Coxsackievirus expression of the murine secretory protein interleukin-4 induces increased synthesis of immunoglobulin G1 in mice

We cloned the sequence encoding murine interleukin-4 (mIL-4), including the secretory signal, into the genome of CVB3/0, an artificially attenuated st rain of coxsackievirus B3, at the junction of the capsid protein 1D and the viral protease 2Apro. Two strains of chimeric CVB3 were constructed using, in one case, identical sequences to encode 2Apro cleavage sites (CVB3/0-mI L4/47) on either side of the inserted coding sequence and? in the other cas e, nonidentical sequences that varied at the nucleotide level without chang ing the amino acid sequences (CVB3-PL2-mIL4/46), Transfection of HeLa tells yielded progeny viruses that replicated with rates similar to that of the parental CVB3/0 strain, although yields of mIL-4-expressing strains were ap proximately 10-fold lower than those of the parental virus. Western blot an alysis of viral proteins isolated from HeLa cells inoculated with either st rain of chimeric virus demonstrated that the chimeric viruses synthesized c apsid protein 1D at approximately twofold-higher levels than the parental v irus. mIL-4 protein was detected by enzyme-linked immunosorbent assay (ELIS A) in HeLa cells inoculated with either strain of chimeric virus. Lysates o f HeLa cells inoculated with either chimeric virus induced the proliferatio n of the mIL-4-requiring murine MC-9 cell line, demonstrating biological ac tivity of the CVB3-expressed mIL-4, Reverse transcription (RT)-PCR analysis of viral RNA derived from sequential passaging of CVB3/0-mIL4/47 in HeLa c ells demonstrated deletion of the mIL-4 coding sequence occurring by the fo urth passage, while similar analysis of CVB3-PL2-mIL4/46 RNA demonstrated d etection of the mIL-4 coding sequence in the virus population through 10 ge nerations in HeLa cells. mIL-4 protein levels determined by ELISA were cons istent with the stability and loss data determined by RT-PCR analysis of th e passaged viral genomes. Studies of insert stability of CVB3-PL2-mIL4/46 d uring replication in mice showed the presence of the viral mIL-4 insert in pancreas, heart, and liver at 14 days postinfection, Comparison of the muri ne antibody responses to CVB3-PL2-mIL4/46 and the parental CVB3/0 strain de monstrated an increased level of CVB3-binding serum immunoglobulin GI in mi ce inoculated with CVB3-PL2-mIL4/46.