bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification of Autographa californica nucleopolyhedrovirus DNA polymerase from infected insect cells
Authors: Hang Xin , Guarino Linda A. 
Published: August 2015

Autographa californicanucleopolyhedrovirus (AcMNPV) DNA polymerase was purified from virus-infected cells using conventional chromatographic methods. The enzymatic activity of fractions eluting from single-stranded agarose gels was found to exactly coincide with a single polypeptide with an apparent molecular mass of approximately 110000 Da on denaturing polyacrylamide gels stained with Coomassie blue. This purification scheme resulted in a 228-fold purification of AcMNPV DNA polymerase with recovery of 3·5% of the initial activity. The specific activity of the most purified fraction of DNA polymerase was 5000 units/mg, which is sufficiently high to eliminate the possibility that contaminants significantly contribute to the polymerase activity. Preparations of purified DNA polymerase had 3′–5′ exonuclease activity, but no 5′–3′ exonuclease activity. The proofreading activity was apparently an intrinsic property of the enzyme as the ratio of nuclease activity to polymerase activity was constant throughout purification. Using a singly-primed M13 DNA template, RF-II DNA was detected within 3 min, indicating a polymerization rate of 40 nt/s. The effects of several DNA polymerase inhibitors on the enzymatic activity of purified DNA polymerase were also determined.
Expression and processing of the canine calicivirus capsid precursor
Authors: Matsuura Yuichi , Tohya Yukinobu , Onuma Mihoko , Roerink Frank , Mochizuki Masami , Sugimura Takaaki 
Published: August 2015

The ORF2 product of canine calicivirus (CaCV) was identified and its processing in mammalian cells was analysed. Immunoblot analysis revealed the presence of the 75 kDa capsid precursor in addition to a 57 kDa capsid protein and a 22 kDa N-terminal polypeptide in CaCV-infected cells treated at an elevated temperature. When the CaCV ORF2 was expressed in a transient mammalian expression system, only the 75 kDa precursor was detected in immunoblot analysis, suggesting that no post-translational processing occurred in this system. However, the precursor was processed to a 57 kDa protein and a 22 kDa polypeptide by the proteinase of feline calicivirus (FCV) when this was co-expressed with ORF2. Processing was blocked by site-directed mutagenesis of the putative cleavage site in the capsid precursor. The results indicate that the proteinase of FCV can cleave the capsid precursor of CaCV to produce the mature capsid protein and that CaCV may have a similar proteinase.
A minimum length of N gene sequence in transgenic plants is required for RNA-mediated tospovirus resistance
Authors: Jan Fuh-Jyh , Fagoaga Carmen , Pang Sheng-Zhi , Gonsalves Dennis 
Published: August 2015

We showed previously that transgenic plants with the green fluorescent protein (GFP) gene fused to segments of the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) displayed post-transcriptional gene silencing of the GFP and N gene segments and resistance to TSWV. These results suggested that a chimeric transgene composed of viral gene segments might confer multiple virus resistance in transgenic plants. To test this hypothesis and to determine the minimum length of the N gene that could trans-inactivate the challenging TSWV, transgenic plants were developed that contained GFP fused with N gene segments of 24–453 bp. Progeny from these plants were challenged with: (i) a chimeric tobacco mosaic virus containing the GFP gene, (ii) a chimeric tobacco mosaic virus with GFP plus the N gene of TSWV and (iii) TSWV. A number of transgenic plants expressing the transgene with GFP fused to N gene segments from 110 to 453 bp in size were resistant to these viruses. Resistant plants exhibited post-transcriptional gene silencing. In contrast, all transgenic lines with transgenes consisting of GFP fused to N gene segments of 24 or 59 bp were susceptible to TSWV, even though the transgene was post-transcriptionally silenced. Thus, virus resistance and post-transcriptional gene silencing were uncoupled when the N gene segment was 59 bp or less. These results provide evidence that multiple virus resistance is possible through the simple strategy of linking viral gene segments to a silencer DNA such as GFP.
The ‘30K’ superfamily of viral movement proteins
Authors: Melcher Ulrich 
Published: August 2015

Relationships among the amino acid sequences of viral movement proteins related to the 30 kDa (‘30K’) movement protein of tobacco mosaic virus – the 30K superfamily – were explored. Sequences were grouped into 18 families. A comparison of secondary structure predictions for each family revealed a common predicted core structure flanked by variable N- and C-terminal domains. The core consisted of a series of β-elements flanked by an α-helix on each end. Consensus sequences for each of the families were generated and aligned with one another. From this alignment an overall secondary structure prediction was generated and a consensus sequence that can recognize each family in database searches was obtained. The analysis led to criteria that were used to evaluate other virus-encoded proteins for possible membership of the 30K superfamily. A rhabdoviral and a tenuiviral protein were identified as 30K superfamily members, as were plant-encoded phloem proteins. Parsimony analysis grouped tubule-forming movement proteins separate from others. Establishment of the alignment of residues of diverse families facilitates comparison of mutagenesis experiments done on different movement proteins and should serve as a guide for further such experiments.
Characterization of the African swine fever virus protein p49: a new late structural polypeptide
Authors: Galindo Inmaculada , Viñuela Eladio , Carrascosa Angel L. 
Published: August 2015

The open reading frame B438L, located within the EcoRI B fragment of the African swine fever virus genome, is predicted to encode a protein of 438 amino acids with a molecular mass of 49·3 kDa. It presents a cell attachment RGD (Arg–Gly–Asp) motif but no other significant similarity to protein sequences in databases. Northern blot and primer extension analysis showed that B438L is transcribed only at late times during virus infection. The B438L gene product has been expressed in Escherichia coli, purified and used as an antigen for antibody production. The rabbit antiserum specific for pB438L recognized a protein of about 49 kDa in virus-infected cell extracts. This protein was synthesized late in infection by all the virus strains tested, was located in cytoplasmic virus factories and appeared as a structural component of purified virus particles.
The UL34 gene product of herpes simplex virus type 2 is a tail-anchored type II membrane protein that is significant for virus envelopment
Authors: Shiba C. , Daikoku T. , Goshima F. , Takakuwa H. , Yamauchi Y. , Koiwai O. , Nishiyama Y. 
Published: August 2015

The UL34 gene of herpes simplex virus type 2 (HSV-2) is highly conserved in the herpesvirus family. The UL34 gene product was identified In lysates of HSV-2-infected cells as protein species with molecular masses of 31 and 32·5 kDa, the latter being a phosphorylated product. Synthesis of these proteins occurred at late times post-infection and was highly dependent on viral DNA synthesis. Immunofluorescence assays revealed that the UL34 protein was localized in the cytoplasm in a continuous net-like structure, closely resembling the staining pattern of the endoplasmic reticulum (ER), in both HSV-2-infected cells and in cells transiently expressing UL34 protein. Deletion mutant analysis showed that this colocalization required the C terminus of the UL34 protein. The UL34 protein associated with virions but not with A, B or C capsids. We treated virions, HSV-2-infected cells and cells expressing the UL34 protein with a protease in order to examine the topology of the UL34 protein. In addition, we constructed UL34 deletion mutant proteins and examined their intracellular localization. Our data strongly support the hypothesis that the UL34 protein is inserted into the viral envelope as a tail-anchored type II membrane protein and is significant for virus envelopment.
Entry of influenza viruses into cells is inhibited by a highly specific protein kinase C inhibitor
Authors: Root Christiana N. , Wills Elizabeth G. , McNair LaShonn L. , Whittaker Gary R. 
Published: August 2015

Following binding to cell surface sialic acid, entry of influenza viruses into cells is mediated by endocytosis. Productive entry of influenza virus requires the low-pH environment of the late endosome for fusion and release of the virus into the cytoplasm and transport of the virus genome into the nucleus. We investigated novel mechanisms to inhibit influenza virus infection using highly specific inhibitors of protein kinase C. We found that one inhibitor, bisindolylmaleimide I, prevented replication of influenza A virus in a dose-dependent manner when added at the time of infection, but had little specific effect when added 2 h after infection had commenced. Virus yields dropped by more than 3 log units in the presence of micromolar levels of bisindolylmaleimide I. Influenza B virus replication was also inhibited by bisindolylmaleimide at micromolar concentrations. We carried out experiments to determine the point in infection that was blocked by bisindolylmaleimide I, and determined that entry of viral ribonucleoproteins (vRNPs) into the nucleus was prevented. Upon drug washout vRNP nuclear entry resumed, showing that bisindolylmaleimide I is reversible. Bisindolylmaleimide I did not affect virus binding and was apparently not acting as a weak base, because its effects were independent of the pH of the external growth medium. These experiments show that bisindolylmaleimide I blocks replication of different types of influenza virus in a dose-dependent and reversible manner, and that virus entry into the cell is inhibited.
The adenovirus E4 ORF6 and E1b 55 kDa proteins cooperate in a p53-independent manner to enhance transduction by recombinant adeno-associated virus vectors
Authors: Trahair Toby N. , Alexander Ian E. , Rowe Peter B. , Smythe Jason A. 
Published: August 2015

The observation that exposure of target cells to genotoxic stress or adenovirus infection enhances recombinant adeno-associated virus (rAAV) transduction is an important lead towards defining the rAAV transduction mechanism, and has significant implications for the exploitation of rAAV in gene therapy applications. The adenovirus-mediated enhancement of rAAV transduction has been mapped to the E4 ORF6 gene, and expression of E4 ORF6 alone has been considered necessary and sufficient to mediate this effect. Since p53 subserves an important function in the cellular response to genotoxic stress, and interacts with the E4 ORF6 gene product during adenovirus infection, we hypothesized that p53 function might be essential to the rAAV enhancement resulting from these cellular insults. In the current study, using the p53-null cell lines H1299 and Saos-2, we find that p53 is not essential to either genotoxic stress or adenovirus-mediated enhancement of rAAV transduction. We further demonstrate using HeLa, H1299 and Saos-2 cells that E4 ORF6 expression alone is not sufficient to enhance rAAV transduction and that coexpression of the adenovirus E1b 55 kDa protein is necessary. Together, these observations indicate that the mechanism by which adenovirus infection enhances rAAV transduction involves cooperative and interdependent functions of the E4 ORF6 and E1b 55 kDa proteins that are p53-independent.
Characterization of two novel Microplitis demolitor polydnavirus mRNAs expressed in Pseudoplusia includens haemocytes
Authors: Trudeau D. , Witherell R. A. , Strand M. R. 
Published: August 2015

The braconid waspMicroplitis demolitorcarriesM. demolitorpolydnavirus (MdPDV) and parasitizes the larval stage of the mothPseudoplusia includens. M. demolitorinjects MdPDV intoP. includenslarvae when it lays an egg and the virus infects various cells including haemocytes. Two new MdPDV transcripts expressed in host haemocytes were characterized in this study. Screening of an MdPDV-infected haemocyte cDNA library identified a 0·4 kb cDNA encoding a predicted protein of 103 amino acids which was named Egf0·4. This protein contained a cysteine-rich epidermal growth factor (EGF)-like motif at its N terminus that was similar to the EGF-like domains in the previously identified MdPDV genesegf1·5andegf1·0. Sequencing of the genomic clone pMd-10 indicated that it contained theegf0·4gene, which consisted of two introns and three exons. This gene was located on MdPDV segment O and appeared to exist in multiple copies. A nucleic acid and expression screen identified a 1·8 kb cDNA encoding a predicted protein of 515 amino acids designated Glc1·8. This protein consisted of a heavily glycosylated central core of six tandemly arranged repeats flanked by hydrophobic N- and C-terminal domains. Northern blotting andin situhybridization studies indicated that bothegf0·4andglc1·8were expressed in MdPDV-infected host haemocytes. Immunocytochemical studies also indicated that Glc1·8 localized to the cell surface.
A novel RNA mycovirus in a hypovirulent isolate of the plant pathogen Diaporthe ambigua
Authors: Preisig O. , Moleleki N. , Smit W. A. , Wingfield B. D. , Wingfield M. J. 
Published: August 2015

Hypovirulent isolates of the fruit tree fungal pathogen Diaporthe ambigua have previously been shown to harbour a double-stranded (ds)RNA genetic element of about 4 kb. In this study, we established the complete cDNA sequence of this dsRNA, which represents a replicative form of a positive-strand RNA virus that we have named D. ambigua RNA virus (DaRV). The nucleotide sequence of the genome is 4113 bp and has a GC content of 53%. Two large ORFs are present in the same reading frame. They are most probably translated by readthrough of a UAG stop codon in the central part of the genome. The longest possible translation product (p125) has a predicted molecular mass of about 125 kDa. A significant homology can be found to the non-structural proteins of carmoviruses of the positive-strand RNA virus family Tombusviridae. These proteins also include the conserved RNA-dependent RNA polymerase (RDRP) domain. In contrast to the genome organization of these plant viruses, no ORF is present at the 3′ end of the DaRV genome that encodes a coat protein. Therefore, it is proposed that DaRV is not encapsidated but that it occurs as RNA–RDRP complexes and/or that it might be associated with cell membranes. Interestingly, six putative transmembrane helices are predicted in the N-terminal part of p56 (translation product of the first ORF, N-terminal part of p125), which might direct and anchor the viral complex to membranes. DaRV is a mycovirus with a unique genome organization and has a distant relationship to the plant virus family Tombusviridae.
Diminished secondary CTL response in draining lymph nodes on cutaneous challenge with herpes simplex virus
Authors: Jones Claerwen M. , Cose Stephen C. , McNally James M. , Jennings Stephen R. , Heath William R. , Carbone Francis R. 
Published: August 2015

We have shown that C57BL/6-derived CD8+CTL specific for an immunodominant herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) determinant express a highly conserved Vβ10/junctional sequence combination. This extreme T cell receptor β-chain bias can be used to track the activation of gB-specific CTL in lymph nodes draining the site of HSV-1 infection. In this report we have examined the accumulation of gB-specific CTL in the primary and secondary or recall CTL responses to HSV-1 infection. We found that gB-specific cytolytic activity present within popliteal lymph nodes draining HSV-infected foot-pads peaked at day 5 post-infection during the primary response. As found previously, this correlates with the accumulation of Vβ10+CD8+CTL in the activated T cell subset. Lymph node-derived cytotoxicity peaked between days 3 and 4 on secondary challenge with virus and, somewhat surprisingly, was considerably below that seen in the primary response. This reduced gB-specific cytolytic activity mirrored a near absence of Vβ10+CD8+T cell enrichment found within the draining lymph nodes during this recall response, consistent with the overall diminution of gB-specific CTL accumulation in this site. Finally, there was a second wave of biased accumulation of Vβ10+CD8+activated T cells within the popliteal lymph nodes well after the resolution of infection in both the primary and secondary responses. These results are discussed in terms of preferential activation of virus-specific memory T cells directly in infected tissues during a secondary CTL response at the expense of draining lymphoid organs.
Molecular characterization of the genome of a partitivirus from the basidiomycete Rhizoctonia solani
Authors: Strauss Ethan E. , Lakshman Dilip K. , Tavantzis Stellos M. 
Published: August 2015

The bisegmented genome of a double-stranded (ds) RNA virus from the fungusRhizoctonia solaniisolate Rhs 717 was characterized. The larger segment, dsRNA 1, is 2363 bases long whereas the smaller segment, dsRNA 2, has 2206 bases. The 5′ ends of the coding strands of dsRNA 1 and dsRNA 2 are highly conserved (100% identity over 47 bases), and contain inverted repeats capable of forming stable stem–loop structures. Analysis of the coding potential of each of the two segments showed that dsRNAs 1 and 2 could code for polypeptides of 730 aa (bases 86–2275; molecular mass 86 kDa) and 683 aa (bases 79–2130; molecular mass 76 kDa), respectively. The 86 kDa polypeptide has all the motifs of dsRNA RNA-dependent RNA polymerases (RDRP), and has significant homology with putative RDRPs of partitiviruses fromFusarium poaeandAtkinsonella hypoxylon. The 76 kDa protein shows homology with the putative capsid proteins (CP) of the same viruses. Northern blot analysis revealed no subgenomic RNA species, consistent with the fact that the long open reading frames encoding the putative RDRP and CP cover the entire length of the respective dsRNAs.
Dengue virus type 1 DNA vaccine induces protective immune responses in rhesus macaques
Authors: Raviprakash Kanakatte , Porter Kevin R. , Kochel Tadeuscz J. , Ewing Daniel , Simmons Monica , Phillips Irving , Murphy Gerald S. , Weiss Walter R. , Hayes Curtis G. 
Published: August 2015

A candidate DNA vaccine expressing dengue virus type 1 pre-membrane and envelope proteins was used to immunize rhesus macaques. Monkeys were immunized intramuscularly (i.m.) or intradermally (i.d.) by three or four 1 mg doses of vaccine, respectively. Monkeys that were inoculated i.m. seroconverted more quickly and had higher antibody levels than those that were inoculated i.d. The sera exhibited virus-neutralizing activity, which declined over time. Four of the eight i.m.-inoculated monkeys were protected completely from developing viraemia when challenged 4 months after the last dose with homologous dengue virus. The other four monkeys had reduced viraemia compared with the control immunized monkeys. The i.d.-inoculated monkeys showed no reduction in viraemia when challenged with the virus. All vaccinated monkeys showed an anamnestic antibody response, indicating that they had established immunological memory. Vaccine-induced antibody had an avidity index similar to that of antibody induced by virus infection; however, no clear correlation was apparent between antibody avidity and virus neutralization titres.
Expression of reporter genes from the defective RNA CD-61 of the coronavirus infectious bronchitis virus
Authors: Stirrups Kathleen , Shaw Kathleen , Evans Sharon , Dalton Kevin , Casais Rosa , Cavanagh David , Britton Paul 
Published: August 2015

The defective RNA (D-RNA) CD-61, derived from the Beaudette strain of the avian coronavirus infectious bronchitis virus (IBV), was used as an RNA vector for the expression of two reporter genes, luciferase and chloramphenicol acetyltransferase (CAT). D-RNAs expressing the CAT gene were demonstrated to be capable of producing CAT protein in a helper-dependent expression system to about 1·6 μg per 106 cells. The reporter genes were expressed from two different sites within the CD-61 sequence and expression was not affected by interruption of the CD-61-specific ORF. Expression of the reporter genes was under the control of a transcription-associated sequence (TAS) derived from the Beaudette gene 5, normally used for the transcription of IBV subgenomic mRNA 5. The Beaudette gene 5 TAS is composed of two tandem repeats of the IBV canonical consensus sequence involved in the acquisition of a leader sequence during the discontinuous transcription of IBV subgenomic mRNAs. It is demonstrated that only one canonical sequence is required for expression of mRNA 5 or for the expression of an mRNA from a D-RNA and that either sequence can function as an acceptor site for acquisition of the leader sequence.
Potato leafroll virus protein P1 contains a serine proteinase domain
Authors: Li X. , Ryan M. D. , Lamb J. W. 
Published: August 2015

The multi-domain potato leafroll virus replicase protein P1 was expressed in insect cells from the polyhedrin promoter of Autographa californica nucleopolyhedrovirus. Using antisera raised against P1, it was shown that P1 was cleaved near the VPg in insect cells in a manner similar to that in plant cells, to produce a ∼27 kDa C-terminal fragment. Furthermore, it was shown that the proposed serine proteinase-like domain within P1 is responsible for this processing and that this can occur in a trans (intermolecular) reaction. Four conserved residues within the serine proteinase domain that are essential for catalysis have been identified, consistent with the proposal that this domain comprises a serine proteinase.
The long terminal repeat is a determinant of cell tropism of maedi-visna virus
Authors: Agnarsdóttir Gudrún , Thorsteinsdóttir Holmfrídur , óskarsson Thórdur , Matthíasdóttir Sigrídur , St. Haflidadóttir Benedikta , Andrésson ólafur S. , Andrésdóttir Valgerdur 
Published: August 2015

Maedi-visna virus (MVV) is a lentivirus of sheep, mainly affecting the lungs and the central nervous system. Long terminal repeat (LTR) sequence variability is common in tissue culture-derived isolates of MVV as well as those of other lentiviruses. The role of this sequence variation in MVV replication has not been explored. PCR amplification of the LTRs of an MVV isolate revealed two product sizes, the larger containing a 53 bp duplication. PCR products containing the two size variants of the LTRs were cloned into an infectious molecular clone of MVV and the resulting chimeric viruses were tested for growth in various cell types. The chimeric virus containing only one copy of the 53 bp sequence was found to grow more slowly in sheep choroid plexus cells, sheep fibroblasts and sheep synovial cells than the virus with the 53 bp duplication. Both viruses grew equally well in macrophages. These results indicate that the LTRs determined the extended cell tropism of MVV.