Characterization of the End Groups of RNA of Rous Sarcoma Virus

Abstract

The 3' terminus of the 30-35S RNA of Rous sarcoma virus is adenosine. Its amount indicates an average molecular weight for that RNA of about 3 X 106. The 5' terminus of 30-35S RNA of Rous sarcoma virus was not di-or triphosphorylated, whether isolated by the standard procedure or from virus collected within 3 min of its release from the cells.The chemical structure of the RNA of the oncornaviruses is still in doubt. Although much evidence favors the existence of subunit RNA molecules of 30-35 S held together by basepaired segments to form the 65-70S genome of these viruses, this concept, as well as the size of the presumed component RNA molecules and the question of whether they are identical or different, are not yet firmly established.It appeared to us that a study of the end groups of the subunits would contribute significant answers to these questions. We previously reported preliminary results (at a meeting at the University of California, Los Angeles, in 1973) that adenosine appeared to be the predominant 3'-terminal residue of the RNA of Rous sarcoma virus (RSV), in contrast to earlier reports that various oncornaviral RNAs terminated in uridine (1-3). Adenosine has been found to be the predominant terminal nucleoside of the RNA of avian myeloblastosis virus (4-6), and the finding that the oncornaviruses contained poly(A) (7), 3'-terminal in avian myeloblastosis virus (5), made this the preferred and presumed end group also for RSV, notwithstanding the reports to the contrary. We have now refined our preparative and analytical procedures sufficiently to yield consistent and reproducible results for the ratio of 3'-terminal nucleosides to internal nucleotides. The results of these analyses are that there is one 3'-unphos- (11) with bicarbonate buffer, supplemented with 1% (v/v) calf and 1% (v/v) chick serum. Typically the label was administered to 40 dishes in a ratio of 2 mCi of adenosine (labeled in the 2 position) to 1 mCi of each of guanosine, cytidine, and uridine; 1 mCi of total label was used per 10-cm petri dish. Medium containing radioactive virus was removed after 4 hr and replaced with 6 ml of the same medium but without addition of more labeled nucleosides. Collections were continued at 4-hr intervals for a period of one or two days. Virus was purified and viral RNAs were extracted as described (10). The 30-35S RNA used was obtained by heating viral RNAs at 1000 for 45 sec, followed by another sucrose gradient fractionation. The RNA, together with carrier RNA of tobacco mosaic virus (TMIV) used as a 30S marker on sucrose gradients, was hydrolyzed in 50,ul of 0.3 M KOH for 18 hr at 370 in a sealed capillary tube, and then spotted directly with absorbance markers (nucleosides and nucleotides) 44.5 cm from the anode side, and 8 cm from one edge of a sheet of Whatman 3 MM paper of 95 X 48 cm. Electrophoresis was in a refrigerated two-phase electrophoresis system, with pyridine-acetate buffer at pH 3.5 (5% v/v glacial acetic acid, 0.5% v/v pyridine, and 1 mM EDTA) at about 3000 V for 4 hr. ...

Identification of the 5' end of Rous sarcoma virus RNA.

“…The terminal position and the 5' linkage of the 7-methylguanosine was shown by #-elimination after periodate oxidation, which was carried out both with 3H-labeled intact poly(A)-minus 35S RSV RNA and with the 32P, 3H doubly-labeled nuclease-and phosphatase-resistant fragment. In the first type of experiment, 3H label was found in the 7-methylguanine and 7-methylguanosine areas, as well as in the adenosine area (data not shown), which, incidentally, confirms our earlier conclusion that adenosine is the 3'-terminal residue of the poly(A)-minus 35S RSV RNA (5). However, the yields were poor in all these analyses for #l-elimination products, and the procedures will have to be improved before quantitative conclusions are possible.…”

“…Since this fragment is now shown to contain four phosphate groups, the approximate chain length of the RNA is 10,000 and the molecular weight based on this terminus is about 3.2 X 106, in good accord with earlier conclusions by us and most others (5,14,15).…”

“…Recently, data were reported showing that a novel blocking group existed on the 5'-triphosphate ends of various mRNAs, namely 5'-linked 7-methylguanosine (6)(7)(8)(9)(10). It appeared to us that such a terminus was possible also in RSV RNA and could account for the radioactivity remaining at the origin of the thin-layer chromatography plates used in our earlier study (5). The present paper presents evidence that all 35S subunits, presumably the viral genome of RSV RNA, have a 5' endgroup similar to that now reported for many cellular and viral mRNAs, namely, 7mG5'ppp5'GmpCp-.…”

“…Specifically, the exceptions to the previously described technique were: (i) Cells infected with RSV (Prague B strain) were grown in 150-cm2 tissue culture flasks and labeled with 10 mCi Of 32p (ICN Pharmaceuticals, Inc.) per flask for 12-14 hr. The virus containing medium was collected and pooled every [4][5][6] (11) except that oxidation was in 0.01-0.02 M NaIO4; After fl-elimination of the enzyme-resistant fragment, the solution was either spotted onto Whatman 3MM and chromatographed in solvent A, where pppNp compounds remain at the origin; or it was passed through a small pasteur pipet containing 6 X 10 mm of AGI-X2 Cl-form (Bio-Rad Lab. ), where the fieliminated oxidized nucleosides were eluted with water; the eluate was concentrated, made 1 M in HC1, and heated at 1000 for 1 hr.…”

“…The report of unphosphorylated adenosine at the 5' end of Rous sarcoma virus (RSV) RNA (4) thus seemed unusual for an animal virus. Yet our attempts to find (p)ppN termini in RSV collected at standard times and within 3 min after its release from the cell also gave negative results (5). Recently, data were reported showing that a novel blocking group existed on the 5'-triphosphate ends of various mRNAs, namely 5'-linked 7-methylguanosine (6)(7)(8)(9)(10).…”