Molecular characterization of a gene from a novel rodent cytomegalovirus (PCMV) and the potential use of PCMV as an expression vector for hantavirus glycoproteins.

摘要

This dissertation describes the characterization and cloning of a virus homolog of a G-protein coupled receptor, from a newly discovered cytomegalovirus from Peromyscus maniculatis (PCMV), the deer mouse. In addition, it describes the construction and characterization of a recombinant PCMV, containing an expression cassette that consists of the GI glycoprotein of Sin Nombre Virus (SNV), fused to the enhanced green fluorescent protein (EGFP). This recombinant vector was constructed to induce an immune response in deer mice against SNV by heterologous expression of the SNV G1 glycoprotein. The UL-33 G-protein homolog from PCMV, designated P33, is a 415 amino acid protein homologous to cellular beta-chemokine receptors. The protein is encoded on a 1330 bp gene that consists of an upstream 33bp exon, separated by an intron of 85 bp from a second exon of 1212bp. Amino acid sequence analysis reveals homology to M33 in murine cytomegalovirus (MCMV), R33 in rat cytomegalovirus (RCMV) and UL-33 in human cytomegalovirus (HCMV) by 62.5%, 59.9% and 39.1%, respectively. Like its homologs in other CMV's, P33 is transcribed as a late gene and sensitive to Phosphonofonnic acid (PFA: Foskarnet). A recombinant PCMV (▴P33:EGFP-NIGI) was constructed, carrying a fusion protein that consists of a section of the M-segment of SNV, fused in frame to EGFP, in place of P33. The M-segment fragment consisted of all of the GI glycoprotein and a portion of the G2 glycoprotein. Embryonic cells from deer mice (PMEC), infected with PCMV (▴P33:EGFP-N1G1), show strong expression of an EGFP of approximately 27 kD, much smaller than 107 kD, the molecular weight of the expected fusion protein. Transcription of mRNA, including the 5 '-end of GI and the 5'-end of EGFP was, shown by RT-PCR. Northern Blots of PCMV(▴P33:EGFP-N1G1) RNA show a predominant EGFP transcript at ≈ 1kb and a minor transcript of approximately 3kb, the latter also being positive for hybridization with a GI probe. These results could represent a naturally occurring event of SNV M-segment transcription and RNA processing. The two different size bands could implicate mRNA cleavage, partial degradation or be indicative of an alternative transcription initiation site.