FIG 7.
Mutations disturbing RNA replication cause instability of CP and prevent its assembly in virion-like particles. (A, C, and D) Analysis of the stability of the CP produced by agroinfiltration (A and C) or agroinoculation (D) of N. benthamiana leaves with the indicated constructs. Samples from infiltrated leaves were collected at 5 days after treatment. Extracts prepared in SDS buffer (SDS) and the time, in minutes, elapsed after extraction with phosphate buffer (0′, 30′, or 60′) are indicated. Protein accumulation was assessed by immunoblot analysis with polyclonal serum specific for PPV CP. Membranes stained with Ponceau red showing the RubisCO large subunit were included as loading controls. (B) Sucrose gradient centrifugation of extracts of N. benthamiana leaves agroinfiltrated with pLONG(CIKS) and pP1HCPro. Extracts, prepared in phosphate buffer, were loaded on the gradient immediately after being prepared (0′) or after 60 min of incubation at 25°C (60′). Sixteen fractions were collected from the bottom, and the 10 upper ones were subjected to CP-specific immunoblot analysis. The asterisk indicates that the membrane was overexposed.