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

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.

Alphavirus RNA synthesis and non-structural protein functions

“…However, it is also possible that both a templated and non-templated mechanism of polyadenylation and terminal addition are at play during an alphavirus infection, allowing repair and restoration of infectivity of damaged or defective viral genomes. Further work by Raju and colleagues has found that, despite the essential nature of the 39 UTR, genomes lacking significant portions of this element are capable of producing progeny by a novel repair mechanism (George & Raju, 2000;James et al, 2007;Raju et al, 1999). Synthetic genomes with a set of deletions to the 39 CSE, UTR and/or poly(A) tail were introduced into cells, and viable progeny were recovered from many of these.…”

Replication of alphaviruses requires a pseudoknot that involves the poly(A) tail

“…Can the pseudoknot model for the 3-CSE explain "Raju's revertants" that were obtained after transfection of severely debilitated mutants of the CSE? (Raju et al, 1999;George & Raju, 2000;James et al, 2007). Many of these mutants acquired alternating blocks of Us and As that were added to the 3'-end of the mutated CSEs by a cellular or the viral polymerase.…”

“…S2) suggests that the pseudoknot or the CSE is not essential for replication. However, the pseudoknot does contribute to viral fitness as wt viruses rapidly outcompeted these mutants and revertants during a co-infection (James et al, 2007).…”

Immunogenicity of a DNA-launched replicon-based canine parvovirus DNA vaccine expressing VP2 antigen in dogs

“…To determine the self-replicating ability of replicase-based CPV DNA vaccine, CPV-VP2 mRNA transcripts were quantified using real-time PCR in pAlpha-CPV-VP2-transfected BHK-21 and compared with non-replicating control plasmid (pAlpha-D3 0 UTR-CPV-VP2). The deletion of 3 0 UTR has made the plasmid non-replicating as the 3 0 UTR of positive sense RNA virus has been reported as essential element for initiation of anti-sense RNA template and replication (Richard and Charles, 2005;James et al, 2007). There were over 24 thousand times more CPV-VP2 mRNA transcripts in pAlpha-CPV-VP2-transfected BHK-21 cells compared to respective non-replicating control indicating the self-replicating ability of replicon-based CPV-VP2 DNA vaccine.…”