An endonuclease switching mechanism in the virion RNA and cRNA promoters of Thogoto orthomyxovirus

Abstract

An in vitro assay was developed to investigate endonuclease activity of Thogoto virus, a tick-borne orthomyxovirus. Endonuclease activity relied on an interaction between the 3' and 5' termini of virion RNA (vRNA) and not those of cRNA. Evidence was obtained that cap structures are cleaved directly from cap donors and that cleavage does not occur after pyrimidines. A 5' hook structure, present in the vRNA promoter but not the cRNA promoter, was introduced into cRNA promoter mutants. These mutants stimulated endonuclease activity, although at levels slightly lower than that of vRNA. The ability of the cRNA promoter to stimulate endonuclease activity when mutated to contain a 5' hook structure indicates that this structure constitutes a switching mechanism for endonuclease activity between the vRNA and cRNA promoters.

FIG. 1

Various promoter structures of THOV, obtained by mixing short model RNAs corresponding to the 3′ and 5′ promoter arms. (A) vRNA (20); (B) cRNA (21); (C) a mutant cRNA promoter containing a hook in the 5′ promoter arm obtained by changing the residue at position 3 from cytosine to uracil; (D) a mutant cRNA promoter containing a hook in the 5′ promoter arm obtained by changing the residue at position 8 from adenine to guanine. Mutations are underlined and in italics.

FIG. 2

Conditions for endonuclease activity with cap donors ³²P labeled in the cap. Reactions were performed with cores plus cap donor supplemented with the 3′ vRNA promoter arm (lane 2), the 5′ vRNA promoter arm (lane 3), the vRNA promoter (lane 4), the 3′ cRNA promoter arm (lane 5), the 5′ cRNA promoter arm (lane 6), and the cRNA promoter (lane 7). Lane 1 contains cap donor alone.

FIG. 3

Conditions for endonuclease activity with cap donors ³²P labeled in the chain. Reactions were performed with viral cores plus cap donor supplemented with the 3′ vRNA promoter arm (lane 2), the 5′ vRNA promoter arm (lane 3), the vRNA promoter (lane 4), and the cRNA promoter (lane 5). Lane 1 contains the cap donor alone; lane M contains end-labeled 13- and 14-nt RNA markers.

FIG. 4

Requirements of the cap donor for endonuclease activity. Cap donors were ³²P labeled in the cap and in the chain. Reactions were performed with viral cores plus the vRNA promoter supplemented with cap donor 6U6A (lane 1), 2U10A (lane 2), 1U11A (lane 3), and 12A (lane 4). Lane 5 contains cap donor 12A alone. See Table 1 for details of cap donors.

FIG. 5

Endonuclease activity stimulated by cRNA promoter mutants. Cap donors were ³²P labeled in the chain. Reactions were performed with viral cores plus cap donor supplemented with the vRNA promoter (lane 2), the mutant cRNA promoters A (lane 3) and B (lane 4), and the wild-type cRNA promoter (lane 5). Lane 1 contains cap donor alone; lane M contains end-labeled 13- and 14-nt RNA markers. See Fig. 1 for details of the promoters and mutants thereof.