Phylogenetic prediction of cis-acting elements: a cre-like sequence in Norovirus genome?

The complete coding sequences of NoV ORF1 were obtained from the DDBJ database and aligned. Shannon entropy calculations and RNAalifold consensus RNA structure prediction identified a discrete, conserved, invariant sequence region with a characteristic AAACG cre motif at positions 240 through 291 of the RNA dependant RNA polymerase (RdRp) sequence (relative to strain [EMBL:EU794713]). This sequence region has a high probability to conform a stem-loop.A new predicted stem-loop has been identified near the 5' end of the RdRp of Human NoV genome. This is the same location recently reported for Hepatovirus cre stem-loop.Internal base pairing that creates stem-loops and other RNA structures places constraints on sequence variability in bases required for structure formation in the genome of RNA viruses. For instance, the Hepatitis C virus (HCV) genome has a marked suppression of synonymous codon variability within several evolutionary conserved stem-loops in the core and NS5B coding regions that demonstrate their role in virus replication [1-3]. Discrete RNA structures such as cis-acting replication elements (cre) in the coding region of human enteroviruses (HEVs) [4] and other viruses also create characteristic suppression of synonymous site variability (SSSV), similar to that observed in HCV [5,6]. Different phylogenetic methods have been developed to predict secondary structures in RNA viruses, like PFOLD [2,7] or Alifold [8], for high-resolution thermodynamic scanning, and like UNAFold [9] for detecting SSSV [2]. These methods have permitted to identify suitable genome regions for an in-depth experimental analysis allowing establishing the role of the identified secondary RNA structures in translation or replication. This approach has permitted to raise the hypothesis that when SSSV (i.e. highly conserved synonymous sites in a RNA virus genome sequence alignment) takes place in a sequence region with a high probability of conforming a secondary structure (i.e. high p