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      RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp


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          Gill-associated virus (GAV) infects Penaeus monodon shrimp and is the type species okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs) using His 6-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N) protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ss)RNAs tested independent of their sequence. The ssRNAs included (+) and (−) sense regions of the GAV genome as well as a (+) sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (ds)RNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa) N-terminal sequence spanning Met 11 to Arg 60. Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M 11PVRRPLPPQPPRNARLI 29) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domain might also play some role in protein/protein interactions stabilizing the helical structure of GAV nucleocapsids.

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          Specific interaction between coronavirus leader RNA and nucleocapsid protein.

          Northwestern blot analysis in the presence of competitor RNA was used to examine the interaction between the mouse hepatitis virus (MHV) nucleocapsid protein (N) and virus-specific RNAs. Our accompanying article demonstrates that anti-N monoclonal antibodies immunoprecipitated all seven MHV-specific RNAs as well as the small leader-containing RNAs from infected cells. In this article we report that a Northwestern blotting protocol using radiolabeled viral RNAs in the presence of host cell competitor RNA can be used to demonstrate a high-affinity interaction between the MHV N protein and the virus-specific RNAs. Further, RNA probes prepared by in vitro transcription were used to define the sequences that participate in such high-affinity binding. A specific interaction occurs between the N protein and sequences contained with the leader RNA which is conserved at the 5' end of all MHV RNAs. We have further defined the binding sites to the area of nucleotides 56 to 65 at the 3' end of the leader RNA and suggest that this interaction may play an important role in the discontinuous nonprocessive RNA transcriptional process unique to coronaviruses.
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            Sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes

            The 3′ end of the 20-kb genome of the Mebus strain of bovine enteric coronavirus (BCV) was copied into cDNA and cloned into the PstI site of the pUC9 vector. Four clones from the 3′ end of the genome were sequenced either completely or in part to determine the sequence of the first 2451 bases. Within this sequence were identified, in order, a 3′-noncoding region of 291 bases, the gene for a 448-amino acid nucleocapsid protein (N) having a molecular weight of 49,379, and the gene for a 230-amino acid matrix protein (M) having a molecular weight of 26,376. A third large open reading frame is contained entirely within the N gene sequence but is positioned in a different reading frame; it potentially encodes a polypeptide of 207 amino acids having a molecular weight of 23,057. A higher degree of amino acid sequence homology was found between the M proteins of BCV and MHV (87%) than between the N proteins (70%). For the M proteins of BCV and MHV, notable differences were found at the amino terminus, the most probable site of O-glycosylation, where the sequence is N-Met-Ser-Ser-Val-Thr-Thr for BCV and N-Met-Ser-Ser-Thr-Thr for MHV. BCV apparently uses two of its six potential O-glycosylation sites.
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              Identification and characterization of a coronavirus packaging signal.

              Previously, a mouse hepatitis virus (MHV) genomic sequence necessary for defective interfering (DI) RNA packaging into MHV particles (packaging signal) was mapped to within a region of 1,480 nucleotides in the MHV polymerase gene by comparison of two DI RNAs. One of these, DIssF, is 3.6 kb in size and exhibits efficient packaging, whereas the other, DIssE, which is 2.3 kb, does not. For more precise mapping, a series of mutant DIssF RNAs with deletions within this 1,480-nucleotide region were constructed. After transfection of in vitro-synthesized mutant DI RNA in MHV-infected cells, the virus product was passaged several times. The efficiency of DI RNA packaging into MHV virions was then estimated by viral homologous interference activity and by analysis of intracellular virus-specific RNAs and virion RNA. The results indicated that an area of 190 nucleotides was necessary for packaging. A computer-generated secondary structural analysis of the A59 and JHM strains of MHV demonstrated that within this 190-nucleotide region a stable stem-loop of 69 nucleotides was common between the two viruses. A DIssE-derived DI DNA which had these 69 nucleotides inserted into the DIssE sequence demonstrated efficient DI RNA packaging. Site-directed mutagenic analysis showed that of these 69 nucleotides, the minimum sequence of the packaging signal was 61 nucleotides and that destruction of the secondary structure abolished packaging ability. These studies demonstrated that an MHV packaging signal was present within the 61 nucleotides, which are located on MHV genomic RNA 1,381 to 1,441 nucleotides upstream of the 3' end of gene 1.

                Author and article information

                Role: Editor
                PLoS One
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                3 August 2011
                : 6
                : 8
                : e22156
                [1 ]CSIRO Livestock Industries, Queensland Bioscience Precinct, St. Lucia, Queensland, Australia
                [2 ]CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, Victoria, Australia
                [3 ]National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Thailand Science Park, Klong Luang, Patumthani, Thailand
                [4 ]CENTEX SHRIMP, Faculty of Science, Mahidol University, Bangkok, Thailand
                Queensland Institute of Medical Research, Australia
                Author notes

                Conceived and designed the experiments: CS JAC PJW. Performed the experiments: CS. Analyzed the data: CS JAC PJW WPM. Contributed reagents/materials/analysis tools: JAC PJW. Wrote the paper: CS JAC PJW WPM.

                Soowannayan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                : 21 November 2010
                : 20 June 2011
                Page count
                Pages: 9
                Research Article
                Nucleic Acids
                RNA processing
                DNA-binding proteins
                Gene Expression
                RNA processing
                Viral Classification
                RNA viruses
                Viral Replication
                Viral Nucleic Acid
                Viral Packaging
                Viral Structure



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