Ryegrass mottle virus complete genome determination and development of infectious cDNA by combining two methods- 3' RACE and RNA-Seq.

Autor: Ina Balke, Ivars Silamikelis, Ilze Radovica-Spalvina, Vilija Zeltina, Gunta Resevica, Davids Fridmanis, Andris Zeltins
Jazyk: angličtina
Rok vydání: 2023
Předmět:
Zdroj: PLoS ONE, Vol 18, Iss 12, p e0287278 (2023)
Druh dokumentu: article
ISSN: 1932-6203
40369145
DOI: 10.1371/journal.pone.0287278
Popis: Ryegrass mottle virus (RGMoV; genus: Sobemovirus) is a single-stranded positive RNA virus with a 30 nm viral particle size. It exhibits T = 3 symmetry with 180 coat protein (CP) subunits forming a viral structure. The RGMoV genome comprises five open reading frames that encode P1, Px, a membrane-anchored 3C-like serine protease, a viral genome-linked protein, P16, an RNA-dependent RNA polymerase, and CP. The RGMoV genome size varies, ranging from 4175 nt (MW411579.1) to 4253 nt (MW411579.1) in the deposited sequences. An earlier deposited RGMoV complete genome sequence of 4212 nt length (EF091714.1) was used to develop an infectious complementary DNA (icDNA) construct for in vitro gRNA transcription from the T7 promoter. However, viral infection was not induced when the transcribed gRNA was introduced into oat plants, indicating the potential absence of certain sequences in either the 5' or 3' untranslated regions (UTR) or both. The complete sequence of the 3' UTR was determined through 3' end RACE, while the 5' UTR was identified using high-throughput sequencing (HTS)-RNA-Seq to resolve the potential absences. Only the icDNA vector containing the newly identified UTR sequences proved infectious, resulting in typical viral infection symptoms and subsequent propagation of progeny viruses, exhibiting the ability to cause repeated infections in oat plants after at least one passage. The successful generation of icDNA highlighted the synergistic potential of utilizing both methods when a single approach failed. Furthermore, this study demonstrated the reliability of HTS as a method for determining the complete genome sequence of viral genomes.
Databáze: Directory of Open Access Journals
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