A Bioinformatic and Biochemical Analysis of Cruciviruses

Abstract

Cruciviruses are novel ssDNA viruses discovered through metagenomics and direct environmental DNA amplification and cloning. The genomes of cruciviruses suggest that gene transfer between RNA and DNA viruses occurred due to the presence of putative protein-encoding genes that are homologous to both ssRNA and ssDNA viruses. In order to gain a better understanding of this group of viruses both bioinformatic analyses and in vitro biochemical experiments were employed. The results of the bioinformatic analyses show that cruciviruses are a highly diverse group of ssDNA viruses. Their placement within established ssDNA phylogenies is difficult due to heterogeneity in their putative replication-associated protein (Rep) that exceeds that of other ssDNA viruses. The results of biochemical experiments show that the putative Rep of the first discovered crucivirus, Boiling Spring Lake RNA-DNA hybrid virus (BSL-RDHV), displays activities consistent with the initiation and completion of rolling circle replication of the ssDNA genome. Specifically, it is demonstrated that recombinant BSL-RDHV Rep is capable of ATP hydrolysis, binding the putative origin of replication, covalently attaching to ssDNA containing a putative nick site, and is released from this covalent attachment in the presence of a pre-formed acceptor ssDNA. Together, these results represent significant progress towards a better understanding of this novel group of viruses. While many questions regarding cruciviruses remain unanswered, this work will enable future research to better characterize the evolution and biochemical capacities of cruciviruses.