Novel expression of mouse adenovirus type 1 early region 3 gp11K at late times after infection

Mutations were introduced into mouse adenovirus type 1 (MAV-1) early region 3 (E3) initiator codons by homologous recombination between viral DNA and a plasmid containing a mutagenized E3 region. The resulting mutant virus, p mE312, contained ATG --> TTA mutations at codon positions 1 and Li and was expected to be null for the expression of the E3 proteins. However, gp11K, an MAV-1 E3 glycoprotein of 14K molecular weight, was detected in mutant-in fected cell lysates at levels about 10-12% of that of wild-type (wt) virus at late limes in infection. The gp11K polypeptide produced by pmE312 at lat e times was immunoprecipitated with two E3-specific antisera prepared again st different regions of the protein. Like gp11K produced by wt virus infect ions, it was sensitive to endoglycosidase H (endo H) and thus resident in t he endoplasmic reticulum (ER). In pmE312-infected cells treated with cytosi ne arabinoside (araC), an inhibitor of DNA replication, the gp11K protein w as not detected by immunoprecipitation. This indicates that gp11K expressio n in pmE312-infected cells at late times was dependent on DNA replication a nd that it was thus translated from a late transcript. In vitro translation of poly(A)(+) RNA from mock-, wild-type-, and pmE312-infected cells showed that gp11K was translated from late mRNA as an similar to 28K fusion betwe en a late protein and gp11K. Our data are consistent with a model in which gp11K is expressed at late times as a late protein-gp11K chimera in both wt - and mutant-infected cells. This chimera is then processed: removal of a l arge N-terminal sequence results in the observed 14K ER-localized gp11K. (C ) 1999 Academic Press.