Synthesis and methyltransferase activity of nonstructural protein nsP1 in Sindbis virus-infected Aedes albopictus...

To investigate the synthesis and turnover of the nonstructure protein, nsP1, in Aedes albopictus cells, we labeled infected cells with [35S]-methionine and immunoprecipitated nsP1 with a polyclonal monospecific rabbit antibody. Synthesis of nsP1 in mosquito cells could be detected 2 hr after infection and continued as long as 24 hr post-infection, regardless whether the infected cells were maintained at 28 degrees C , 34.5 degrees C , or 37 degrees C. Whereas the time pattern of nsP1 synthesis varied with temperature. Nonstructural protein 1 synthesis at 28 degrees C was maximal at 4 hr, then decreased. At 37 degrees C synthesis reached a high level at 2 hr and remained constant for 24 hr. Pulse-chase experiments showed that nsP1 in mosquito cells, whether made as early as 5 hr post-infection or as late as 24 hr, were stable during that time, the longest period tested. Enzyme activity reached a maximum at 10 hr and remained almost the same level until 24 hr. The yield of Sindbis virus from mosquito cells was higher at 34.5 degrees C and 37 degrees C than at 28 degrees C. Methyltransferase activity was needed for modification of positive-strand genomic and subgenomic RNAs. The activity of methyltransferase observed until late in the replication cycle probably accounted for both the continued synthesis and minimal turnover of nsP1.