Efficient replication, and evolution of Sindbis virus genomes with non-canonical 3′A/U-rich elements (NC3ARE) in neonatal mice

• Published in: Virus genes 2007-12, Vol.35 (3), p.651-662
• Main Authors: James, Frederick D., Hietala, Katie A., Eldar, Dganit, Guess, Tiffany E., Cone, Cecil, Mundall, Nathan, Barnett, Joey V., Raju, Ramaswamy
• Format: Article
• Language: English
• Subjects: Animals; Biotechnology; Cell culture; Disease transmission; Genomes; Neonates; Plaques; Polyadenine; Polyadenylation; Replication; RNA viruses; Viremia
• ISSN: 0920-8569; 1572-994X
• DOI: 10.1007/s11262-007-0130-z

Sindbis virus (SIN) is a mosquito-transmitted animal RNA virus. We previously reported that SIN genomes lacking a canonical 19 nt 3[variant prime]CSE undergo novel repair processes in BHK cells to generate a library of stable atypical SIN genomes with non-canonical 3[variant prime]A/U-rich elements (NC3AREs) adjacent to the 3[variant prime] poly(A) tail [1]. To determine the stability and evolutionary pressures on the SIN genomes with NC3AREs to regain a 3[variant prime]CSE, five representative SIN isolates and a wild type SIN were tested in newborn mice. The key findings of this study are: (a) all six SIN isolates, including those that have extensive NC3AREs in the 3[variant prime]NTRs, replicate well and produce high titer viremia in newborn mice; (b) 7-9 successive passages of these isolates in newborn mice produced comparable levels of viremia; (c) while all isolates produced only small-sized plaques during primary infection in animals, both small- and large-sized plaques were generated in all other passages; (d) polymerase stuttering occurs on select 3[variant prime] oligo(U) motifs to add more U residues within the NC3AREs; (e) the S3-8 isolate with an internal UAUUU motif in the 3[variant prime]poly(A) tail maintains this element even after 9 passages in animals; (f) despite differences in 3[variant prime]NTRs and variable tissue distribution, all SIN isolates appear to produce similar tissue pathology in infected animals. Competition experiments with wt SIN and atypical SIN isolates in BHK cells show dominance of wt SIN. As shown for BHK cells in culture, the 3[variant prime]CSE of the SIN genome is not required for virus replication and genome stability in live animals. Since the NC3AREs of atypical SIN genomes are not specific to SIN replicases, alternate RNA motifs of alphavirus genome must confer specificity in template selection. These studies fulfill the need to confirm the long-term viability of atypical SIN genomes in newborn mice and offer a basis for exploring the use of atypical SIN genomes in biotechnology. [PUBLICATION ABSTRACT]