Evidence forsplicing ofaviansarcomavirus5'-terminal genomic sequences ontoviral-specific RNA ininfected cells

The5'-terminal nucleotide sequences ofthe avian sarcoma virus (ASV) genome aretranscribed bythereverse transcriptase invitro into aDNAtranscript that represents the entire distance ('100 nucleotides) between thetRNATrP primer molecule andthe5'terminus. We haveusedthese DNAioo transcripts inhybridization reactions withASV-specific RNA frominfected avian cells andfind nucleotide sequences com- plementary tothese transcripts onall ofthevarious size classes ofviral mRNA identified. Similar hybridization results were obtained with aspecific DNAtranscript complementary toviral genomic nucleotide sequences between thetRNATrP primer molecule andupto,butnotincluding, theterminal redundant sequences (DNA70), indicating that theobserved hybridization ofDNAIo0 toall size classes ofviral RNAininfected cells did notreflect hybridization ofDNA10o totheterminal redundant sequences atthe3'endoftheviral genome. Escheichia coli RNaseHhydrolysis ofRNA-DNAhybrids consisting ofgenomic 35SRNAobtained fromvirus andDNAIoo transcripts indicated that viral genomic sequences comr lementary tothese DNA transcripts werenotpresent atsites distal totheendsoftheRNA genomeandtherefore notadjacent tothecorresponding gene sequences representing thevarious species ofviral mRNAfrom infced cells. These studies suggest that the5'-terminal genomic nucleotide sequences, oraportion thereof, aresomehow added or"spliced" ontoeachASV-specific mRNAspecies ininfected cells either during orafter transcription ofproviral DNA for someasyetundetermined purpose. The5'terminus oftheavian sarcoma virus (ASV) genome ap- pears toplay animportant role inthereplication ofthevirus forseveral reasons. First, initiation ofreverse transcription occurs onatRNATrP primer molecule located near(s100 nu- cleotides) this endoftheviral genome (refs. 1-10). Second, a portion ofthe5'terminus (i.e., 21nucleotides) isrepeated at the3'endoftheviral genome andpresumably isused tofa- cilitate continued, uninterrupted transcription oftheviral ge- nomeduring proviral DNAsynthesis (5,11-14). Finally, the initiator codon, AUG,andnucleotide sequences implicated in thebinding ofeukaryotic ribosomes tomRNA arepresent within this region ofthegenome, suggesting apossible role in theinitiation oftranslation oftheviral genome (6). Inthis communication wepresent aseries ofexperiments indicating that the5'-terminal genomic nucleotide sequences, oraportion thereof, arepresent oneach size class ofASV-spe- cific mRNA identified ininfected cell cultures. Furthermore, wedemonstrate that these nucleotide sequences arenotpresent adjacent tothecorresponding genesequences ingenomic RNA obtained fromvirus. These data suggest that the5'-terminal nucleotide sequences oftheASVgenomearesomehow "spliced" ontotheindividual species ofviral-specific mRNA present ininfected cells toprovide someasyetunknown Thecosts ofpublication ofthis article weredefrayed inpart bythe payment ofpage charges. This article musttherefore behereby marked "advertisement" inaccordance with 18U.S.C.§1734 solely toindicate this fact.