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The phosphoprotein gene of a dolphin morbillivirus isolate exhibits genomic variation at the editing site
G Bolt1, S Alexandersen, M Blixenkrone-Møller
1Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark.
Abstract:
The nucleotide sequence of the phosphoprotein (P) gene of a dolphin morbillivirus (DMV) isolate was determined. Like those of other morbilliviruses the DMV P gene encoded P and C proteins in overlapping open reading frames and V protein by editing the P gene transcript. Among P mRNA based clones the editing site variants GGGC, GGGG, GAGC and GGGGGGC predicting a P protein, and the variants GGGGC and GGGGGG predicting a V protein, were found. Surprisingly, the three variants GGGC, GGGG and GAGC were also found among clones generated from genomic RNA of the DMV isolate. Thus, more than one viral genome type appeared to be present in cells infected with the DMV isolate. By a similar analysis of the virus genomes in the tissue from which the DMV isolate was obtained, only the GGGC type was found, indicating that the GGGG and GAGC types arose during adaptation of the virus to growth in cell cultures. No editing site variants likely to have arisen by editing the GAGC type were encountered, and it remains ot be determined whether mRNA encoding V protein can be transcribed from genomes with this editing site. Using antisera raised against the common N terminus and unique C termini of the predicted P and V proteins, the in vivo expression of these proteins was demonstrated.
Insights
Dolphin morbillivirus (DMV) P gene analysis revealed multiple genome types in cell culture, with variants arising during adaptation. In vivo expression of P and V proteins was confirmed, but the role of GAGC variants requires further study.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Morbilliviruses, including dolphin morbillivirus (DMV), possess a phosphoprotein (P) gene encoding multiple proteins.
- The P gene is known to produce P, C, and V proteins through overlapping reading frames and RNA editing.
Purpose of the Study:
- To determine the nucleotide sequence of the DMV P gene.
- To investigate the P gene variants and their expression in DMV.
- To understand the origin of different P gene variants during virus adaptation.
Main Methods:
- Nucleotide sequencing of the DMV P gene from both cell culture isolates and original tissue.
- Analysis of P mRNA and genomic RNA clones to identify editing site variants.
- Western blot analysis using specific antisera to detect in vivo protein expression.
Main Results:
- The DMV P gene encodes P, C, and V proteins, with V protein production via editing.
- Multiple P gene editing site variants (GGGC, GGGG, GAGC, GGGGGGC, GGGGC, GGGGGG) were identified.
- Unexpectedly, GGGC, GGGG, and GAGC variants were found in genomic RNA, suggesting mixed viral populations.
- The GGGG and GAGC variants appear to arise during cell culture adaptation, as only GGGC was found in the original tissue.
- In vivo expression of predicted P and V proteins was confirmed.
Conclusions:
- DMV exhibits genetic diversity in its P gene, with evidence of mixed genome types in cell culture.
- Cell culture adaptation can lead to the emergence of specific P gene variants.
- Further research is needed to determine if the GAGC variant can be edited to produce V protein.
- The study confirms the in vivo expression of DMV P and V proteins.