1887

Abstract

RNA-dependent RNA polymerase (RdRp) was solubilized from crude extracts of infected by (HCRSV), a member of the . After treatment of the extracts with micrococcal nuclease to remove the endogenous templates, the full-length genomic RNA and the two subgenomic RNAs were efficiently synthesized by the partially purified RdRp complex . When the full-length RNAs of , , and were used as templates, no detectable RNA was synthesized. Synthesis of HCRSV minus-strand RNA was shown to initiate opposite the 3′-terminal two C residues at the 3′ end and . The CCC-3′ terminal nucleotide sequence was optimal and nucleotide variations from CCC-3′ diminished minus-strand synthesis. In addition, two putative stem–loops (SLs) located within the 3′-terminal 87 nt of HCRSV plus-strand RNA were also essential for minus-strand RNA synthesis. Deletion or disruption of the structure of these two SLs severely reduced or abolished RNA synthesis. HCRSV RNA in which the two SLs were replaced with the SLs of could replicate in kenaf protoplasts, indicating that functionally conserved structure, rather than nucleotide sequence, plays an important role in the minus-strand synthesis of HCRSV. Taken together, the specific sequence CCC at the 3′ terminus and the two SLs structures located in the 3′UTR are essential for efficient minus-strand synthesis of HCRSV.

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2004-06-01
2019-10-15
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