Evolution of coxsackie B virus during in vitro persistent infection: detection of protein mutations using two-dimensional polyacrylamide gel electrophoresis

Abstract

Serotype 5 coxsackie B virus (CBV5) can establish in vitro persistent infections in human rhabdomyosarcoma (RD) cells. This paper describes the characterisation of the virus released from the persistently infected RD cell line designated piRD-3673. Although infectious virus was released for 42 sequential passages of piRD-3673 cells, no gross virus-specific cytopathic effect was detected when the cells were examined by light microscopy. Two-dimensional polyacrylamide gel electrophoresis was used to compare the virus released from piRD-3673 cells with the CBV5 isolate (CBV-3673) used to initiate the persistent virus infection. Two of the virus intracellular proteins (apparent molecular weights 33,000 and 39,000, designated p33 and p39, respectively) increased in their net basic charge for the virus released from piRD-3673 cells compared to CBV-3673; a reduction in the apparent molecular weights of p33 and p39 was also observed. The charge alteration for both p33 and p39 was a two-stage process, the accumulative effect of which resulted in p33 increasing in pI from 6.14 to 6.53 and p39 increasing in pI from 6.29 to 6.63. The first mutation of p33 and p39 occurred between passages 7 and 10 of piRD-3673 cells and affected both the charge and apparent molecular weight of these two proteins. The second mutation at passage 15 of piRD-3673 cells caused only a change in the charge of p33 and p39. Two other virus proteins (p54 and p75) showed no evidence of mutation over the same passage history of piRD-3673 cells. The virus released from piRD-3673 cells also differed from CBV-3673 by two further criteria, a reduction in plaque-forming efficiency in HEp-2 cells and increased virus replication in RD cells. These data on virus evolution are discussed in relation to the maintenance of persistent CBV infections and the presence of naturally occurring CBV variants.