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The number and diversity of viral sequences that are identified in metagenomic data far exceeds that of experimentally characterized virus isolates. In a recent workshop, a panel of experts discussed the proposal that, with appropriate quality control, viruses that are known only from metagenomic data can, and should be, incorporated into the official classification scheme of the International Committee on Taxonomy of Viruses (ICTV). Although a taxonomy that is based on metagenomic sequence data alone represents a substantial departure from the traditional reliance on phenotypic properties, the development of a robust framework for sequence-based virus taxonomy is indispensable for the comprehensive characterization of the global virome. In this Consensus Statement article, we consider the rationale for why metagenomic sequence data should, and how it can, be incorporated into the ICTV taxonomy, and present proposals that have been endorsed by the Executive Committee of the ICTV.

ABSTRACTThe single-stranded adeno-associated virus type 2 (AAV) genome is flanked by terminal repeats (TRs) that fold back on themselves to form hairpinned structures. During AAV DNA replication, the TRs are nicked by the virus-encoded Rep proteins at the terminal resolution site (trs). This origin function apparently requires three sequence elements, the Rep binding element (RBE), a small palindrome that comprises a single tip of an internal hairpin within the TR (RBE'), and the trs. Previously, we determined the sequences at the trsrequired for Rep-mediated cleavage and demonstrated that the trsendonuclease reaction occurs in two discrete steps. In the first step, the Rep DNA helicase activity unwinds the TR, thereby extruding a stem-loop structure at thetrs. In the second step, Rep transesterification activity cleaves the trs. Here we investigate the contribution of the RBE and RBE' during this process. Our data indicate that Rep is tethered to the RBE in a specific orientation duringtrsnicking. This orientation appears to align Rep on the AAV TR, allowing specific nucleotide contacts with the RBE' and directing nicking to the trs. Accordingly, alterations in the polarity or position of the RBE relative to the trsgreatly inhibit Rep nicking. Substitutions within the RBE' also reduce Rep specific activity, but to a lesser extent. Interestingly, Rep interactions with the RBE and RBE' during nicking seem to be functionally distinct. Rep contacts with the RBE appear necessary for both the DNA helicase and trscleavage steps of the endonuclease reaction. On the other hand, RBE' contacts seem to be required primarily for TR unwinding and formation of thetrsstem-loop structure, not cleavage. Together, these results suggest a model of Rep interaction with the AAV TR during origin nicking through a tripartite cleavage signal comprised of the RBE, the RBE', and the trs.

Sequence Read Archive submissions to the National Center for Biotechnology Information often lack useful metadata, which limits the utility of these submissions. We describe the Sequence Taxonomic Analysis Tool (STAT), a scalable k-mer-based tool for fast assessment of taxonomic diversity intrinsic to submissions, independent of metadata. We show that our MinHash-based k-mer tool is accurate and scalable, offering reliable criteria for efficient selection of data for further analysis by the scientific community, at once validating submissions while also augmenting sample metadata with reliable, searchable, taxonomic terms.

ABSTRACTThe single-stranded adeno-associated virus (AAV) genome is flanked by terminal hairpinned origins of DNA replication (terminal repeats [TRs]) that are nicked at the terminal resolution site (trs) by the AAV Rep protein in an ATP-dependent, site-specific manner. Here we determine the minimal trssequence necessary for Rep cleavage, 3'-CCGGT/TG-5', and show that this 7-base core sequence is required only on the nicked strand. We also identify a potential stem-loop structure at thetrs. Interestingly, Rep nicking on a TR substrate that fixes this trsstem-loop in the extruded form no longer requires ATP. This suggests that ATP-dependent Rep helicase activity is necessary to unwind the duplex trsand extrude the stem-loop structure, prior to the ATP-independent Rep transesterification reaction. The extrusion of origin stem-loop structures prior to nicking appears to be a general mechanism shared by plant and animal viruses and bacterial plasmids. In the case of AAV, this mechanism of TR nicking would provide a possible regulatory function.