Time course of virus-specific macromolecular synthesis during rubella virus infection in vero cells

Abstract

Virus specific macromolecular synthesis was studied in Vero cells infected with plaque-purified rubella virus under one-step multiplication conditions. Under these conditions, the rate of virus production was found to increase rapidly until 24 hr postinfection after which time the rate of virus production rose more slowly, reaching a peak level at 48 hr postinfection. This peak rate of virus production was maintained through 72 hr postinfection. A majority of the cells remained alive through 96 hr postinfection, although a 20 to 30% decrease in the number of living cells occurred between 24 and 48 hr postinfection, the time period at which cytopathic effect was first observed. The virus structural proteins were first detected intracellularly at 16 hr postinfection. The rate of synthesis of these proteins was already maximal at 16 hr postinfection and remained constant through 48 hr postinfection. By immunofluorescence, cells expressing virus proteins were first observed at 12 hr postinfection. At 24 hr postinfection, 35 to 50% of the cells in the infected culture were exhibiting immunofluorescence, at 36 hr postinfection, 65 to 90% of the cells were exhibiting immunofluorescence, and at 48 hr postinfection, all of the cells were exhibiting immunofluorescence. The virus genomic and subgenomic RNA species were first detectable by 12 hr postinfection. The rate of synthesis of both of these species peaked at 26 hr postinfection. Rubella virus infection was found to have no effect on total cell RNA synthesis. However, a modest inhibition of total cell protein synthesis which reached 40% by 48 hr postinfection was observed. When Northern analysis of RNA extracted from infected cells was performed, a negative-polarity, virus-specific RNA probe hybridized only to the virus genomic and subgenomic RNA species. A positive-polarity, virus-specific RNA probe hybridized predominantly to a negative-polarity RNA of genome length indicating that both the genomic and subgenomic RNAs are synthesized from a genome-length negative-polarity template. Defective interfering (DI) RNAs were not detected in infected cells through 96 hr postinfection or in cells onto which virus released through 96 hr postinfection was passaged. Thus, the generation of DI particles by rubella virus appears to play no role in the slow, noncytopathic replication of this virus or in the ability of rubella virus-infected cells to survive for extended periods of time.