During FY2004, we continued our exploration of the biology and evolution of the RNase A family of ribonucleases, an unusual enzyme family that is restricted to vertebrate species. While the chemistry of these enzymes has been carefully elucidated, the biological role of this highly divergent family remains for the most part unexplored. Our laboratory remains in the forefront of these biological studies, building on our past history which includes such highlights as the molecular cloning of human EDN/RNase 2 (PNAS 1989), human ECP/RNase 3 (J Exp Med 1989), elucidation of the unusual evolution of this lineage in primates (Nature Genetics 1995) and in rodents (PNAS 2000), structure function analysis of human EDN (PNAS 2002), identification and molecular cloning of human RNase 6 (NAR 1996), human RNase 8 (NAR 2002), and characterization of human EDN and ECP as antiviral ribonucleases (JID 1998; NAR 1998a, NAR 1998b). The first of our three major papers on the RNase A superfamily for this reporting year (ref # 17, Human RNase 7 a new cationic ribonuclease of the RNase A superfamily) detailed the molecular cloning and characterization of human RNase 7. We found that the human RNase 7 gene is expressed in various somatic tissues including the liver, kidney, skeletal muscle and heart. Recombinant RNase 7 is ribonucleolytically active against yeast tRNA, as expected from the presence of eight conserved cysteines and the catalytic histidine-lysine- histidine triad which are signature motifs of this superfamily. The protein is atypically cationic with an isoelectric point (pI) of 10.5. Expression of recombinant RNase 7 in Escherichia coli completely inhibited the growth of the host bacteria, similar to what has been observed for the cationic RNase, eosinophil cationic protein (ECP/RNase 3, pI 11.4). An in vitro assay demonstrates dose-dependent cytotoxicity of RNase 7 against bacteria E.coli, Pseudomonas aeruginosa and Staphylococcus aureus. While RNase 7 and ECP/RNase 3 are both cationic and share this particular aspect of functional similarity, their protein sequence identity is only 40%. Of particular interest, ECP/RNase 3's cationicity is based on an (over)abundance of arginine residues, whereas RNase 7 includes an excess of lysine. This difference, in conjunction with the independent origins and different expression patterns, suggests that RNase 7 and ECP/RNase 3 may have been recruited to target different pathogens in vivo. The second paper documented the gene regulation of mouse eosinophil-associated ribonuclease 2 (mEar 2; ref #6 Identification of a purine-rich intronic enhancer element in the mouse eosinophil-associated ribonuclease 2 (mEar 2) gene), the prototypical ribonuclease of the mouse RNase 2/3 cluster. In this work, we demonstrated that the presence of non-coding exon 1 and the intron in tandem with a 361-bp 5' promoter of mEar 2 results in enhanced reporter gene expression, as much as 6-to 10-fold over the activity observed with the 5' promoter alone. We identified a conserved purine-rich element in the intron of the mEar 2 gene that was necessary for maximum transcription and that interacted specifically with NFAT-binding proteins in nuclear extracts derived from the mouse LA4 epithelial cell line. Similar intronic enhancers have been described as regulating transcription of the human EDN gene, suggesting an overall conservation of an important regulatory strategy. We have also evaluated the expression of mEar 2 in response to respiratory virus infection in vivo (ref #10 Diminished expression of an antiviral ribonuclease in response to pneumovirus infection in vivo; see Project Number AI000943-01LAD for more on this virus model). In the third paper under this project heading, we demonstrated that mEar 2 had antiviral properties simiar to its human ortholog, EDN, as micromolar concentrations promoted an approximately sixfold reduction in the infectivity of pneumonia virus of mice (PVM) for target respiratory epithelial cells in vitro. Although initially identified as a component of eosinophilic leukocytes, mEar 2 mRNA and protein were also detected in lung tissue accompanied by enzymatically active mEar 2 in bronchoalveolar lavage fluid (BALF). At t=3 days post-inoculation with PVM (strain J3666), we observed the characteristic inflammatory response accompanied by diminished expression of total mEar mRNA and protein in lung tissue and a corresponding fivefold drop in ribonuclease activity in BALF. No change in mEar expression was observed in response to infection with PVM strain 15, a replication-competent strain of PVM that does not elicit a cellular inflammatory response. However, mEar expression was not directly dependent on inflammation per se, as diminished expression of mEar mRNA and BAL ribonuclease activity were also observed in PVM-infected, inflammation-deficient, MIP-1alpha -/- mice. We proposed that this mechanism might represent a novel virus-mediated evasion strategy, with a mechanism that is linked in some fashion to virus-specific pathogenicity.
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