5.6.1. Full-length viral RNA synthesis strategies
Full-length cloning of viral genomes has been a common tool in virology since the 1980’s (Taniguchi et al., 1978; Lowry et al., 1980; Racaniello and Baltimore, 1981), but it is often a long and tedious process, mostly due to the frequent instability of the full-length clones in bacteria (Boyer and Haenni, 1994). It is thought that cryptic bacterial promoters and secondary structures within the viral sequences encourage the bacteria to excise problematic viral regions from the plasmid clone. Full-length clones therefore have to be monitored constantly for possible deletions and re-arrangements as part of their maintenance. The process has been made easier by improvements in cloning techniques and the stability of the cloning vectors with respect to accepting and maintaining long (~10kb) inserts. There are several alternatives to cloning full-length genomes. Sometimes it is easier to clone the genome in several partial clones first, and then recombine these afterwards into a single full-length clone (Rodriguez et al., 2006).
Another strategy, used for viruses that prove impossible to clone full-length, is to clone the genome in two halves, which are maintained independently and then recombined in vitro prior to RNA transcription using a suitably engineered restriction site (Jakab et al., 1997). This improves the stability of the clones, the engineered restriction site can be used for recombining different virus strains for gene function analysis and also serves as a useful marker for tracing the virus through experiments.
A third alternative is to dispense with cloning altogether and generate infectious RNA transcripts directly off full-length PCR products that have a suitable recognition site for the T3, T7 or SP6 RNA polymerase incorporated into the full-length amplification primers, for transcript synthesis. This avoids the instability problems of cloned full-length clones but limits the extent to which the genomes can be manipulated genetically. This approach was successful for synthesizing full-length, infectious transcripts of BQCV after it proved impossible to successfully clone full-length BQCV (Benjeddou et al., 2002).