Construction and characterization of CV-1P cell lines which constitutively express the simian virus 40 agnoprotein: alteration of plaquing phenotype of viral agnogene mutants

Abstract

The simian virus 40 (SV40) agnoprotein is a 61-amino-acid polypeptide encoded in the leader region of some late viral mRNAs. Its function is unclear, although previous investigations suggest that the agnoprotein may function in late transcriptional regulation and virus assembly. To define the specific role(s) of agnoprotein in the SV40 lytic cycle, CV-1P cell lines were constructed in which the agnogene was stably integrated and constitutively expressed under the control of a retroviral long terminal repeat. Two types of cell lines were isolated. One group, typified by the cell line Ag18, produced low levels of agnogene-specific mRNA and agnoprotein. The other type, represented by a single isolate named Ag8, produced high levels of agnogene-specific mRNA and correspondingly high levels of agnoprotein. By indirect immunofluorescence, the agnoprotein was located predominantly in the cytoplasmic and perinuclear region of both cell lines; this is its site of localization in wild-type (WT)-infected CV-1P cells. Viruses that were agnoprotein-minus formed small plaques on normal CV-1P cells, but produced nearly WT-sized plaques on Ag18 cells. Conversely, the plaques formed on Ag8 cells infected with agnoprotein-minus mutants of WT SV40 were markedly smaller than the plaques formed by these viruses when they were grown on control cells. Overall, our results suggest that the agnoprotein is a trans-effector of virus production. The opposite effects on plaquing that were observed with Ag8 and Ag18 cells correlated with the very different levels of agnogene expression in the two cell lines. This suggests that the nature of the effect of the agnoprotein on virus production may vary depending on its intracellular concentration.