Analysis of splice junctions and in vitro and in vivo translation potential of the small, abundant B19 parvovirus RNAs.
Two parvovirus B19 cDNA libraries have been constructed; one from COS-7 cells transfected with a B19/pSVOd hybrid vector and the other from B19-infected human erythroid leukemic cells. We have used these libraries to investigate the expression of the abundant classes of polyadenylated B19 RNAs; the 700- and 800-nt class which terminates in the middle of the genome and the 500- and 600-nt class which contains an ORF from the extreme right-hand end of the genome. The 700- and 800-nt RNA species were not found in the COS cell library, suggesting that a variant polyadenylation signal (ATTAAA or AATAAC) in the middle of the genome is not efficiently recognized in these cells. In contrast, the 700- and 800-nt class was highly represented in the human library, confirming the use of this variant polyadenylation signal in the normal host cell of the virus. In COS cells the middle exon of the 500- and 600-nt class of RNA exhibited variability in both splice donor and acceptor sites. However, in human cells there were only two splice acceptor sites nt 1910 and 2030, and a single splice donor site nt 2183 for this exon. Antisera, prepared against a peptide derived from the 94-aa potential protein encoded by the 500- and 600-nt class of RNA, recognized, on a Western blot, a polypeptide of approximately 11 kDa that was translated in vitro from these cDNAs and in vivo in pSVOd/B19 transfected COS cells. Immunoprecipitation revealed that two proteins were made from this ORF, suggesting the use of internal translation initiation site(s). Another antisera, raised against a second peptide corresponding to an antigenic region of the potential protein encoded by the 700- and 800-nt class of RNA, failed to detect a 15-kDa protein by Western blotting or immunoprecipitation of labeled proteins both in vitro and in vivo in COS cells.
St Amand
J
J
Department of Biochemistry, University of British Columbia, Vancouver, Canada.
Beard
C
C
Humphries
K
K
Astell
C R
CR
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010321:Parvoviridae / Q000235:genetics* / Q000276:immunology; D014176:Protein Biosynthesis; D012326:RNA Splicing; D012367:RNA, Viral / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes; D012995:Solubility; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics
D000956:Antigens, Viral; D004279:DNA, Viral; D012367:RNA, Viral; D014764:Viral Proteins