With these precedents in mind, in the present study, we investigated the effect of CPO on RSV biology. Because of the formal possibility that CPO might result in increased RSV virulence, due to the expected increased viral protein synthesis, these studies were performed in a viral backbone that was strongly attenuated by a genetically stabilized attenuating ts mutation in the L polymerase. CPO had no effect on multicycle virus replication However, replication was marginally reduced in two rodents models. In hamsters, CPO RSVs induced lower levels of serum RSV-neutralizing antibodies. Thus, CPO of an RNA virus for a mammalian host has paradoxical effects on virus replication and the adaptive humoral immune response. and/or family of the order. Its genome is a single-stranded negative-sense 15.2-kb RNA carrying 10 genes in the order 3-NS1-NS2-N-P-M-SH-G-F-M2-L-5, preceded by a short leader region and followed by a short trailer region. The M2 mRNA encodes Pyrroloquinoline quinone two proteins, M2-1 and M2-2, expressed from overlapping ORFs. The genes are each flanked by short gene start and gene end transcription signals and are transcribed as individual mRNAs by sequential transcription initiating at a single promoter in the leader region. As is typical for MMP13 (13). The I1314L mutation genetically stabilizes the 1313 mutation against deattenuation. The presence of this stabilized attenuating mutation thus provided safety against potentially increased virulence. The effects of CPO of RSV ORFs on virus biology were investigated. RESULTS Design of CPO rRSVs. We used gene synthesis and reverse genetics to produce 4 rRSVs, named Max A, Maximum B, Maximum L, and Maximum FLC (Fig. 1A), in which, respectively, 6, 2, 1, and 9 of the 11 RSV ORFs were recoded using a computer algorithm to achieve the most positive CPB based on utilization in the human being ORFeome (Fig. 1B). The patterns of ORFs subjected to codon pair optimization (CPO) in these four viruses were the same as were chosen for CPD in our earlier study (11). This large-scale recoding by codon pairs overrepresented in the human being ORFeome was done with no changes to amino acid coding and included only a small number of post-CPO Pyrroloquinoline quinone manual changes in synonymous codon utilization to remove excessive homopolymer tracts and sequences resembling RSV (GenScript). Each contained a single ORF encoding wt or CPO G or F protein (Fig. 6A) flanked 5 by a T7 promoter, the viral innovator region, and a gene start transcription signal and flanked 3 by a gene end transcription signal, the viral trailer region, and a self-cleaving ribozyme cloned into a pBluescript plasmid vector. The minigenome sequences were completely confirmed by Sanger sequencing. Transcription by T7 RNA Pyrroloquinoline quinone polymerase yielded a positive-sense copy of the minigenome. Open Pyrroloquinoline quinone in a separate windowpane FIG 6 Manifestation of G and F proteins from wt and CPO ORFs contained in minigenomes. Four RSV minigenomes were constructed that every contained a single gene having a wt or CPO ORF encoding RSV G or F. (A) Gene maps of the four cDNAs used to evaluate the manifestation of F and G. Each cDNA contained a single G or F ORF, either wt or CPO, under the control of wt G or wt F gene start (Gs) and gene end (Ge) transcription signals, with an upstream RSV innovator region (Le) and downstream trailer region (Tr). Each cDNA was cloned into a pBluescript plasmid vector, with the Le region preceded by a T7 promoter and the Tr region followed by a self-cleaving ribozyme (not shown), such that expression from the T7 RNA polymerase yielded a positive-sense RNA copy with right 3 and 5 ends. (B to F) The ability of the CPO versus wt F and G ORFs to express F and G was tested on BSR T7/5 cells that constitutively express the Pyrroloquinoline quinone T7 RNA polymerase. BSR T7/5 cells were transfected having a plasmid combination encoding the indicated minigenome, together with the four RSV support plasmids expressing the N, P, M2-1, and L proteins, which are necessary to reconstitute the disease polymerase complex that directs viral transcription and RNA replication. Mock-transfected cells were used as regulates. Cells were incubated.
