- Volume 92, Issue 1, 2011
Volume 92, Issue 1, 2011
- Animal
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- DNA viruses
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Lysine residues of interferon regulatory factor 7 affect the replication and transcription activator-mediated lytic replication of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8
More LessKaposi's sarcoma-associated herpesvirus (KSHV) infection goes through latent and lytic phases, which are controlled by the viral replication and transcription activator (RTA). Upon KSHV infection, the host responds by suppressing RTA-activated lytic gene expression through interferon regulatory factor 7 (IRF-7), a key regulator of host innate immune response. Lysine residues are potential sites for post-translational modification of IRF-7, and were suggested to be critical for its activity. In this study, we analysed the 15 lysine residues for their effects on IRF-7 function by site-directed mutagenesis. We found that some mutations affect the ability of IRF-7 to activate interferon (IFN)-α1 and IFN-β promoters, to suppress RTA-mediated lytic gene expression and to repress KSHV reactivation and lytic replication. However, other mutations affect only a subset of these four functions. These findings demonstrate that the lysine residues of IRF-7 play important roles in mediating IFN synthesis and modulating viral lytic replication.
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Covalent modification by SUMO is required for efficient disruption of PML oncogenic domains by Kaposi's sarcoma-associated herpesvirus latent protein LANA2
The multifunctional Kaposi's sarcoma-associated herpesvirus (KSHV) latent protein latency-associated nuclear antigen 2 (LANA2) has a critical role in KSHV-induced B-cell malignancies. LANA2 increases the level of small ubiquitin-like modifier (SUMO)2-ubiquitin-modified PML and induces the disruption of PML oncogenic domains (PODs) by a process that requires a non-covalent SUMO interaction domain (SIM) in LANA2. We now demonstrate that LANA2 is covalently conjugated to SUMO1 and SUMO2 both in vitro and in latently KSHV-infected B-cells. We show that a LANA2 SIM mutant exhibits a slightly altered sumoylation pattern, which suggests that non-covalent SUMO interactions represent a mechanism for determining SUMO substrate recognition and modification. In addition, several lysine residues were mapped as SUMO conjugation sites. A sumoylation-deficient mutant shows impaired ability to induce disruption of PODs, which suggests that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interaction between LANA2 and other SUMO-modified or SUMO-interacting proteins required for disruption of PODs.
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Intraventricular injection of myxoma virus results in transient expression of viral protein in mouse brain ependymal and subventricular cells
More LessOncolytic viruses that selectively infect and lyse cancer cells have potential as therapeutic agents. Myxoma virus, a poxvirus that is known to be pathogenic only in rabbits, has not been reported to infect normal tissues in humans or mice. We observed that when recombinant virus was injected directly into the lateral ventricle of the mouse brain, virally encoded red fluorescent protein was expressed in ependymal and subventricular cells. Cells were positive for nestin, a marker of neural stem cells. Rapamycin increased the number of cells expressing the virally encoded protein. However, protein expression was transient. Cells expressing the virally encoded protein did not undergo apoptosis and the ependymal lining remained intact. Myxoma virus appears to be safe when injected into the brain despite the transient expression of virally derived protein in a small population of periventricular cells.
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Mass identification of transcriptional units expressed from the Bombyx mori nucleopolyhedrovirus genome
In order to identify the transcriptional units expressed from an entire nucleopolyhedrovirus (NPV) genome during infection, we constructed a full-length-enriched cDNA library from Bombyx mori NPV (BmNPV)-infected BmN cells. We randomly sequenced 11 520 clones from both ends to obtain a total of 4679 BmNPV-derived transcriptional units. The data revealed a number of novel transcripts, including putative non-coding RNAs, most of which are expressed from recognized baculovirus early or late promoter motifs. These findings provide new insights into the complex transcriptional regulation of an NPV genome and suggest roles for as-yet-uncharacterized transcripts.
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- Plant
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An RGG sequence in the replication-associated protein (Rep) of Tomato yellow leaf curl Sardinia virus is involved in transcriptional repression and severely impacts resistance in Rep-expressing plants
Truncated versions of the replication-associated protein (Rep) of Tomato yellow leaf curl Sardinia virus (TYLCSV) can interfere with various viral functions and the N-terminal 130 aa are sufficient for strongly inhibiting C1-gene transcription and virus replication and confer resistance in transgenic plants. In this work, we analysed the relevance of an RGG sequence at aa 124–126, highly conserved in begomoviruses, in these inhibitory functions as well as in the subcellular localization of Rep. Although no role of this RGG sequence was detected by cell fractionation and immunogold labelling in Rep localization, this sequence appears relevant for the transcriptional control of the C1-gene and for the inhibition of viral replication and dramatically impacts resistance in transgenic plants. These results are discussed in the context of the model of Rep-mediated resistance against TYLCSV.
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Evolutionary analysis of tomato Sw-5 resistance-breaking isolates of Tomato spotted wilt virus
More LessTomato spotted wilt virus (TSWV) causes severe economic losses in many crops worldwide and often overcomes resistant cultivars used for disease control. Comparison of nucleotide and amino acid sequences suggested that tomato resistance conferred by the gene Sw-5 can be overcome by the amino acid substitution C to Y at position 118 (C118Y) or T120N in the TSWV movement protein, NSm. Phylogenetic analysis revealed that substitution C118Y has occurred independently three times in the studied isolates by convergent evolution, whereas the substitution T120N was a unique event. Analysis of rates of non-synonymous and synonymous changes at individual codons showed that substitution C118Y was positively selected.
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- Other Agents
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Differentiation of ruminant transmissible spongiform encephalopathy isolate types, including bovine spongiform encephalopathy and CH1641 scrapie
More LessWith increased awareness of the diversity of transmissible spongiform encephalopathy (TSE) strains in the ruminant population, comes an appreciation of the need for improved methods of differential diagnosis. Exposure to bovine spongiform encephalopathy (BSE) has been associated with the human TSE, variant Creutzfeldt–Jakob disease, emphasizing the necessity in distinguishing low-risk TSE types from BSE. TSE type discrimination in ruminants such as cattle, sheep, goats and deer, requires the application of several prion protein (PrP)-specific antibodies in parallel immunochemical tests on brain homogenates or tissue sections from infected animals. This study uses in a single incubation step, three PrP-specific antibodies and fluorescent Alexa dye-labelled anti-mouse Fabs on a Western blot. The usual amount of brain tissue needed is 0.5 mg. This multiplex application of antibodies directed towards three different PrP epitopes enabled differential diagnosis of all established main features of classical scrapie, BSE and Nor98-like scrapie in sheep and goats, as well as the currently known BSE types C, H and L in cattle. Moreover, due to an antibody-dependent dual PrP-banding pattern, for the first time CH1641 scrapie of sheep can be reliably discriminated from the other TSE isolate types in sheep.
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- Phage
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The complete genome sequence and proteomics of Yersinia pestis phage Yep-phi
Yep-phi, a lytic phage of Yersinia pestis, was isolated in China and is routinely used as a diagnostic phage for the identification of the plague pathogen. Yep-phi has an isometric hexagonal head containing dsDNA and a short non-contractile conical tail. In this study, we sequenced the Yep-phi genome (GenBank accession no. HQ333270) and performed proteomics analysis. The genome consists of 38 616 bp of DNA, including direct terminal repeats of 222 bp, and is predicted to contain 45 ORFs. Most structural proteins were identified by proteomics analysis. Compared with the three available genome sequences of lytic phages for Y. pestis, the phages could be divided into two subgroups. Yep-phi displays marked homology to the bacteriophages Berlin (GenBank accession no. AM183667) and Yepe2 (GenBank accession no. EU734170), and these comprise one subgroup. The other subgroup is represented by bacteriophage ΦA1122 (GenBank accession no. AY247822). Potential recombination was detected among the Yep-phi subgroup.
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Volumes and issues
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