Bunyamwera virus can repair both insertions and deletions during RNA replication

Abstract

The genomic termini of RNA viruses contain essential cis-acting signals for such diverse functions as packaging, genome translation, mRNA transcription, and RNA replication, and thus preservation of their sequence integrity is critical for virus viability. Sequence alteration can arise due to cellular mechanisms that add or remove nucleotides from terminal regions, or, alternatively, from introduction of sequence errors through nucleotide misincorporation by the error-prone viral RNAdependent RNA polymerase (RdRp). To preserve template function, many RNA viruses utilize repair mechanisms to prevent accumulation of terminal alterations. Here we show that Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family of segmented negative-sense RNA viruses, also can repair its genomic termini. When an intact nontranslated region (NTR) was added to the anti-genomic 39 end, it was precisely removed, to restore both length and RNA synthesis function of the wild-type template. Furthermore, when nucleotides were removed from the anti-genome 39 end, and replaced with a duplicate and intact NTR, both the external NTR were removed, and the missing nucleotides were restored, thus, indicating that the BUNV RdRp can both remove and add nucleotides to the template. We show that the mechanism for repair of terminal extensions is likely that of internal entry of the viral RdRp during genome synthesis. Possible mechanisms for repair of terminal deletions are discussed.

Nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and RNA polymerization

“…This strategy would seem risky, given the functional sensitivity of terminal promoter sequences and the cellular response to dsRNA. However, BUNV is known to be able to repair partial deletions at its termini that could arise through nuclease attack (54), thus restoring wild-type sequence and functionality.…”

Attenuation of Bunyamwera Orthobunyavirus Replication by Targeted Mutagenesis of Genomic Untranslated Regions and Creation of Viable Viruses with Minimal Genome Segments

“…In order to understand the mechanism behind the regain of fitness, the entire nucleotide sequences of a number of viral genomes were determined to assess the extent of mutation. A previous in vitro study using a minigenome system showed that BUNV was able to repair both insertions and deletions in its UTRs (28). Therefore, the first concern was to ensure that the UTRs were unchanged and the deletions were still present after 10 passages.…”

Bunyavirus: Structure and Replication

“…Walter and Barr recently discovered that the RdRp of BUNV is capable of correcting sequence alternations to the NTRs (Walter and Barr 2010 ) . When an intact NTR was introduced into the antigenomic 3 ¢ end, it was precisely removed, and the impaired RNA synthesis that resulted from the NTR addition was restored.…”

“…Likewise, when nucleotides were deleted from the antigenomic 3 ¢ end and replaced with a duplicate and intact NTR, the inserted NTR was removed and the missing nucleotides were reinstated. Furthermore, Walter and Barr demonstrated that the repair of NTRs is likely accomplished by direct entry of the RdRp into the internal NTRs during replication (Walter and Barr 2010 ) . However, the precise repair mechanism employed by RdRp remains unclear at this point.…”