Genome 3'-end repair in dengue virus type 2.
Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3'-end repair in dengue virus-2 in mammalian cells, a series of 3'-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7-10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3' end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3'-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3' stem-loop (3' SL) structure was abrogated by mutations, viruses eventually restored the 3' SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3'-end sequences based on the best-fit RNA structure that can support viral replication.
Teramoto
Tadahisa
T
Laboratory of Vector-Borne Virus Diseases, Center for Biologics Evaluation and Review, Food and Drug Administration, Bethesda, Maryland 20892, USA.
Kohno
Yukari
Y
Mattoo
Pravina
P
Markoff
Lewis
L
Falgout
Barry
B
Padmanabhan
Radhakrishnan
R
D000818:Animals; D001483:Base Sequence; D003716:Dengue Virus / Q000235:genetics*; D016679:Genome, Viral; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000737:chemistry; D014779:Virus Replication
D012367:RNA, Viral