FIG 9.

Mutations of the IAPV hinge region disrupt virus translation in vitro and infection. (A) A hybrid CrPV/IAPV infectious clone was produced by replacing the CrPV IGR IRES in the CrPV infectious clone with the IAPV IGR IRES. (B) In vitro transcribed hybrid CrPV/IAPV containing the wild type (WT, hinge is UA), a mutant M2 (SLVI mutation) that disrupts IAPV IRES activity (25), a stop codon mutation in ORF1 (ORF1 Stop), or mutant hinge sequences AA or AU were incubated in an SF-21 insect cell lysate (2 h) in the presence of S³⁵ labeled methionine/cysteine. Translation products were visualized by SDS-PAGE analysis and autoradiography. (C) Immunoblots of CrPV VP2 and tubulin or (D) viral titers of lysates of S2 cells transfected (48 h) with in vitro transcribed RNA (3 μg) of the indicated hybrid CrPV/IAPV RNAs. The ORF1 stop contains a stop codon within the N-terminus of ORF1, thus preventing ORF1 expression (25). Viral titers were calculated from endpoint dilution analysis. Shown are a representative immunoblot and viral titers from at least three independent experiments ± SD.