Conservation of the pentanucleotide motif at the top of the yellow fever virus 17D 3′ stem–loop structure is not required for replication

Abstract

The pentanucleotide (PN) sequence 59-CACAG-39 at the top of the 39 stem-loop structure of the flavivirus genome is well conserved in the arthropod-borne viruses but is more variable in flaviviruses with no known vector. In this study, the sequence requirements of the PN motif for yellow fever virus 17D (YFV) replication were determined. In general, individual mutations at either the second, third or fourth positions were tolerated and resulted in replication-competent virus. Mutations at the fifth position were lethal. Base pairing of the nucleotide at the first position of the PN motif and a nucleotide four positions downstream of the PN (ninth position) was a major determinant for replication. Despite the fact that the majority of the PN mutants were able to replicate efficiently, they were outcompeted by parental YFV-17D virus following repeated passages in double-infected cell cultures. Surprisingly, some of the virus mutants at the first and/ or the ninth position that maintained the possibility of forming a base pair were found to have a similar fitness to YFV-17D under these conditions. Overall, these experiments suggest that YFV is less dependent on sequence conservation of the PN motif for replication in animal cells than West Nile virus. However, in animal cell culture, YFV has a preference for the wt CACAG PN sequence. The molecular mechanisms behind this preference remain to be elucidated.

Functions of the 3′ and 5′ genome RNA regions of members of the genus Flavivirus

“…In contrast, only the G nt was found to be essential in the YFV-17D PN sequence 5′ CACAG 3′ (Silva, Molenkamp et al 2007). Although individual substitution of the other YFV virus PN nts did not impair virus replication, the mutant viruses were outcompeted by wild type virus in co-infected cells.…”

“…Mutational analysis of individual nts in the top loop of the 3′ SL in a WNV infectious clone or in a replicon showed that the majority were cis acting and that three of the nts (underlined, PN sequence in bold, 5′ cA C A G UG C 3′) were essential for virus replication but did not affect translation (Khromykh, Kondratieva et al 2003, Elghonemy, Davis et al 2005, Tilgner, Deas et al 2005, Silva, Molenkamp et al 2007). In contrast, only the G nt was found to be essential in the YFV-17D PN sequence 5′ CACAG 3′ (Silva, Molenkamp et al 2007).…”

Structure Determination of the Top‐Loop of the Conserved 3′‐Terminal Secondary Structure in the Genome of Flaviviruses

“…Extensive mutation studies are available on the top-loop in mosquito-borne viruses (WNV [7] and YFV [8] ). They demonstrate that the identity of the partially conserved U immediately downstream of the pentanucleotide is not essential for viral replication in eukaryotic cells.…”

“…The stem of Watson-Crick base pairs of this hairpin ends with a conserved GC base pair that closes the loop part of the secondary structure though it can efficiently be replaced by a UA in YFV. [8] Because the sequence of the pentanucleotide motif in this conserved 3'-terminal secondary structure of flaviviruses is considered as a viral determinant that is responsible for vector specificity, we performed a solution structure determination by NMR spectroscopy of this hairpin in the RNA genome for the three clusters of flaviviruses.…”

“…Mutation studies on the flavivirus-conserved pentanucleotide in the 3'-stem-loop of the West Nile [7] (WNV) and YFV, [8] which both belong to mosquito-borne viruses, were performed to determine the importance of the conserved pentanucleotide for replication in eukaryotic cells. The study revealed that the second, third, and fifth position in the sequence are a prerequisite for RNA synthesis, but not for viral translation of the WNV genome.…”