Ryegrass mottle virus complete genome determination and development of infectious cDNA by combining two methods - 3' RACE and 5' RACE-seq

Sobemovirus ryegrass mottle virus (RGMoV) is a single-stranded positive virus with a 30 nm viral particle size. It exhibits T=3 symmetry, with 180 coat protein (CP) subunits forming the virus structure. The RGMoV genome comprises five open reading frames, encoding P1, Px, a membrane-anchored 3C-like serine protease, a virus genome-linked protein, P16, an RNA-dependent RNA polymerase, and a coat protein. The RGMoV genome size varies, ranging from 4175 nt (MW411579.1) to 4253 nt (MW411579.1) in deposited sequences. An earlier deposited RGMoV complete genome sequence of 4212 nt length (EF091714.1) was utilized to develop an infectious complementary DNA (icDNA) construct for in vitro gRNA transcription from the T7 promoter. However, when the transcribed gRNA was introduced to oat plants, it failed to induce viral infection. This indicated the potential absence of certain sequences in either the 5 or 3 untranslated regions (UTR) or both. To resolve this, the complete sequence of the 3 UTR was determined through 3 end RACE, while the 5 UTR was identified using high-throughput sequencing (HTS) - 5 RACE-seq. Only the icDNA vector containing both newly identified UTR sequences proved infectious, resulting in classical viral infection symptoms and subsequent propagation of progeny viruses, exhibiting the ability to cause repeated infection in oat plants after at least one passage. The successful generation of the icDNA highlights the synergistic potential of utilizing both methods when one approach alone fails. Furthermore, this study demonstrates the reliability of HTS as a method for determining the complete genome sequence of viral genomes.