We have previously observed that a sequence in coat protein (CP) ORF of Turnip yellow mosaic virus (TYMV) is required for efficient replication of the virus. The sequence was predicted to take a stem-loop structure, thus termed SL2. While examining various SL2 mutants, we observed that all the modifications resulting in extension of translation beyond the CP ORF significantly suppressed subgenomic RNA accumulation. The genomic RNA level, in contrast, was not affected. Introduction of an in-frame stop codon in the CP ORF of these constructs restored the level of subgenomic RNA. Overall, the results suggest that the read-through makes the subgenomic RNA unstable.
Animals ; Brassica napus ; Codon, Terminator ; Ecthyma, Contagious ; RNA ; Tymovirus ; Viruses

Turnip yellow mosaic virus (TYMV) is a non-enveloped icosahedral virus composed of 20 kDa single coat proteins. In this study, we modified the TYMV coat protein (CP) ORF by inserting an oligonucleotide linker corresponding to T7, HSV, Tat, (Arg)9, or (RxR)4 peptide at the 5'-end of the CP ORF and examined its effect on replication, RNA packaging, and virion assembly. The results showed that the constructs containing (Arg)9 and (RxR)4 sequences were barely capable of replication. The TYMV constructs containing T7 and Tat peptide produced virions that co-migrated with wild-type virions. However, the insertion of T7 and Tat sequences impaired genomic RNA (gRNA) accumulation and packaging, respectively. When only the CP gene was expressed, CPs with (Arg)9 or (RxR)4 successfully produced virus-like particles whose mobility was comparable to that of wild type. In the case of CP having a HSV tag, the virion band was not detected, although a sufficient amount of CP was produced. This indicates that CP with the HSV tag failed to assemble into virions. Overall, the results suggest that TYMV replication, RNA packaging and virion assembly are strongly influenced by the insertion sequence.
Animals ; Brassica napus* ; Capsid Proteins ; Ecthyma, Contagious ; Product Packaging* ; RNA* ; Tymovirus* ; Virion*

The primary clue for locating protein-coding regions is the open reading frame and the determination of ORFs (Open Reading Frames) is the first step toward the gene prediction, especially for prokaryotes. In this respect, we have developed a web-based ORF search tool called ORF Miner. The ORF Miner is a graphical analysis utility which determines all possible open reading frames of a selectable minimum size in an input sequence. This tool identifies all open reading frames using alternative genetic codes as well as the standard one and reports a list of ORFs with corresponding deduced amino acid sequences. The ORF Miner can be employed for sequence annotation and give a crucial clue to determination of actual protein-coding regions.
Amino Acid Sequence ; Animals ; Ecthyma, Contagious ; Genetic Code ; Open Reading Frames ; Resin Cements

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA genome. Information for TYMV replication is limited, except that the 3'-terminal sequence and 5'-untranslated region are required for genome replication. When a foreign sequence was inserted at the position upstream of the coat protein (CP) open reading frame (ORF), replication of the recombinant TYMV was comparable to wild type, as long as an RNAi suppressor was provided. In contrast, when the foreign sequence was inserted between the CP ORF and the 3'-terminal tRNA-like structure, replication of the recombinant virus was not detected. This result suggests that the CP ORF contains an essential replication element which should be appropriately spaced with respect to the 3'-end. Analysis of TYMV constructs containing a part or a full additional CP ORF indicates that the 3' quarter of the CP ORF is required for TYMV replication.
Animals ; Brassica napus ; Ecthyma, Contagious ; Genome ; Open Reading Frames ; Plant Viruses ; RNA ; Tymovirus ; Viruses

PURPOSE: Human herpesviruses have been associated with the etiology of several human cancers. The role of these viruses in carcinogenesis has not yet been clarified. This study focused on identifying and characterizing the transforming potential of cloned DNA fragments from human cytomegalovirus (HCMV). MATERIALS AND METHODS: Multiple DNA fragments of HCMV were applied to cells for transformation. Morphological transforming region II (mtrII) of HCMV strain Towne has been identified to a 3.0kb XbaI-BamHI DNA fragment which was retained in transformed cells. The transforming activity was induced by a 980 bp BaII-Xho I subfragment (pBS980) containing both promoter/ regulatory elements as well as three open reading frames (ORFs), i.e., 79ORF, 83ORF, and 34ORF. The ORFs have been evaluated for transforming potential in NIH3T3 cells. RESULTS: MtrII (pBS980) has BglII restriction enzyme site which divides into two subfragments, pBS440 and pBS540, the latter has whole 83ORF, 34ORF, and fragment of 79ORF, the former has only fragment of 79ORF. Among three ORFs, 83ORF and 34ORF were not functional in transformation, because in pBS540 these ORFs were not truncated. CONCLUSION: The 79ORF (79-aa transforming peptide) has allowed a better approach to determine the role of HCMV in human carcinogenesis.
Animals ; Carcinogenesis ; Clone Cells ; Cytomegalovirus* ; DNA ; Ecthyma, Contagious ; Herpesviridae ; Humans* ; Open Reading Frames

HExDB is a database for analyzing exon and splicing pattern information in Homo sapiens. HExDB is useful for specific purposes: 1) to design primers for exon amplification from cDNA and 2) to understand the change of ORFs by alternative splicing. HExDB was constructed by integrating data from AltExtron which is the computationally predicted exon database, Ensemble cDNA annotation, and Affymetrix genome tile published recently. Although it may contain false positive data, HExDB is good starting point due to its sensitivity. At present, there are as many as 2,046,519 exons stored in the HExDB. We found that 16.8% of the exons in the database was constitutive exons and 83.1% were novel gene exons.
Alternative Splicing* ; Animals ; DNA, Complementary ; Ecthyma, Contagious ; Exons* ; Genome ; Humans* ; Open Reading Frames

Turnip yellow mosaic virus (TYMV) is a non-enveloped icosahedral virus that has a single 6.3 kb positive-strand RNA as a genome. Previously, it was observed that the recombinant construct TY-eGFP2, where an eGFP gene was inserted at the position downstream of the coat protein (CP) ORF of TYMV genome, barely replicated. The inhibition of replication was relieved by insertion of an additional copy of the 3' quarter of the CP ORF after the foreign sequence. In this study, we have examined if the 3' quarter of the CP ORF contains any replication elements. M-fold analysis predicted three stem-loop structures in this region. Analysis of the TY-eGFP2 constructs containing one or two of these stem-loop structures indicates that the secondary structure predicted in the region between nt-6139 and nt-6181, termed SL2, is essential for TYMV replication. The critical role of SL2 was confirmed by the observation that deletion of the 3' quarter of the CP ORF from the wild-type TYMV genome nearly abolished replication and that insertion of SL2 into the deletion mutant restored the replication. Mutations disrupting the stem of SL2 greatly reduced viral RNA replication, indicating that the secondary structure is essential for the enhancing activity.
Animals ; Brassica napus ; Coat Protein Complex I ; Ecthyma, Contagious ; Genome ; RNA ; RNA, Viral ; Tymovirus ; Viruses

Adult ; Animals ; Ecthyma, Contagious ; diagnosis ; virology ; Female ; Humans ; Microscopy, Electron, Transmission ; methods ; Young Adult

Varicella-zoster virus (VZV) is a causative agent for shingles and herpes zoster. The genomes of VZV contain five reiteration (R) sequences and an origin of replication (ORI) sequences composed of tandem repeats whose numbers vary among different strains. Variation of the genome lengths among VZV strains could be attributed by the lengths of R sequences. There was a strong correlation between the lengths of VZV genome and R sequences, while variation of ORI did not contribute the variation of VZV genome length. The high G+C contents of The R sequences in ORF11, 14 and 22 influenced the codon usage of VZV in these ORFs. None of the most frequent 5 codons in R sequences was included in the top 5 most frequent codon in ORF11-14-22 or VZV genome, and vice versa.
Animals ; Base Composition ; Codon ; Ecthyma, Contagious ; Genome ; Herpes Zoster ; Herpesvirus 3, Human* ; Open Reading Frames ; Replication Origin ; Tandem Repeat Sequences

Norovirus (NoV), which belongs to the family Caliciviridae, is one of the major causes of nonbacterial acute gastroenteritis in the world. In this study, we purified proteins from the epitope region of norovirus for development of the rapid diagnosis system using polyclonal antibodies. As antigens, parts of the ORF (open reading frame) 2, ORF2-P domain, ORF2-Epi, and ORF3 regions were selected and their expressions were induced. The antigenicity of the purified proteins was identified by Western blotting. Each of the purified proteins was injected into mice for the production of novel antibodies and after 3 months of immunization, sera from the mice were obtained. The polyclonal antibody titer was tested by enzyme-linked immunosorbent assay (ELISA) and antibody against ORF2-Epi showed the highest titer. Those polyclonal antibodies can be used in further immunoassay for the rapid detection of NoVs from food and clinical specimens.
Animals ; Antibodies ; Blotting, Western ; Caliciviridae ; Ecthyma, Contagious ; Enzyme-Linked Immunosorbent Assay ; Gastroenteritis ; Humans ; Immunization ; Immunoassay ; Mice ; Norovirus ; Proteins