In vitro and in vivo characterization of a mouse adenovirus type 1 early region 3 null mutant

Previous attempts to construct a mouse adenovirus type 1 early region 3 (E3 ) null mutant by initiator codon mutagenesis were unsuccessful because one of the E3 proteins, gp11K, is synthesized as a fusion protein from a late v iral mRNA (A. N. Cauthen and K. R. Spindler, Virology 259:119-128, 1999). T herefore, a different mutagenesis strategy was employed that inserted termi nation codons into all three reading frames of the E3 proteins. This strate gy produced a mutant, pmE314, that was null for the expression of E3 protei ns as determined by immunoprecipitation with E3-specific antisera, This mut ant grew as well as wild-type (wt) virus in both 3T6 mouse fibroblasts and mouse brain microvascular endothelial cells. However, the 50% lethal dose f or pmE314 in adult NM Swiss outbred mice was approximately 6 log units high er than that of wt virus, indicating that pmE314 was less virulent in mice, In situ hybridization experiments revealed that the absence of the E3 prot eins did not alter the tropism of the mutant virus from that of wt virus. W hen the histopathology was evaluated, the characteristics of the pmE314 inf ection at both doses administered were strikingly different from those exhi bited by wt virus, The central nervous system of wt-infected mice exhibited damage to the endothelium and recruitment of inflammatory cells, whereas t he central nervous system of pmE314-infected mice showed no inflammatory re sponse and only mild signs of endothelial damage.