium. On the basis of one-step growth experiments, the average virus yield was calculated as approximately 10-25 phage particles for phages Mlo30, Mam12 and Mam20, and as many as 100-120 for phage Mlo1. The rate of phage adsorption to heat-treated cells showed differences in the nature of their receptors, which seemed to be thermal sensitive, thermal resistant, or a combination of the two. Only the receptor for phage Mlo30 was likely to be an LPS molecule, which was supported by a neutralization test. The smooth LPS with O-antigenic chains of the phage-sensitive M. loti strain completely reduced the bactericidal activity of virions at a concentration of 1 µg/ml. The molecular weights of phage DNAs estimated from restriction endonuclease cleavage patterns were in the range from approximately 39 kb for group C phages to approximately 80 kb for B2.

Protein kinase D-dependent trafficking of the large Herpes simplex virus type 1 capsids from the TGN to plasma membrane.

The biosynthetic pathway carries cargos from the endoplasmic reticulum (ER) to the trans Golgi network (TGN) via a typical passage through the Golgi. Interestingly, large particles such as procollagen, chylomicrons and some viruses all reach the TGN by atypical routes. Given this dichotomy, we anticipated that such cargos might rely on non-classical machineries downstream of the TGN. Using Herpes simplex virus type 1 (HSV-1) as a model and a synchronized infection protocol that focuses on TGN to plasma membrane transport, the present study revealed the surprising implication of the cellular serine-threonine protein kinase D in HSV-1 egress. These findings, confirmed by a variety of complementary means [pharmacological inhibitors, dominant negative mutant, RNA interference and electron microscopy (EM)], identify one of possibly several cellular factors that modulate the egress of viruses transiting at the TGN. Moreover, the involvement of this kinase, previously known to regulate the transport of small basolateral cargos, highlights the trafficking of both small and exceptionally large entities by a common machinery downstream of the TGN, in sharp contrast to earlier steps of transport. Conceptually, this indicates the TGN is not only a sorting station from which cargos can depart towards different destinations but also a meeting point where conventional and unconventional routes can meet along the biosynthetic pathway. Lastly, given the apical release of HSV-1 in neurons, it opens up the possibility that this kinase might regulate some apical sorting.

Identification and characterization of a novel Tritimovirus species isolated from wild Trisetum flavescens L., family Poaceae.

Yellow oat-grass plants (Trisetum flavescens L.) with mild mosaic and pronounced dwarfing symptoms were observed at different locations in the Czech Republic. Electron microscope observations of symptomatic plants revealed the presence of filamentous particles and inclusion bodies characteristic of the family Potyviridae. The virus was readily mechanically transmitted to its original host plus a narrow host range of monocot species. Serological assays of infected plant extracts using antiserum specific to the closest species in the family Potyviridae were negative. The 3' end of the viral genome was cloned, sequenced and compared to sequences of species in the family Potyviridae. The virus is more closely related to viruses in the genus Tritimovirus than to other genera within the Potyviridae. Based on phylogenetic analyses of the coat protein cistron and flanking genomic regions, we propose this is a distinct viral species of the genus Tritimovirus, tentatively named Yellow oat-grass mosaic virus (YOgMV).

Characterization of phages virulent for Robinia pseudoacacia Rhizobia.

Three lytic phages (PhiRP1, PhiRP2, and PhiRP3) specific for Robinia pseudoacacia rhizobia were isolated from the soil under black locust. They were characterized by their morphology, host range, and some other properties including DNA molecular weights. Studied phages have been found to belong to Siphoviridae family that comprises viruses with long, and noncontractile tails. They had broad host ranges and effectively lysed not only Robinia pseudoacacia microsymbionts but also different Mesorhizobium species. The phages were homogenous in latent periods (300 min) but heterogeneous in burst sizes (100-200 phage particles per one infected cell) and rise periods (90-120 min). They showed a distinct adsorption rate to Robinia pseudoacacia rhizobia (70.4-93.94%). The molecular weights of phage DNAs estimated from restriction enzyme digests were in the range from ca. 82 kb to ca. 105 kb.

Immunodrugs: therapeutic VLP-based vaccines for chronic diseases.

Worldwide, the prevalence of noncommunicable chronic diseases is increasing. The use of vaccines to induce autoantibodies that neutralize disease-related proteins offers a means to effectively and affordably treat such diseases. Twenty vaccines designed to induce therapeutic autoantibodies were clinically tested in the past 12 years. Immunodrugs are therapeutic vaccines comprising virus-like particles (VLPs) covalently conjugated with self-antigens that induce neutralizing autoantibody responses. Four such VLP-based vaccines have been clinically tested and one has achieved proof of principle: a reduction of blood pressure in hypertensive patients. To facilitate preliminary clinical testing, novel nonclinical study programs have been developed. Safety study designs have considered the underlying B and T cell immunology and have examined potential toxicities of vaccine components and primary and secondary pharmacodynamic action of the vaccines.

The carboxy-terminal two-thirds of the cowpea chlorotic mottle bromovirus capsid protein is incapable of virion formation yet supports systemic movement.

Previous investigations into recombination in cowpea chlorotic mottle bromovirus (CCMV) resulted in the recovery of an unusual recombinant virus, 3-57, which caused a symptomless infection of cowpeas but formed no detectable virions. Sequence analysis of cDNA clones derived from 3-57 determined that mutations near the 5' terminus of the capsid protein gene introduced an early translational termination codon. Further mutations introduced a new in-frame start codon that allowed translation of the 3' two-thirds of the capsid protein gene. Based on the mutations observed in 3-57, wild-type CCMV clones were modified to determine if the carboxyl two-thirds of the capsid protein functions independently of the complete protein in long-distance movement. Analysis of these mutants determined that while virion formation is not required for systemic infection, the carboxy-terminal two-thirds of the capsid protein is both required and sufficient for systemic movement of viral RNA. This indicates that the CCMV capsid protein is multifunctional, with a distinct long-distance movement function in addition to its role in virion formation.