Virus T1 Research Articles

Genomic and Proteomic Characterizations of Sfin-1, a Novel Lytic Phage Infecting Multidrug-Resistant Shigella spp. and Escherichia coli C.

Shigellosis is a public health threat in developed as well as developing countries like “India.” While antibiotic therapy is the mainstay of treatment for shigellosis, current emergence of multidrug-resistant strains of Shigella spp. has posed the problem more challenging. Lytic bacteriophages which destroy antibiotic resistant Shigella spp. have great potential in this context and hence their identification and detailed characterization is necessary. In this study we presented the isolation and a detailed characterization of a novel bacteriophage Sfin-1, which shows potent lytic activity against multidrug-resistant isolates of Shigella flexneri, Shigella dysenteriae, Shigella sonnei obtained from clinical specimens from shigellosis patients. It is also active against Escherichia coli C. The purified phage is lytic in nature, exhibited absorption within 5–10 min, a latent period of 5–20 min and burst size of ∼28 to ∼146 PFU/cell. The isolated phage shows stability in a broad pH range and survives an hour at 50°C. Genome sequencing and phylogenetic analyses showed that Sfin-1 is a novel bacteriophage, which is very closely related to T1-like phages (89.59% identity with Escherichia virus T1). In silico analysis indicates that Sfin-1 genome consists of double stranded linear DNA of 50,403 bp (GC content of 45.2%) encoding 82 potential coding sequences, several potential promoters and transcriptional terminators. Under electron microscopy, Sfin-1 shows morphology characteristics of the family Siphoviridae with an isometric head (61 nm) and a non-contractile tail (155 nm). This is most likely the first report of a lytic bacteriophage that is active against three of the most virulent multidrug-resistant Shigella species and therefore might have a potential role in phage therapy of patients infected with these organisms.

Genetic Transformation of a Mouse Cell Line with Genes of the Receptor for Equine Infectious Anemia Virus (ELR1) and Cyclin T1 (eCT1)

The complexity of the pathogenesis and insufficient knowledge of the equine infectious anemia virus (EIAV) make it necessary to find an adequate laboratory model for studying the infectious process and the immune response. One of the ways to solve this problem is the production of cell cultures with the desired properties using genetic transformation. The aim of the present study was to obtain mouse embryonic fibroblasts with genes of the receptor of the equine infectious anemia virus (ELR1) and cyclin T1 (eCT1). For this purpose, cells were transfected with pcDNA3.1-ELR1 and pcDNA3.1-cycT1 plasmids containing nucleotide sequences of the target genes using the calcium-phosphate method. Eight stable genetically transformed clones of STO cells with [ELR1eCT1neo]+ phenotype were obtained using the selection of cells on G418. The frequency of genetic STO transformation was 2.8 × 10–5. The integration of genes into the cell genome was detected by PCR using gene-specific primers with subsequent nucleotide sequencing using DNA isolated from cells as the matrix. The expression of equine ELR1 and eCT1 genes in mouse fibroblasts was confirmed at the transcriptional level by the presence of the mRNA of the target genes in the RT-PCR reaction. The obtained stable STO cell line was deposited in the cell culture collection of the All-Russia Scientific Research Institute of Experimental Veterinary Medicine Federal Scientific Center.

Pepper necrotic spot virus, a new tospovirus infecting solanaceous crops in Peru

Two virus isolates, T1 and T2, causing necrotic spots on leaves and stems of pepper and tomato, respectively, were isolated in the La Joya valley, Arequipa, Peru, in 2010. These two isolates were inoculated to differential hosts for tospoviruses and showed differential fitness: T1 induced necrotic local lesions in Vigna unguiculata, whereas T2 produced only chlorotic spots. The complete nucleotide sequence of the small (S) RNA from T2 and 1863bp of the S RNA from T1 were determined. The deduced N protein sequence showed high amino acid identity (97%) between the isolates, indicating that the T1 and T2 are isolates of the same virus. Sequence comparisons indicated that the amino acid sequence of the N protein shared 53.49-87.98% identity with known American tospoviruses. Phylogenetic analysis of both the NSs and N proteins revealed that this new tospovirus belongs to the American group. We conclude that this tospovirus should be considered a member of a new species. The name Pepper necrotic spot virus (PNSV) is proposed.

The Use of a Microporous Polyvinylidene Fluoride (PVDF) Membrane Filter to Separate Contaminating Viral Particles from Biologically Important Proteins

Abstract.Viral agents (influenza A virus, 80—120 nm; phage T1, 50 nm head, 150 nm head, 150 nm tail; phage PR772, 53 nm; poliovirus, 28—30 nm; and phage PP7, 25 nm) were used to determine the ability of a newly developed, modified polyvinylidene fluoride (PVDF) membrane filter to remove viruses from several fluids. These included ultrapure water, Dulbecco's modified Eagle minimum essential medium (DMEM) and DMEM with 10% fetal bovine serum (DMEM—10). Small volume (10 ml) filtration experiments were done with 47–mm disks while larger volumes (1 litre) were done with virus suspended in DMEM–10, using cartridge filters with a surface area of 1·63 m 2. With 47–mm disks, influenza A virus and phage T1 were removed to below detectable limits in all fluids tested (titre reduction [Tr] >2·0 × 10 6and >5·8 × 10 8, respectively). The retention of phage PP7 and poliovius was consistent but fluid dependent. The greatest concentration of phage PP7 and poliovirus was removed from ultrapure water (phage PP7, Tr = 2·1 × 10 7; poliovirus, TR > 3·2 × 10 4), while the removal efficency from DMEM–10 was substantially lower (phage PP7, Tr = 2·3; poliovirus, Tr = 2?1 × 10 2). Results of cartridge challenges in DMEM–10 were comparable to the corresponding small disk challenges. These results demonstrate that this PVDF membrane filter was very effective (Tr > 10 6) in removing viral particles (> 50 nm); smaller viruses (< 50 nm) were also consistently removed, but the level of removal depended on the virus and type of fluid tested. In separate experiments, the recovery of purified albumin (69 000 Da) and IgG (150 000 Da) in the filtrate was also determined at approx. 0·015 mg/ml and approx. 10 mg/ml. Recovery of albumin and IgG was >90%. Efficient virus retention coupled with high recovery of protein <150 000 Da suggest potential applications of this membrane filter, when protection against adventitious viral contaminants is desired.

Post-transcriptional inhibition of the interleukin-1 binding protein B15R of Vaccinia virus after coexpression of the related T1 protein

The interleukin-1 binding B15R protein of Vaccinia virus and murine T1 are related extracellular glycoproteins with similarity to the extracellular domain of interleukin-1 receptors. In cells infected with a recombinant Vaccinia virus directing the overexpression of T1, production of the endogenous viral B15R protein is abrogated. T1 synthesis specifically interferes with the production of B15R, but not of other secretory viral proteins. Inhibition of B15R expression occurs at the posttranscriptional level, is exerted in trans and requires the presence of T1 protein in the infected cell. These results suggest a common maturation pathway for the B15R and T1 proteins which might also apply to other members of the interleukin-1 receptor family.

Identification of a Satellite Double-Stranded RNA in the Parasitic Protozoan Trichomonas vaginalis Infected with T. vaginalis Virus T1

Co-infection by a 0.5-kb small double-stranded (ds) RNA together with Trichomonas vaginalis virus (TVV) genomic 4.6-kb dsRNA is commonly observed in a number of T. vaginalis isolates. By molecular cloning and primer extension experiments, the 497-bp cDNA sequence of a 0.5-kb dsRNA co-infecting with TVV-T1 in T. vaginalis T1 isolate was elucidated. Consistent with the replication cycle of a typical dsRNA virus, a plus-strand viral RNA beginning at +1 of the 0.5-kb dsRNA was identified in infected T. vaginalis T1 cells by primer extension and Northern hybridization studies. The 0.5-kb dsRNA was separately encased in TVV capsits from the viral genomic dsRNA, as shown by protein analysis and electron microscopic examination of viral particles purified by multiple rounds of CsCl gradient centrifugation. The riboprobes transcribed from a cloned cDNA of the 0.5-kb dsRNA exhibited strong hybridization to a small dsRNA in a T. vaginalis T9 isolate, which harbors a TVV-T9 distantly related to TVV-T1, but the same probes showed very little hybridization to the viral genomic dsRNA of both TVV-T1 and TVV-T9. Very little sequence homology between the 0.5-kb dsRNA and the 4.6-kb dsRNA in TVV-T1 was found by computer-assisted analysis, suggesting that the small dsRNA in T. vaginalis T1 is not derived from the genome of TVV-T1 or other distantly related T. vaginalis viruses. These results suggest that the small dsRNAs in T. vaginalis are satellite RNAs of T. vaginalis virus.

Primary structure of a DNA (N6-adenine)-methyltransferase from Escherichia coli virus T1. DNA sequence, genomic organization, and comparative analysis.

Escherichia coli virus T1 encodes a DNA (N6-adenine)-methyltransferase (M.T1) with the same sequence specificity as the E. coli DNA (N6-adenine)-methyltransferase (M.Eco dam). This enzyme was purified to homogeneity and a partial amino acid sequence determined. Oligonucleotides were constructed and used not only as probes to map the gene on the T1 genome, but also as primers in sequencing reactions to establish the nucleotide sequence of the M.T1 locus by primer extension. These data represent the first analysis of the genomic organization of bacterial virus T1 on a molecular level. Significant homology to E. coli consensus transcription and translation-initiation signals suggest that the gene for M.T1 is most probably under control of its own promoter. It may be transcribed as a polycistronic mRNA, together with a downstream open reading frame which codes for a polypeptide containing 83 amino acids (HP 83). Both the deduced primary and the secondary structure of the M.T1 were compared to those of other known DNA methyltransferases, especially those recognizing the sequence, GATC; there is little similarity of the T1 enzyme to the other members of this family.

Identification, purification, and characterization of Escherichia coli virus T1 DNA methyltransferase.

An Escherichia coli virus T1-induced DNA methyltransferase was identified by activity gel analysis in homogenates of infected E. coli DNA-adenine-methylation-deficient strains. Although the Mr of this protein (31,000) is in the same range as that of the E. coli DNA adenine methyltransferase, the two proteins are not closely related; the E. coli dam gene does not hybridize with T1 DNA. Selective conditions for measurement of the T1 activity were developed, and the enzyme was purified to functional homogeneity, as shown by activity analysis in polyacrylamide gels. Requirements for optimal activity of the viral enzyme were determined to be pH 6.9, ionic strengths below 0.1 M KCl, and a temperature between 40 and 43 degrees C. The Km for S-adenosyl-L-methionine is 4.9 microM. The purified T1 DNA methyltransferase is capable of methylating adenine in 5'-GATC-3' sites in vitro.

Evidence that Escherichia coli virus T1 induces a DNA methyltransferase.

DNA of Escherichia coli virus T1 is resistant to MboI cleavage and appears to be heavily methylated. Analysis of methylation by the isoschizomeric restriction enzymes Sau3AI and DpnI revealed that recognition sites for E. coli DNA adenine methylase (dam methylase) are methylated. The same methylation pattern was found for virus T1 DNA grown on an E. coli dam host, indicating a T1-specific DNA methyltransferase.

Development of escherichia coli virus T1. ATP-mediated discrimination of gene expression.

The mechanism of host shut-off following virus T1 infection was studied using Escherichia coli wild type and ATPase deficient (unc-) cells. Host protein synthesis measured either as amino acid incorporation into proteins or as enzyme synthesis is immediately inhibited in T1-infected wild type cells. In contrast, host repression in the ATPase-deficient cells is almost unaffected after T1 infection. The continuation of host macromolecule synthesis in the unc- cells is due to constant ATP concentrations after infection, whereas an immediate drop in intracellular ATP levels in T1-infected wild type cells causes repression of host protein synthesis. This result is confirmed when host protein synthesis is determined at decreasing ATP concentrations following the starvation of cells.

Development of Escherichia coli virus T1. The role of the proton-motive force.

In the interaction between Escherichia coli virus T1 and its host cell, which leads to reorientation of macromolecule synthesis, the alteration of the host cell membrane is an important step: The proton-motive force is rapidly reduced. This became apparent from selective changes in energy-coupled transports: proton-motive force- and ATP-dependent transports are inhibited in wild type cells. However, in ATPase-deficient (unc-) cells the ATP-driven transports are not affected by T1. The membrane potential is reduced by T1 as was measured by triphenylmethylphosphonium ion distribution. The potassium gradient is dissipated and calcium ions are accumulated by the cells. However, the residual (but reduced) membrane energy is essential for T1 development since the addition of uncouplers prevents any viral production. Consistently, the cell membrane remains intact after T1 infection: proteins, like beta-galactoside, amino acids, and alpha-methylglucoside cannot passively penetrate the membrane of T1-infected cells.

Development of Escherichia coli virus T1: escape from host restriction

The bacterial virus T1 grows interchangeably on different Escherichia coli strains (C, B, and K). This implies that T1 has an efficient mechanism to overcome the host restriction barrier. The DNA of T1 was found to be methylated independently of the hosts. The percentage of N6-methyladenine varied from 1.6 to 1.8, and the 5-methylcytosine content varied from 0.1 to 0.4%. In contrast, the range in percentage of N6-methyladenine and 5-methylcytosine found in the hosts was 0.7 to 2.4 and 0.0 to 1.1, respectively.

Large T1 oligonucleotides of Moloney leukemia virus missing in an env gene recombinant, HIX, are present on an intracellular 21S Moloney viral RNA species.

HIX, a recombinant derived from Moloney leukemia virus, has an envelope glycoprotein different from that of the Moloney virus. HIX and Moloney viruses share the majority of the large T1 oligonucleotides derived from their genomes but each possesses a set of distinctive oligonucleotides that lie clustered in corresponding regions in the 3' halves of their oligonucleotide maps. These regions presumably contain envelope glycoprotein coding sequences. The type C viral envelope glycoprotein is believed to be translated from a 21S RNA. Thus, at least part of the region of the Moloney virus genome that is altered relative to HIX was expected to be present on such a species. To test this prediction, we purified an intracellular 21S Moloney viral RNA species and analyzed its large T1 oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. This RNA contains one T1 oligonucleotide that is probably derived from the 5' end of the Moloney virus genome, the Moloney virus T1 oligonucleotides that are missing in HIX, and those that lie to their 3' side on the Moloney virus T1 oligonucleotide map.

Development of Escherichia coli Virus T1: Repression of Host Gene Expression

Host protein synthesis, measured either as amino acid incorporation into proteins or as enzyme synthesis, is inhibited rapidly after infection Escherichia coli with T1. Analysis of this inhibition, using a technique which distinguishes between translation and transcription, revealed that translation of host mRNA is specifically blocked. Comparison of the time course of T1-induced host repression with inhibition by the drugs rifampicin, nitrofurantoin and chloramphenicol showed that T1 affects the initiation step of host translation. Intact membranes are apparently essential for host repression, suggesting a membrane-mediated process. Concomitant viral protein synthesis is not required. The membrane-altering principle is a constituent of the viral particle.

Development ofE. coli virus T1: The pattern of gene expression

T1 infected bacteria exhibit a distinct pattern of gene expression. The control of this expression is accessible to biochemical analysis. T1 induces the synthesis of 31 proteins in E. coli. The virion contains 15 proteins. By means of T1 amber mutants, 10 gene products have been assigned to specific T1 genes. Three classes of T1 proteins are defined by the kinetics of their syntheses: early, early-late and late proteins. The regulation of protein synthesis involes at least three mechanisms: for cessation of host gene expression, for discontinuation of the early class during the late phase and for induction of the late T1 proteins. The positive control of late gene expression is not coupled to replication. The host RNA-polymerase transcribes the viral genome throughout the infectious cycle. No virus coded RNA-polymerase is induced.

Changes in the Latent Period of a Bacterial Virus

Studies of the inactivation of T1 bacteriophage by deuterons, ultraviolet light, and temperature have indicated that the loss of ability to form plaques is not necessarily due to one single process (1, 2). A careful investigation into the effects of X-rays on T2 phage has been made by Watson (3), and it has been shown that, for example, the effect of X-rays on the killing power of a virus differs from that on the ability to multiply. The serum-blocking power of T1 bacteriophage is also inactivated thermally quite differently from the infectivity (4), and further studies show that this is also true for the action of ionizing radiation (5). It appeared worth while to see whether any change in the latent period of a bacterial virus occurred in virus which could otherwise be considered as viable. Studies of this kind made by Luria (6) showed that for ultraviolet light there is a definite increase in the latent period for viruses which have undergone a certain amount of inactivation. Luria did not observe any effect of ionizing radiation. In the experiments here described, the bacterial virus T1 acting on E. coli was subjected to drying, drying in vacuum, deuteron and a-particle bombardment, X-irradiation, and heat. We then investigated whether the latent period was increased in those particles which actually survived. The results show that such an .ncrease in latent period does take place. 1