Sequences derived from parvoviruses (family Parvoviridae) are relatively common in animal genomes, but the functional significance of these endogenous parvoviral element (EPV) sequences remains unclear. In this study, we used a combination of in silico and molecular biological approaches to investigate a fusion gene carried by guinea pigs (genus Cavia) that is partially derived from an EPV. This gene, named enRep-M9l, encodes a predicted polypeptide gene product comprising a partial myosin9-like (M9l) gene fused to a 3′ truncated, EPV-encoded replicase. We examined the genomic and phylogenetic characteristics of the EPV locus (enRep) that encodes the viral portions of enRep-M9l, revealing that it derives from an ancient dependoparvovirus (genus Dependoparvovirus) that was incorporated into the guinea pig germ line between approximately 22 and 35 million years ago (MYA). Despite these ancient origins, the regions of the enRep locus that are expressed in the enRep-M9l gene are conserved across multiple species in the family Caviidae (guinea pigs and cavies), consistent with a potential function at the amino acid level. Using molecular biological approaches, we further demonstrated that (i) enRep-M9l mRNA is broadly transcribed in guinea pig cells, (ii) the cloned enRep-M9l transcript can express a protein of the expected size in guinea pig cells in vitro, and (iii) the expressed protein localizes to the cytosol. Our findings demonstrate that, consistent with a functional role, the enRep-M9l fusion gene is evolutionarily conserved, broadly transcribed, and capable of expressing protein. IMPORTANCE DNA from viruses has been “horizontally transferred” to mammalian genomes during evolution, but the impact of this process on mammalian biology remains poorly understood. The findings of our study indicate that a novel gene has evolved in guinea pigs through fusion of host and virus genes.

word count: 239 17 Text word count: 3128 18 2 Abstract 19Sequences derived from parvoviruses (family Parvoviridae) are relatively common in 20 animal genomes, but the functional significance of these endogenous parvoviral element (EPV) 21 sequences remains unclear. In this study we use a combination of in silico and molecular 22 biological approaches to investigate a fusion gene encoded by guinea pigs (genus Cavia) that is 23 partially derived from an EPV. This gene, named enRep-Myo9, encodes a predicted polypeptide 24 gene product comprising a partial myosin9 (Myo9)-like gene fused to a 3' truncated, EPV-25 encoded replicase. We first examined the genomic and phylogenetic characteristics of the EPV 26 locus that encodes the viral portions of enRep-Myo9. We show that this locus, named enRep, is 27 specific to guinea pigs and derives from an ancient representative of the parvoviral genus 28 Dependoparvovirus that integrated into the guinea pig germline 22-35 million years ago. Despite 29 these ancient origins, however, the regions of enRep that are incorporated into the coding 30 sequence of the enRep-Myo9 gene are conserved across multiple species in the family Caviidae 31 (guinea pigs and cavies) consistent with purifying selection. Using molecular biological 32 approaches, we further demonstrate that: (i) enRep-Myo9 mRNA is broadly transcribed in guinea 33 pig cells; (ii) the cloned enRep-Myo9 transcript can express a protein of the expected size in 34 guinea pig cells in vitro, and; (iii) the expressed protein localizes to the cytosol. Our findings 35 demonstrate that, consistent with a functional role, the enRep-Myo9 fusion gene is evolutionarily 36 conserved, broadly transcribed, and capable of expressing protein. 37 38 Importance 39DNA from viruses has been 'horizontally transferred' to mammalian genomes during 40 evolution, but the impact of this process on mammalian biology remains poorly understood. The 41 3 findings of our study indicate that in guinea pigs a novel gene has evolved through fusion of host 42 and virus genes. 43 44