Uncoating kinetics of hepatitis A virus virions and provirions

Abstract

When the growth kinetics of immature hepatitis A virus provirions and mature virions were monitored, distinct eclipse phases were noted for both types of particles. Strikingly, uncoating of virions occurred around 4 h postinfection, while uncoating of provirions occurred predominantly between 8 and 10 h postinfection. It is proposed that the heterogeneous mixture of infectious hepatitis A virus particles (virions and provirions) typically present in inocula is responsible for the normally asynchronous nature of hepatitis A virus uncoating kinetics.

FIG. 1

Kinetics of HAV and PV replication. Monolayers of BS-C-1 cells were infected for 90 min at 4°C with HAV or PV before being shifted to 37°C. Infectious virus was assayed throughout the respective growth cycles by RIFA for HAV or plaque assay for PV. Cytopathic effect (CPE) was graded on a scale of 0+ to 4+, where 0+ represents a healthy cell monolayer and 4+ is destruction of more than 60% of cells. Much of the PV yield would be in the supernatant from 12 h postinfection.

FIG. 2

Kinetics of HAV and PV uncoating. Cultures of BS-C-1 cells were infected with serial dilutions of light-sensitive virus at 4°C in the dark. Cells were shifted to 37°C for various times and then exposed to light. After exposure, overlay medium was added to allow the number of particles uncoated during the incubation period to be estimated by RIFA for HAV or plaque assay for PV. RIFU, radioimmunofocus-forming units.

FIG. 3

Relationship between VP0 self-cleavage and specific infectivity. In five separate experiments, provirions were purified from infected cells and duplicate aliquots were placed at −70 or 37°C for various times. The percent conversion of VP0 to VP2 was determined, and the corresponding increase in infectivity was determined (Table 1). The percent decrease in VP0 content was then calculated and plotted against the increase in infectivity. It should be noted that the percent decrease in VP0 content of these preparations correlated absolutely with the increase in VP2 content in all cases. The lighter line indicates an exponential curve fit.

FIG. 4

Single-cycle growth curves of HAV virions and provirions. In two separate experiments, pools of provirions were used to infect BS-C-1 cells with or without prior self-cleavage of VP0 at 37°C. (A) VP0 content of particles was approximately 52% before cleavage and 27% after self-cleavage at 37°C for 3 days. (B) VP0 content of particles was 65% before cleavage and 22% after self-cleavage at 37°C for 4 days. Virions in panel A were diluted 1:5 prior to infection, providing an approximately equal input titer. RIFU, radioimmunofocus-forming units.