Nucleotide sequence at the 5' terminus of the avian sarcoma virus genome.

The initiation of DNA s nthesis in vitro by RNA-directed DNA polymerase (deoxynucreosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7) of avian oncornaviruses requires a tRNAtrP primer molecule located close to the 5' end of the viral RNA genome. DNA transcripts, 100 nucleotides in length, initiated on the tRNAtrP primer molecule contain nucleotide sequences complementary to a large (25 nucleotides) RNase T, oligonucleotide, T-13, After infection of cells by RNA tumor viruses, proviral DNA is transcribed from the viral RNA genome by the virion-associated DNA polymerase and subsequently integrated into the genome of the host cell (1). Proviral DNA can be found in the form of covalently closed circular molecules that are presumably required intermediates for integration (2-4). Although molecular weight estimates suggest that proviral DNA isolated from infected cells represents a complete transcript of 35S RNA (2-4), the mechanism by which the viral RNA genome is converted into the circular form of proviral DNA is presently unknown.The in vitro synthesis of DNA from avian tumor virus 70S RNA by purified RNA-directed DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) is dependent on the presence of a tRNAtrP primer molecule that is located near the 5' end of the avian RNA tumor virus genome and serves as the major initiation site for DNA synthesis in vitro (5-9). Evidence for the 5'-terminal location of tRNAtrp-initiated DNA transcripts has been obtained by determining the location of nucleotide sequences in the viral RNA genome that are complementary to, and therefore transcribed into, these DNA transcripts (6, 10, 11). Recently, a large T1-RNase-resistant oligonucleotide containing the modified structure 7mGpppGmpCp has been identified as the 5' terminus of the avian sarcoma virus (ASV) genome (11). We have used this observation to confirm the location of tRNAtrP-initiated DNA transcripts at the 5' end of the viral genome and to define the approximate distance between the tRNAtrp primer and the 5' terminus. In addition, we demonstrate that DNA transcripts synthesized at the 3' terminus of the viral RNA contain nucleotide sequences complementary to genomic sequences at the 5' end of the molecule. The possible role of this observed terminal redundancy in provirus replication is discussed. MATERIALS AND METHODSReagents and Virus. The sources and preparation of most of the pertinent materials have been described (12)(13)(14). Purified avian myeloblastosis virus RNA-directed DNA polymerase was obtained from J. W. Beard of Life Sciences, Inc., St. Petersburg, FL., through the auspices of the Viral Cancer Program of the National Cancer Institute. ASV, Bratislava strain, subgroup C (B77), was propagated in duck embryo fibroblasts (15,16). ASV (Prague C) was also obtained through the Viral Cancer Program of the National Cancer Institute from University Laboratories Inc., Highland Park, NJ.Purification of Viral RNA. RNA was extracted from virus with sodium dodecyl ...

Section: Resultssupporting

confidence: 59%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Terminally repeated sequences in the avian sarcoma virus RNA genome.

Collett¹,

Dierks²,

Cahill³

et al. 1977

“…1, Fig. 1), because it appears from a variety of evidence (12)(13)(14)(15)(16)(17) (18) have found an inhibitory effect of poly(A) and poly(O-methyl-A) on murine oncornavirus production in tissue culture, and inhibition largely due to impairment of penetration of virus into the host cell. Milleir et al (19) report that the trinucleotide complement to the -C-C-A terminus of tRNA inhibits protein synthesis by 40% when added to mammalian cells in tissue culture.…”

Section: Resultsmentioning

confidence: 99%

Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide.

Zamecnik¹,

Stephenson²

1978

1,552

622

The tridecamer d(A-A-T-G-G-T-A-A-A-A-T-G-G), which is complementary to 13 nucleotides of the 3'- and 5'-reiterated terminal sequences of Rous sarcoma virus 35S RNA, was added to chick embryo fibroblast tissue cultures infected with Rous sarcoma virus. Inhibition of virus production resulted. The inference emerges that the tridecamer and its counterpart with blocked 3'- and 5'-hydroxyl termini enter the chick fibroblast cells, hybridize with the terminal reiterated sequences at the 3' and 5' ends of the 35S RNA, and interfere with one or more steps involved in viral production and cell transformation. Likely sites of action are (i) the circularization step of the proviral DNA intermediate, and (ii) the initiation of translation, the latter being described in the following communication [Stephenson, M. L. & Zamecnik, P. C. (1978) Proc. Natl. Acad. Sci. USA 75, 285--288].

“…and to prime the synthesis of minus strand DNA (15)(16)(17)(18)(19). The plus strong stop DNA for M-MuLV and MSV is a complementary copy of the 5' end of the minus strand DNA.…”

mentioning

confidence: 99%

Nucleotide sequences of integrated Moloney sarcoma provirus long terminal repeats and their host and viral junctions.

Dhar¹,

McClements²,

Enquist³

et al. 1980

293

166

Integrated Moloney murine sarcoma provirus (MSV) has direct terminal repeat sequences (TRS). We determined the nucleotide sequence of both 588base-pair TRS elements and the adjacent host and viral junctions of an integrated MSV cloned in bacterophage A Sequences were identified corresponding to the tRNAO primer binding site in genomic RNA and the reverse-transcribed minus strong stop DNA. Each 588-base-pair repeat contains putative sites for promoting RNA sythesis and RNA polyadenylylation. The first and last 11 nuclotides of the TRS are inverted with respect to each other, and the same four-nucleotide host sequence is found bracketing integrated MSV. Some similarities of TRS and prokaryotic insertion sequence elements are discussed.