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

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.

VPg Impact on Ryegrass Mottle Virus Serine-like 3C Protease Proteolysis and Structure.

Sobemoviruses encode serine-like 3C proteases (Pro) that participate in the processing and maturation of other virus-encoded proteins. Its cis and trans activity is mediated by the naturally unfolded virus-genome-linked protein (VPg). Nuclear magnetic resonance studies show a Pro-VPg complex interaction and VPg tertiary structure; however, information regarding structural changes of the Pro-VPg complex during interaction is lacking. Here, we solved a full Pro-VPg 3D structure of ryegrass mottle virus (RGMoV) that demonstrates the structural changes in three different conformations due to VPg interaction with Pro. We identified a unique site of VPg interaction with Pro that was not observed in other sobemoviruses, and observed different conformations of the Pro ß2 barrel. This is the first report of a full plant Pro crystal structure with its VPg cofactor. We also confirmed the existence of an unusual previously unmapped cleavage site for sobemovirus Pro in the transmembrane domain: E/A. We demonstrated that RGMoV Pro in cis activity is not regulated by VPg and that in trans, VPg can also mediate Pro in free form. Additionally, we observed Ca2+ and Zn2+ inhibitory effects on the Pro cleavage activity.

Identification and Full Genome Analysis of the First Putative Virus of Sea Buckthorn (Hippophae rhamnoides L.).

The agricultural importance of sea buckthorn (SBT; Hippophae rhamnoides L.) is rapidly increasing. Several bacterial and fungal pathogens infecting SBT have been identified and characterized; however, the viral pathogens are not yet known. In this study, we identified, isolated, and sequenced a virus from a wild plantation of SBT for the first time. Sequence analysis of the obtained viral genome revealed high similarity with several viruses belonging to the genus Marafivirus. The genome of the new virus is 6989 nucleotides (nt) in length according to 5', 3' RACE (without polyA-tail), with 5' and 3' 133 and 109 nt long untranslated regions, respectively. The viral genome encoded two open reading frames (ORFs). ORF1 encoded a polyprotein of 1954 amino acids with the characteristic marafivirus non-structural protein domains-methyltransferase, Salyut domain, papain-like cysteine protease, helicase, and RNA-dependent RNA polymerase. ORF1 was separated from ORF2 by 6 nt, encoding the coat protein (CP) with typical signatures of minor and major forms. Both CP forms were cloned and expressed in a bacterial expression system. Only the major CP was able to self-assemble into 30 nm virus-like particles that resembled the native virus, thus demonstrating that minor CP is not essential for virion assembly.

Neutralization of MERS coronavirus through a scalable nanoparticle vaccine.

MERS-CoV continues to cause human outbreaks, so far in 27 countries worldwide following the first registered epidemic in Saudi Arabia in 2012. In this study, we produced a nanovaccine based on virus-like particles (VLPs). VLPs are safe vaccine platforms as they lack any replication-competent genetic material, and are used since many years against hepatitis B virus (HBV), hepatitis E virus (HEV) and human papilloma virus (HPV). In order to produce a vaccine that is readily scalable, we genetically fused the receptor-binding motif (RBM) of MERS-CoV spike protein into the surface of cucumber-mosaic virus VLPs. The employed CuMVTT-VLPs represent a new immunologically optimized vaccine platform incorporating a universal T cell epitope derived from tetanus toxin (TT). The resultant vaccine candidate (mCuMVTT-MERS) is a mosaic particle and consists of unmodified wild type monomers and genetically modified monomers displaying RBM, co-assembling within E. coli upon expression. mCuMVTT-MERS vaccine is self-adjuvanted with ssRNA, a TLR7/8 ligand which is spontaneously packaged during the bacterial expression process. The developed vaccine candidate induced high anti-RBD and anti-spike antibodies in a murine model, showing high binding avidity and an ability to completely neutralize MERS-CoV/EMC/2012 isolate, demonstrating the protective potential of the vaccine candidate for dromedaries and humans.