Analysis of ISVP-to-ISVP* conversion.

ISVPs (2 × 10¹² particles/ml) of the indicated viral strains were divided into aliquots of equivalent volumes and heated at the indicated temperatures for 20 min. The reaction mixtures were cooled on ice and then digested with 0.10 mg/ml trypsin (Sigma-Aldrich) for 30 min on ice. Following addition of the SDS-PAGE loading dye, the samples were subjected to SDS-PAGE analysis. For analysis by quantitative infectivity assay, P2 stocks or purified virus stocks of the indicated viruses were diluted 1:10 in virion storage buffer (10 mM Tris-HCl [pH 7.4], 15 mM MgCl₂, and 150 mM NaCl). A 200-μg/ml concentration of TLCK-treated chymotrypsin (Worthington Biochemical) was added to each sample. Samples were heated to 37°C for 30 min. The reaction was quenched by the addition of 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich) and cooled on ice for 10 min. The reaction mixtures were divided into equivalent volumes and incubated at 4°C or 40°C for 20 min. These mixtures were used to initiate infection of L929 cells, and infectivity was determined by plaque assay. The change in infectivity at a given temperature (T) was calculated using the following formula: log₁₀(PFU/ml)_T − log₁₀(PFU/ml)_4°C.