FIG 6.

Growth and biochemical properties of rCan viruses. (A) Photomicrograph of foci produced under agarose by parental (top) and K33S-GPC (bottom) rCan viruses. Foci were immunochemically stained using the anti-NP MAb AG12. Scale marker, 2 mm. (B and C) Growth kinetics following low-multiplicity infection (MOI, 0.01) of Vero cells with parental rCan (black circles), K33S-GPC rCan (red squares), and K33S/A168T/ΔFP-GPC rCan (blue triangle). Cell culture supernatants were harvested at indicated times, and virion accumulation was assessed by NP ELISA (B) and by enumeration of focus-forming units (FFU) (C). Input viral titers were confirmed contemporaneously. The relative specific infectivity of the virus was calculated as the ratio of FFU/ml and NP ELISA values. Virus spread through the culture was qualitatively assessed by immunochemical staining of the monolayer; all viruses spread comparably, and cultures were completely infected by 72 h postinfection. (D) Purified rCan and K33S-GPC rCan virions harvested at 48 h were subjected to immunoblot analysis using either GP2-directed MAbs G3 and G5 (38) or the NP-directed MAb AG12. Only the proteolytically mature GPC is incorporated into virions. More slowly migrating NP bands have been shown to represent ubiquitinated forms of the protein (NP-Ubi; unpublished data). A frequently observed NP degradation product is indicated as NP-degn. The results presented in this figure were representative of three replicate studies.