Synthetic Viral Genomics: Risks and Benefits for Science and Society Ralph S. Baric University of North Carolina at Chapel Hill
A second solution to large genome instability was developed using coronaviruses as models. Seven contiguous cDNA clones that spanned the 31.5 kilobase (kb) coronavirus
genome (e.g., mouse hepatitis virus [MHV] or SARS-CoV) were amplified, isolated and
ligated into standard polymerase chain reaction (PCR) cloning vectors (PCR is one
technique used to amplify sequences that are rare and/or not available in large quantities,
to provide enough material for subsequent experiments). The ends of the cDNAs were
engineered with unique junctions, generated by class IIS restriction endonucleases like
BglI or Esp3I. These enzymes leave asymmetric ends, which are designed to seamlessly
reproduce the exact virus sequence, allow directional assembly of adjacent cDNA
subclones, and direct the production of an intact full length cDNA construct of ~31.5 Kb
in length. With enzymes like Esp3I, interconnecting restriction site junctions can be
located at the ends of each cDNA and systematically removed during the assembly of the
complete full-length cDNA product (Figure 4a). The availability of a contiguous set of
DNAs containing unique interconnecting junctions provides for the systematic assembly
of large DNA molecules greater than 1,000,000 base pairs by in vitro ligation (85). In the
case of coronaviruses (Figure 4b), full length cDNAs are assembled that contain a T7
transcription site at the 5’ end of the genome. RNA transcripts driven from the full
length cDNA were infectious upon delivery into susceptible cells (85, 87). Alternatively,
coronavirus genomes can be stably cloned into BAC vectors. T7 or eukaryotic
promoters encoded upstream of the viral sequences allow for the synthesis of full length
RNA genome sequences, which are infectious upon introduction into cells (1). Seamless assembly (also called No See’m Sites (85)) cascades have been used to
assemble full length cDNAs of the coronaviruses mouse hepatitis virus, transmissible
gastroenteritis virus, infectious bronchitis virus and the SARS-CoV (85,86,87). Because
certain type IIS restriction endonucleases (e.g., Esp3I, AarI, Sap1) recognize asymmetric
binding sites and leave asymmetric ends, these enzymes can be used to create the unique
interconnecting junctions, which can be subsequently removed from the final assembly
product allowing for the seamless reconstruction of an exact sequence (Figure 4b). This
approach avoids the introduction of nucleotide changes that are normally associated with
building a full-length cDNA product of a viral genome. These non-palindrome restriction sites will also provide other novel recombinant DNA applications. For example, by PCR it will be possible to insert Esp3I or a related non-palindromic restriction site at any given
nucleotide in a viral genome and use the variable domain for simple and rapid site-
specific mutagenesis. By orientating the restriction sites as “No See’m”, the sites are
removed during reassembly, leaving only the desired mutation in the final DNA product.
The dual properties of strand specificity and a variable end overhang that can be tailored
to match any sequence allow for Esp3I sites to be engineered as “universal connectors”
that can be joined with any other four nucleotide restriction site overhangs (e.g. EcoRI,
PstX1, BamH1). Alternatively, “No See’m” sites can be used to insert foreign genes into
viral, eukaryotic, or microbial genome or vector, simultaneously removing all evidence of
the restriction sites that were used in the recombinant DNA manipulation. …………
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