On the stability of sequences inserted into viral genomes

Willemsen, Anouk; Zwart, Mark P

Poliovirus is a good candidate as a live viral vector for the expression of foreign genes, since the attenuated Sabin strains of poliovirus elicit strong protective immune responses without causing disease (Sabin 1957) . Insertions of up to 534 bp of the rotavirus VP7 gene into Sabin 3 poliovirus gave rise to infectious viruses that expressed portions of the VP7 outer capsid protein (Mattion et al. 1994 ). This is promising as antibodies generated to VP7 are able to neutralize the virus. Nevertheless the size of the insert in this construction is limited as only inserts of about 300 bp or smaller were stable upon serial passages in tissue culture, whereas larger insertions failed to produce infectious viruses (Mattion et al. 1994 ). One of the major limitations appears to be the polyprotein nature of the genome. The recombinant viruses expressing the inserted gene were found to be slower in the assembly of infectious virus particles, and showed smaller plaques and lower virus titers. This is possibly due to slow cleavage at the artificial cleavage sites around the insert (Mattion et al. 1994) . Sindbis virus, another ssRNA(þ) virus genome that encodes for a single ORF, accepted relatively large inserts of 3.2 kbp in the 11.8 kbp genome (Pugachev et al. 1995) . However, the recombinant Sindbis viruses appeared unstable and especially inserts at the 3 0 end were rapidly lost during serial passages, suggesting a positional effect.