A general strategy to inhibiting viral -1 frameshifting based on upstream attenuation duplex formation

Hu, Hao-Teng; Cho, Che-Pei; Lin, Ya-Hui; Chang, Kung-Yao

The −1 PRF elements used in this study, containing different viral downstream −1 PRF stimulators and upstream sequences flanking the slippery sites, were constructed by assembling different pieces of chemically synthesized DNA oligonucleotides with partially overlapping sequences via the polymerase chain reaction (PCR)-based ligation approach (32) . Forward and reverse DNA primers, respectively carrying SalI and BamHI restriction sites and appropriately designed annealing sequences, were used for PCR amplification of the cDNAs encoding viral −1 PRF elements of interest. The amplified inserts encoding distinct viral −1 PRF signals were then cloned into the SalI/BamHI sites of appropriate −1 PRF reporters. Cloning was performed using standard procedures and the resultant recombinant −1 PRF reporters were trans-formed into the DH5␣ strain of Escherichia coli cells for maintenance and selection by ampicillin. Mutagenesis was introduced into the desired position using the quick-change mutagenesis kit from Stratagene according to the manufacturer's instructions. Identities of all cloned and mutated −1 PRF elements were confirmed by DNA sequencing analysis.