Virus Genes

, Volume 35, Issue 3, pp 651–662 | Cite as

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

  • Frederick D. James
  • Katie A. Hietala
  • Dganit Eldar
  • Tiffany E. Guess
  • Cecil Cone
  • Nathan Mundall
  • Joey V. Barnett
  • Ramaswamy Raju
Article
First Online:
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Accepted:

Abstract

Sindbis virus (SIN) is a mosquito-transmitted animal RNA virus. We previously reported that SIN genomes lacking a canonical 19 nt 3′CSE undergo novel repair processes in BHK cells to generate a library of stable atypical SIN genomes with non-canonical 3′A/U-rich elements (NC3AREs) adjacent to the 3′ poly(A) tail [1]. To determine the stability and evolutionary pressures on the SIN genomes with NC3AREs to regain a 3′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′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′ oligo(U) motifs to add more U residues within the NC3AREs; (e) the S3–8 isolate with an internal UAUUU motif in the 3′poly(A) tail maintains this element even after 9 passages in animals; (f) despite differences in 3′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′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.

Keywords

Sindbis RNA genome A/U-rich 3′NTR Alphavirus Replication Neonatal mice 
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Notes

Acknowledgements

This work was supported by NIH grant GM57439, and MBRS-SCORE institutional program. We acknowledge the use of Molecular Biology Core Facility at Meharry, RCMI supported institutional facilities at Meharry, Vanderbilt-Meharry Alliance resources, and Vanderbilt University core facilities including DNA Core facility. We thank Lee Limbird, Diana Marver, Joel Trupin of Meharry and Grady Dwar, Louise Trent, and Palani Nalliappan of RNA-Net for their helpful comments. We also acknowledge the idea from Amiya K. Banerjee (Cleveland) that alphavirus and other RNA replicases carry A/U-rich RNAs as prosthetic components to use them as templates, to synthesize, and add A/U-rich motifs to RNA genomes.

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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Frederick D. James
    • 1
  • Katie A. Hietala
    • 1
  • Dganit Eldar
    • 1
  • Tiffany E. Guess
    • 1
  • Cecil Cone
    • 2
  • Nathan Mundall
    • 3
  • Joey V. Barnett
    • 3
  • Ramaswamy Raju
    • 1
  1. 1.Department of Biomedical Sciences, Division of Microbial Pathogenesis and Immune ResponseMeharry Medical College, School of MedicineNashvilleUSA
  2. 2.Department of PathologyMeharry Medical College, School of MedicineNashvilleUSA
  3. 3.Department of PharmacologyVanderbilt University School of MedicineNashvilleUSA

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