Rescue of a 7502-nucleotide (49.3% of full-length) synthetic analog of respiratory syncytial virus genomic RNA

Abstract

A cDNA was constructed to encode an internally-truncated version of negative-sense genomic RNA (vRNA) of respiratory syncytial virus (RSV) containing (in 3' to 5' order) the 3' vRNA leader region, the complement of the open reading frame for bacterial chloramphenicol acetyl transferase (CAT) under the control of RSV gene-start and gene-end signals, an intergenic nucleotide, the complete L gene including the gene-start and gene-end signals, and the 5' vRNA trailer region. The encoded vRNA analog (RSV-CAT-L) would be 7502 nucleotides (nt) in length, 49.3% of the complete 15,222-nt parental vRNA. RSV-CAT-L vRNA was synthesized in vitro from HgaI-digested cDNA and transfected into RSV-infected cells. The vRNA was "rescued" such that it was expressed to yield CAT and was packaged into particles that could be passaged to express CAT in fresh cells. The efficiency of rescue was greatly improved by a single nucleotide substitution in the leader region that had been found to increase the efficiency of rescue and passage of the previously described 935-nt RSV-CAT vRNA. Compared to the 8-fold smaller RSV-CAT vRNA, and adjusted to molar equivalence of transfected RNA, RSV-CAT-L vRNA was 119- to 347-fold less efficient in expressing CAT upon transfection, whereas RSV-CAT-L vRNA containing the above-mentioned nucleotide substitution was 15.8-fold less efficient.