Investigation of a unique short open reading frame within the 3' untranslated region of the canine distemper virus matrix messenger RNA.
Increasing evidence suggest that the long "untranslated" region (UTR) between the matrix (M) and the fusion (F) proteins of morbilliviruses has a functional role. In canine distemper virus (CDV), the F 5' UTR was recently shown to code for a long F signal peptide (Fsp). Subsequently, it was reported that the M/F UTRs combined with the long Fsp were synergistically regulating the F mRNA and protein expression, thereby modulating virulence. Unique to CDV, a short putative open reading frame (ORF) has been identified within the wild-type CDV-M 3' UTR (termed M2). Here, we investigated whether M2 was expressed from the genome of the virulent and demyelinating A75/17-CDV strain. An expression plasmid encoding the M2 ORF tagged both at its N-terminal (HA) and C-terminal domains (RFP), was first constructed. Then, a recombinant virus with its putative M2 ORF replaced by HA-M2-RFP was successfully recovered from cDNA (termed recA75/17(green)-HA-M2-RFP). M2 expression in cells transfected or infected with these mutants was studied by immunoprecipitation, immunofluorescence, immunoblot and flow cytometry analyses. Although fluorescence was readily detected in HA-M2-RFP-transfected cells, absence of red fluorescence emission in several recA75/17(green)-HA-M2-RFP-infected cell types suggested lack of M2 biosynthesis, which was confirmed by the other techniques. Consistent with these data, no functional role of the short polypeptide was revealed by infecting various cell types with HA-M2-RFP over-expressing or M2-knockout recombinant viruses. Thus, in sharp contrast to the CDV-F 5' UTR reported to translate a long Fsp, our data provided evidence that the CDV-M 3' UTR does not express any polypeptides.
Wiener
Dominique
D
Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Bremgartenstrasse 109a, 3001 Bern, Switzerland.
Vandevelde
Marc
M
Zurbriggen
Andreas
A
Plattet
Philippe
P
D020413:3' Untranslated Regions; D000818:Animals; D004217:Distemper Virus, Canine / Q000235:genetics*; D004285:Dogs; D005434:Flow Cytometry; D015870:Gene Expression; D017930:Genes, Reporter; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D014760:Viral Fusion Proteins / Q000235:genetics; D014763:Viral Matrix Proteins / Q000235:genetics*; D000097573:Red Fluorescent Protein
D020413:3' Untranslated Regions; D006388:Hemagglutinins; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; D014763:Viral Matrix Proteins; D000097573:Red Fluorescent Protein