Insertion position as well as the inserted TRS and gene sequences differentially affect the retention of foreign gene expression by simian hemorrhagic fever virus (SHFV)

Abstract

Recombinant SHFV infectious cDNA clones expressing a foreign gene from an additional sg mRNA were constructed. Two 3′ genomic region sites, between ORF4′ and ORF2b and between ORF4 and ORF5, were utilized for insertion of the myxoma M013 gene with a C-terminal V5 tag followed by one of the three inserted transcription regulatory sequences (TRS), TRS2′, TRS4′ or TRS7. M013 insertion at the ORF4′/ORF2b site but not the ORF4/ORF5 site generated progeny virus but only recombinant viruses with an inserted TRS2′ retained the entire M013 gene through passage four. Insertion of an auto-fluorescent protein gene, iLOV, with an inserted TRS2′ at the ORF4′/ORF2b site generated viable progeny virus and iLOV expression was maintained through passage eight. Although regulation of SHFV subgenomic RNA synthesis is complex, the ORF4′/ORF2b site, which is located between the two sets of minor structural proteins, is able to tolerate foreign gene insertion.

Insertion of Exogenous Genes within the ORF1a Coding Region of Porcine Astrovirus

“…Other advantages of iLOV include pH-stability and oxygen-independence. The expression of the small iLOV protein from many positive-sense single-stranded RNA viruses with small, compact, or overlapping genomes allows tracking of viral infection and replication in living cells [36][37][38]. In the present study, when iLOV was inserted into the sites identified in the CC, Vpg, and HVR domains, respectively; the only replicative and infectious recombinant clones found were the ones fused with HVR.…”

Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

“…The current location of the eGFP-expressing cassette is not ideal as even a few passages of rSHFV-eGFP resulted in loss of fluorescence, likely due to a compromise of the eGFP-encoding ORF. A study by Di et al indicated that such compromise can be circumvented by shuffling the ORF to different locations in the SHFV genome, replacing the type of encoded reporter, or both [ 52 ]. In addition to the creation of a more stable reporter-encoding SHFV for advanced in vitro studies, it would be useful to determine the pathogenicity of such a virus in vivo in a macaque host.…”