Replicative form of Semliki Forest virus RNA contains an unpaired guanosine

Abstract

The mechanism of replication of the genome of animal viruses containing infectious single-stranded RNA is not understood in detail. Alphaviruses contain an infectious single-stranded genome RNA of about 4.2 × 10⁶ molecular weight, which sediments at about 42S on sucrose density gradients^1–4. Accordingly they are designated as (+)strand RNA viruses⁵. Most studies on the structure or replication of alphaviruses have been made using either Sindbis (SIN) or Semliki Forest (SF) viruses. Besides the infectious 42S plus-strand RNA, a 26S single-stranded RNA of positive polarity is synthesised in alphavirus-infected cells^3,4,6. The 26S RNA sequences are identical to the sequences present in the 3′terminal region of the 42S RNA^7,8. The 42S RNA functions as mRNA for the synthesis of nonstructural proteins including an RNA poly–merase^9–11. The 26S RNA is the mRNA for all viral structural proteins^12–15. The 42S SIN virus genome RNA has the 5′terminal structure m⁷GpppApUpYpGp (ref. 16), and a cap structure containing m⁷₁, m^2,7₂ G or m^2,2,7₃G is present in SIN virus-specific 26S RNA (ref. 17). Analogous studies on the 5′-termini of the corresponding SF-virus-specific nucleic acids have not been reported. The only species of virus-specific RNA of negative polarity detected in alphavirus-infected cells sediments at about 42S on sucrose density gradients and is complementary to the infectious viral genome RNA^18–21. The poly(A) sequences present at the 3′-termini of the virus-specific 42S and 26S RNA molecules^22–24 are transcribed from a complementary poly(U) sequence²⁰. We now report that we have identied the structures of cap containing oligonucleotides of the SF virus-specific (+)strand RN As, and the sequence of 23 nucleotides at the 3′-terminus of the (−)strand RNA synthesised in infected cells. Our studies show that the replicative form of SF virus contains an unpaired guanosine at the 3′-end of the (−)strand.