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At least 235 records · Page 13Linked to original sources
PubMed1992

Identification and mapping of origins of DNA replication within the DNA sequences of the genome of insect iridescent virus type 6.

The origins of DNA replication of the genome (209 kbp) of Chilo iridescent virus (CIV), which is circularly permuted and terminally redundant, were identified. The defined genomic library of CIV, which represents 100% of DNA sequences of the viral genome (e.g., all 32 EcoRI CIV DNA fragments), was used for transfection of Choristoneura fumiferana insect cell cultures (CF-124) that were previously infected with CIV. The plasmid rescue experiments were carried out to select those recombinant plasmids that were amplified during viral replication in CIV-infected cell cultures. It was found that six recombinant plasmids harboring the EcoRI DNA fragments C [13.5 kbp, 0.909-0.974 map units (m.u.)], H (9.8 kbp, 0.535-0.582 m.u.), M (7.25 kbp, 0.310-0.345 m.u.), O (6.5 kbp, 0.196-0.228 m.u.), Q (5.9 kbp, 0.603-0.631 m.u.), and Y (2.0 kbp, 0.381-0.391 m.u.) were able to be amplified under the conditions used. This indicates that the CIV genome possesses six DNA replication origins. Subclones of the EcoRI CIV DNA fragments C and H were screened under the same conditions. It was found that DNA sequences within the EcoRI DNA fragments C and H at the genome coordinates 0.924-0.930 and 0.535-0.548, respectively, contain origins of viral DNA replication. The DNA nucleotide sequences of the EcoRI CIV DNA fragment Y (1986 bp) were determined for identifying the DNA sequence of the corresponding origin of DNA replication. The computer-aided analysis revealed the presence of a 15-mer inverted repeat at nucleotide positions 661-675 and 677-691 (661-TAAATTTAATGAGAA-G-TTCTCATTAAATTTA-692). The analysis of the DNA sequence of the EcoRI DNA fragment H corresponding to the particular region at the genome coordinates 0.535-0.548 (1) showed that this region contains a 16-mer inverted repeat at the nucleotide positions 1315 and 1332 (1315-TAAATTTTAATGGTTA-A-TAACCATTAAAATTTA-1347), which is very similar to the inverted repetition found within the EcoRI DNA fragment Y. The successful recognition and amplification of the single-stranded synthetic DNA sequences of both strands of CIV-ori-Y (nucleotide position 661-691) using phage M13 system in CIV-infected cells is strong evidence that the CIV-ori-Y is bidirectionally active, and this DNA sequence is considered to be the origin of DNA replication within the EcoRI CIV DNA fragment Y.

Base Sequence↗
PubMed2003

[Fusion expression of the ORF5 gene of porcine reproductive and respiratory syndrome virus in insect cells].

A 0.75kb fragment containing the complete cDNA of glutathione-S-transferase (GST) and a modified thrombin cleavage sites were amplified and cloned into pFastBacl, the donor plasmid of Bac-to-Bac baculovirus expression systems, downstream from the polyhedrin promoter (pPolh), resulting in the GST fusion transposition plasmid pFGST. After transposition and transfection, SDS-PAGE analysis showed that the developed GST fusion expression system can highly express GST. The signal sequences of the major structural protein gene ORF5 of porcine reproductive and respiratory syndrome virus (PRRSV) strain YA was removed by PCR and the truncated ORF5 gene was inserted into pFGST and fused with GST to generate the transposition plasmid expressing GST-ORF5 fusion protein. The recombinant baculovirus rvBacGST53 was obtained by transposition and transfection. GST-ORF5 fusion protein was identified by SDS-PAGE and Western blot. The fusion protein is 45kD and is specific to the monoclonal antibody against the E protein of PRRSV. The fusion protein was directly injected into mice and the anti-sera can react with the PRRSV-infected MARC-145 cells by indirect immunofluorescent assay (IFA), indicating that the fusion protein have better immunogenicity.

Animals↗
PubMed1992

The complete nucleotide sequence of cell fusing agent (CFA): homology between the nonstructural proteins encoded by CFA and the nonstructural proteins encoded by arthropod-borne flaviviruses.

Cell fusing agent (CFA) is an RNA virus originally isolated from a line of Aedes aegypti mosquito cells. Although our characterization of the virus many years ago showed that it resembled the flaviviruses, there was no detectable serological cross-reaction with members of the genus flavivirus. Furthermore, unlike the well-studied members of the genus flavivirus, CFA did not replicate in any of several vertebrate cell lines tested. We have now determined the nucleotide sequence of the CFA genome. Comparison of the predicted amino acid sequence of the CFA polyprotein with viral protein sequences in Genbank, has made it apparent that CFA should now be assigned to the family Flaviviridae, genus flavivirus. The homology between CFA proteins and those of other flaviviruses was highest for NS5 (45%) and NS3 (34%). Little homology was found for the structural proteins. Thus, CFA is only distantly related to the other flaviviruses for which there is sequence information; nevertheless, with respect to their hydrophobicity plots, the CFA polyprotein and the polyproteins of other flaviviruses are remarkably similar. We suggest that CFA is an insect virus, which was present in the embryos from which the Ae. aegypti cell line was established. Thus, CFA seems to be the first member of the family Flaviviridae, genus flavivirus, to be identified as an insect virus.

Amino Acid Sequence↗
PubMed2005

A cross-kingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry.

Rhopalosiphum padi virus (RhPV) is an insect virus of the Dicistroviridae family. Recently, the 579-nucleotide-long 5' untranslated region (UTR) of RhPV has been shown to contain an internal ribosome entry site (IRES) that functions efficiently in mammalian, plant, and insect in vitro translation systems. Here, the mechanism of action of the RhPV IRES has been characterized by reconstitution of mammalian 48S initiation complexes on the IRES from purified components combined with the toeprint assay. There is an absolute requirement for the initiation factors eIF2 and eIF3 and the scanning factor eIF1 to form 48S complexes on the IRES. In addition, eIF1A, eIF4F (or the C-terminal fragment of eIF4G), and eIF4A strongly stimulated the assembly of this complex, whereas eIF4B had no effect. Although the eIF4-dependent pathway is dominant in the RhPV IRES-directed cell-free translation, omission of either eIF4G or eIF4A or both still allowed the assembly of 48S complexes from purified components with approximately 23% of maximum efficiency. Deletions of up to 100 nucleotides throughout the 5'-UTR sequence produced at most a marginal effect on the IRES activity, suggesting the absence of specific binding sites for initiation factors. Only deletion of the U-rich unstructured 380-nucleotide region proximal to the initiation codon resulted in a complete loss of the IRES activity. We suggest that the single-stranded nature of the RhPV IRES accounts for its strong but less selective potential to bind key mRNA recruiting components of the translation initiation apparatus from diverse origins.

5' Untranslated Regions↗
PubMed2005

Kelp fly virus: a novel group of insect picorna-like viruses as defined by genome sequence analysis and a distinctive virion structure.

The complete genomic sequence of kelp fly virus (KFV), originally isolated from the kelp fly, Chaetocoelopa sydneyensis, has been determined. Analyses of its genomic and structural organization and phylogeny show that it belongs to a hitherto undescribed group within the picorna-like virus superfamily. The single-stranded genomic RNA of KFV is 11,035 nucleotides in length and contains a single large open reading frame encoding a polypeptide of 3,436 amino acids with 5' and 3' untranslated regions of 384 and 343 nucleotides, respectively. The predicted amino acid sequence of the polypeptide shows that it has three regions. The N-terminal region contains sequences homologous to the baculoviral inhibitor of apoptosis repeat domain, an inhibitor of apoptosis commonly found in animals and in viruses with double-stranded DNA genomes. The second region contains at least two capsid proteins. The third region has three sequence motifs characteristic of replicase proteins of many plant and animal viruses, including a helicase, a 3C chymotrypsin-like protease, and an RNA-dependent RNA polymerase. Phylogenetic analysis of the replicase motifs shows that KFV forms a distinct and distant taxon within the picorna-like virus superfamily. Cryoelectron microscopy and image reconstruction of KFV to a resolution of 15 A reveals an icosahedral structure, with each of its 12 fivefold vertices forming a turret from the otherwise smooth surface of the 20-A-thick capsid. The architecture of the KFV capsid is unique among the members of the picornavirus superfamily for which structures have previously been determined.

Amino Acid Sequence↗
PubMed1992

Characterization of the third origin of DNA replication of the genome of insect iridescent virus type 6.

The structure of the third origin of DNA replication (CIV-ori-M) of the genome (209 kbp) of Chilo iridescent virus (CIV) was determined by DNA nucleotide sequence analysis. The CIV-ori-M is located within the DNA sequences of the EcoRI CIV DNA fragment M (7 kbp; 0.310-0.345 viral map units) between the genome coordinates 0.310 (EcoRI site) and 0.317 (NcoI site). The DNA nucleotide sequence of the EcoRI/NcoI CIV DNA fragment (1601 bp) was determined for identifying the DNA sequence of the corresponding origin of DNA replication. The analysis of the DNA sequences of this region revealed the presence of a 12-mer inverted repeat at nucleotide positions 485-496 and 503-513 (485-AGATATTTGACT-496-TATGT-503-AGTCAAATATCT-513) that are able to form a hairpin-loop structure. A double-stranded DNA fragment was synthesized that corresponds to the nucleotide positions 485-513 that were cloned into the phages M13mp18 and M13mp19, and were screened for their ability to be amplified in CF-124 cell cultures infected with CIV. The successful amplification of the DNA sequence of the CIV-ori-M is strong evidence that this particular region of the CIV genome indeed serves as the origin of DNA replication.

Base Sequence↗
PubMed1987

Molecular cloning and physical mapping of the genome of insect iridescent virus type 6: further evidence for circular permutation of the viral genome.

A defined and complete gene library of the Chilo iridescent virus (CIV) genome was established. The CIV DNA was cleaved with restriction endonucleases EcoRI, NcoI, SphI, and BamHI or double digested with BamHI/SalI and the resulting DNA fragments were inserted into the corresponding sites of the bacterial vectors pACYC184, pKm2, pL-ES-C3, and pAT153 using T4 DNA ligase. All cloned fragments were identified by digestion of the recombinant plasmids with different restriction enzymes and checked by hybridization of recombinant plasmid to viral DNA. This analysis revealed that sequences representing 100% of the viral genome were cloned into the EcoRI site of pACYC184. Although the CIV genome is linear, all 32 EcoRI fragments have been cloned directly. This suggests that the CIV genome is circularly permuted. In addition, NcoI(72%), SphI(40.7%), BamHI (11.6%), and BamHI/SalI(39.7%) DNA fragments of the viral genome were inserted into the corresponding sites of pKm2, pL-ES-C3, and pAT153, respectively. The physical map of the viral genome was constructed using the established gene library for restriction enzymes ApaI, BamHI, EcoRI, NcoI, SalI, and SmaI. Although the CIV genome is linear, this analysis revealed that the restriction maps of the viral genome are circular. This finding supports the hypothesis that the CIV genome is circularly permuted.

Cloning, Molecular↗
PubMed1998

Evolution of viral DNA-dependent DNA polymerases.

DNA viruses as their host cells require a DNA-dependent DNA polymerase (Pol) to faithfully replicate their genomic information. Large eukaryotic DNA viruses as well as bacterial viruses encode a specific Pol equipped with a proofreading 3'-5'-exonuclease, and other replication proteins. All known viral Pol belong to family A and family B Pol. Common to all viral Pol is the conservation of the 3'-5'-exonuclease domain manifested by the three sequence motifs Exo I, Exo II, and Exo III. The polymerase domain of family A and B Pol is clearly distinguishable. Family A Pol share 9 distinct consensus sequences, only two of them are convincingly homologous to sequence motif B of family B Pol. The putative sequence motifs A, B, and C of the polymerase domain are located near the C-terminus in family A Pol and more central in family B Pol. Thus, family A Pol show a significant greater spacing between the Exo III motif and the Pol motif A that is especially extended in the case of the mitochondrial Pol gamma. From each host and virus family whenever possible the consensus sequences of two distantly related polymerase species were aligned for assessment of phylogenetic trees, using both maximum parsimony and distance methods, and evaluated by bootstrap analysis. Three alternative methods yielded trees with identical major groupings. A subdivision of viral family B Pol was achieved resulting in a branch with Pol carrying out a protein-primed mechanism of DNA replication, including adenoviruses, bacteriophages and linear plasmids of plant and fungal origin. Archaebacterial Pol and cellular Pol epsilon were consistently found at the base of this branch. Another major branch comprised alpha- and delta-like viral Pol from mammalian herpesviruses, fish lymphocystis disease virus, insect ascovirus, and chlorella virus. Due to a lower branch integrity Pol of T-even bacteriophages, poxviruses, African swine fever virus, fish herpesvirus, and baculoviruses were not clearly resolved and placed in alternate groupings. A composite and rooted tree of family A and B Pol shows that viral Pol with a protein-priming requirement represent the oldest viral Pol species suggesting that the protein-primed mechanism is one of the earliest modes of viral DNA replication.

Consensus Sequence↗
PubMed1993

Identification of the gene encoding the major capsid protein of insect iridescent virus type 6 by polymerase chain reaction.

The gene encoding the major capsid protein of Chilo iridescent virus (CIV) has been identified by PCR using oligonucleotide primers corresponding to different regions of the major capsid proteins of Tipula iridescent virus (TIV) and iridescent virus 22 (IV22). A DNA fragment of 0.5 kbp was amplified using two oligonucleotide primers corresponding to the amino acid positions 146 to 153 and 304 to 313 of the major capsid protein of TIV, respectively. The radioactively labelled DNA fragment derived from PCR was hybridized to a CIV gene library. This analysis revealed that only the EcoRI CIV DNA fragment X [2.85 kbp; 0.589 to 0.603 viral map units (m.u.)] hybridized to the amplified DNA fragment. An RNA transcript of about 1.5 kb was identified when the PCR product was used as a hybridization probe. The same RNA transcript was detected when the EcoRI fragments X and Q (5.9 kbp; 0.603 to 0.631 viral m.u.) were used as probes. This indicates that the expected gene is located within map coordinates 0.589 to 0.631 and harbours part of the DNA sequences of fragments Q and X. The analysis of the DNA sequences of this particular region of the CIV genome revealed the presence of one open reding frame of 1401 bp. The DNA sequences of this region encode a protein of 467 amino acid residues with an M(r) of 51.4K. A high degree (64.7%) of amino acid sequence identity was detected between the major capsid protein of TIV and/or IV22 and the amino acid composition of the identified CIV protein.

Amino Acid Sequence↗