- Volume 77, Issue 4, 1996
Volume 77, Issue 4, 1996
- Animal
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- DNA viruses
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Identification and characterization of BICP27, an early protein of bovine herpesvirus 1 which may stimulate mRNA 3′ processing
More LessSequence analysis of the left genomic terminus of bovine herpesvirus 1 (BHV-1) revealed two convergently transcribed genes with 3′ ends about 300 bp apart. The gene on the left is the previously described circ gene; that on the right was found to encode a protein of 400 amino acids which was designated BICP27 because of its homology to ICP27 (Vmw63) of herpes simplex virus 1 (HSV-1) and related proteins from other alpha-, beta- and gammaherpesviruses. Rabbit antisera raised against a synthetic oligopeptide representing the amino terminus of the predicted polypeptide demonstrated the presence of BICP27 in the nuclei of infected cells by in situ immunoadsorbent assays. In Western immunoblots, BICP27 was detected as a 50 kDa BHV-1 specific protein expressed with early kinetics, in contrast to HSV-1 ICP27 which is an immediate early (IE) protein. A DNA fragment containing BICP27 coding sequences was inserted into a baculovirus genome. The recombinant BICP27 protein, identified by its reactivity with the antipeptide sera, exhibited the same electrophoretic mobility as BICP27 specified by BHV-1. Transient expression assays using target genes differing only in their poly(A) sites showed that BICP27, like its HSV-1 counterpart, may be involved in 3′ processing of mRNA.
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Murine gammaherpesvirus-induced splenomegaly: a critical role for CD4 T cells
More LessMurine gammaherpesvirus (MHV-68) causes an acute respiratory infection followed by a latent infection in B lymphocytes. In the first 2–3 weeks after infection mice develop a marked splenomegaly, where the spleen cell number increases by 2–3-fold. Cytofluorimetric analysis during splenomegaly revealed an increase in numbers of B lymphocytes and of both CD4+ and CD8+ T lymphocytes. The largest increase relative to uninfected spleens was in the CD8+ population. The number of latently infected cells in the spleen peaked at day 10 post-intraperitoneal infection, then declined to 1/106–1/107 cells per spleen. Depletion of CD4+ T lymphocytes prevented the splenomegaly and greatly reduced the peak infective centre level, while having no effect on the long-term level of latently infected cells. Given the similarity between MHV-68-induced splenomegaly and Epstein-Barr virus-induced infectious mononucleosis, these data highlight the usefulness of MHV-68 as a mouse model for the study of gammaherpesvirus immunology and pathobiology.
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- Insect
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Isolation and characterization of a member of the cysteine-rich gene family from Campoletis sonorensis polydnavirus
More LessThe endoparasitic wasp Campoletis sonorensis injects a symbiotic polydnavirus into its host Heliothis virescens. Viral gene expression protects the wasp egg and larva from encapsulation by host haemocytes. Three related C. sonorensis polydnavirus (CsPDV) genes, which are expressed in parasitized H. virescens, have been previously isolated and grouped into a cysteine-rich gene family. In this report, a CsPDV gene encoding an abundant 1.4 kb mRNA expressed in parasitized insects was isolated and mapped to viral segment V (15.2 kb) by Southern blotting and PCR. The VHv1.4 cDNA is 1338 bp long and has an ORF that encodes 322 amino acids with two complete and one partial cysteine motifs. Similar to other characterized CsPDV cysteine motifs, the VHv1.4 motifs are also characterized by six cysteines at conserved positions and variable inter-cysteine amino acids. DNA sequence analyses show that the VHv1.4 gene shares regions of significant identity (73–97%) with the VHv1.1 gene, a member of the cysteine-rich gene family. The VHv1.4 and the VHv1.1 proteins are 62% identical overall; at the N termini including the signal peptide and the N-terminal cysteine motif the identity is greater (90%). Unlike other CsPDV cysteinerich genes, the VHv1.4 cDNA has two conserved domains (77% identical in nucleotides, 55% identical in amino acids) that presumably result from the duplication of a portion of the gene. The VHv1.4 gene has four introns with splicing sites located at positions similar to VHv1.1 introns. Introns 2 and 3, located in the first and second domains respectively, have greater identity (97%) than the flanking exon sequences (77%). We propose, based on the evidence presented in this paper, that the VHv1.4 gene is a new member of the cysteinerich polydnavirus gene family.
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- Plant
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Purification, characterization, assembly and crystallization of assembled alfalfa mosaic virus coat protein expressed in Escherichia coli
More LessThe coat protein of alfalfa mosaic virus (AMV) was cloned and expressed in Escherichia coli as a fusion protein containing a 37 amino acid extension with a (His)6 region for affinity purification. About half of the expressed recombinant coat protein (rCP) was soluble upon extraction and half was insoluble in inclusion bodies. Western blot analysis confirmed the identity of the rCP and protoplast infectivity assays indicated that the rCP was biologically active in an early event of AMV infection, called genome activation. The rCP assembled into T = 1 empty icosahedral particles, as described previously for native coat protein. Empty particles formed hexagonal crystals that diffracted X-rays to 5.5 Å resolution. The crystals of trypsin-treated particles of rCP appear to be isomorphous with crystals of trypsintreated particles of native coat protein. Spherical particles containing RNA assembled when the rCP was combined with in vitro transcripts of AMV RNA4, the smallest naturally encapsidated AMV RNA. Bacilliform particles that resembled native virions assembled when the rCP was combined with transcripts of RNA1, the largest genomic RNA.
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Genomic reassortment of barley mild mosaic virus: evidence for the involvement of RNA1 in pathogenicity
More LessA reverse transcription-polymerase chain reaction (RT-PCR) was developed for specific detection of RNA1 and RNA2 of two barley mild mosaic virus strains (BaMMV-Ka1 and BaMMV-Na1) and a barley yellow mosaic virus strain (BaYMV-II-1). Mechanical inoculation of barley cultivars with a mixture of BaMMV-Ka1 and BaMMV-Na1, followed by RT-PCR to detect RNA components in infected plants, revealed that the RNAs of the two strains were exchangeable in vivo to generate all nine possible combinations containing at least one RNA1 and one RNA2. Infected plants with mixed or reassorted RNAs were selected and used as inocula for further analysis of cultivar reactions. The results demonstrate that the pathogenicity and symptomatology are determined solely by RNA1. In contrast, following inoculation with mixtures of BaYMV-II-1 and either BaMMV-Ka1 or BaMMV-Na1, no heterologous combinations of their RNAs were observed.
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Cooperative binding to nucleic acids by barley yellow mosaic bymovirus coat protein and characterization of a nucleic acid-binding domain
More LessThe capacity of several coat protein (CP) mutants of a German isolate of barley yellow mosaic bymovirus (BaYMV) to bind to nucleic acids was studied in vitro. Recombinant CP, produced by overexpression in Escherichia coli, was purified from inclusion bodies and subsequently renatured. Binding to single-stranded (ss) RNA and ssDNA oligonucleotides was found to be cooperative and sequence non-specific. By deletion mutagenesis, several truncated CP derivatives were created and their nucleic acid-binding capacity was investigated in order to define a protein domain responsible for RNA- and DNA-binding. The nucleic acid-binding domain consists of a core which was located to an internal 23 amino acid peptide (aa 125–147) and an adjacent domain (aa 148–184) which stimulates binding.
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- Short communication
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Nucleotide sequence analysis of RNA-5 of five isolates of beet necrotic yellow vein virus and the identity of a deletion mutant
More LessThe nucleotide sequences of RNA-5 from two laboratory isolates (D-5 and D-6) and three field isolates (SH1, S43 and R83) of beet necrotic yellow vein virus (BNYVV) were determined. Isolates D-5 and D-6, derived from a D field culture during mechanical inoculation, contained RNA-5 of molecular size 1.4 kb and 1.0 kb, respectively. The sequences of D-5, SH1, S43 and R83 were found to be at least 98% identical and from 1342 to 1347 nucleotides in length, excluding the poly(A) tail. Each contained a single open reading frame (ORF) encoding a 228 amino acid protein with a molecular mass of 26189 Da (P26). The coding sequence was bordered by a long leader of 443 to 448 nucleotides and a 3′-terminal non-coding region of 215 nucleotides. In isolate D-6, containing the smaller ∼ 1.0 kb RNA species referred to as RNA-5a, the ORF had undergone an internal deletion of 303 nucleotides. No sequence identity was found between RNA-5 and either RNA-3 or RNA-4, except for the 5′-terminal nine residues and for approximately the 3′-terminal 200 residues. Thus, the genome organization of BNYVV RNA-5 is very similar to that of RNA-3 and RNA-4, both of which are essential for survival of BNYVV in nature. Although RNA-5 is not essential, it may be associated with symptom expression of BNYVV.
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- Other
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Exposure to autoclaving or sodium hydroxide extends the dose-response curve of the 263K strain of scrapie agent in hamsters
More LessAn analysis was made of incubation period data from experiments in which samples of brain-tissue infected with the 263K strain of scrapie agent were injected intracerebrally into hamsters following exposure of the tissue to autoclaving or sodium hydroxide. Where there was survival of infectivity, this often produced extended mean incubation periods compared with the maximal incubation periods in controls injected with untreated agent. These results confirmed that, after chemical or physical treatment, infectivity titre should not be calculated by comparing the incubation period from a single dilution-group against a standard dose-response curve for untreated agent.
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