Replication of semliki forest virus: polyadenylate in plus-strand RNA and polyuridylate in minus-strand RNA

D L Sawicki, P J Gomatos
1976 Journal of Virology  
The 42S RNA from Semliki Forest virus contains a polyadenylate [poly(A)] sequence that is 80 to 90 residues long and is the 3'-terminus of the virion RNA. A poly(A) sequence of the same length was found in the plus strand of the replicative forms (RFs) and replicative intermediates (RIs) isolated 2 h after infection. In addition, both RFs and RIs contained a polyuridylate [poly(U)] sequence. No poly(U) was found in virion RNA, and thus the poly(U) sequence is in minus-strand RNA. The poly(U)
more » ... m RFs was on the average 60 residues long, whereas that isolated from the RIs was 80 residues long. Poly(U) sequences isolated from RFs and RIs by digestion with RNase Ti contained 5'-phosphorylated pUp and ppUp residues, indicating that the poly(U) sequence was the 5'terminus of the minus-strand RNA. The poly(U) sequence in RFs or RIs was free to bind to poly(A)-Sepharose only after denaturation of the RNAs, indicating that the poly(U) was hydrogen bonded to the poly(A) at the 3'-terminus of the plus-strand RNA in these molecules. When treated with 0.02 g.g of RNase A per ml, both RFs and RIs yielded the same distribution of the three cores, RFI, RFII, and RFIII. The minus-strand RNA of both RFI and RFIII contained a poly(U) sequence. That from RFII did not. It is known that RFI is the double-stranded form of the 42S plus-strand RNA and that RFIII is the experimentally derived double-stranded form of 26S mRNA. The poly(A) sequences in each are most likely transcribed directly from the poly(U) at the 5'-end of the 42S minus-strand RNA. The 26S mRNA thus represents the nucleotide sequence in that one-third of the 42S plus-strand RNA that includes its 3'-terminus. Isolation of poly(U) sequences. The column material was poly(A)-Sepharose 4B, which was the generous gift of S. L. Marcus (27). The washing J. VIROL. on May 9, 2020 by guest
doi:10.1128/jvi.20.2.446-464.1976 fatcat:ieocp2nyrrbdndyp2qgtcz2qpu