Translational in vitro activity of the 3a gene and the coat protein gene derived from brome mosaic virus RNA 3 by site-specific cleavage with RNase H

AbstractTwo translationally active fragments were derived from the dicistronic Brome Mosaic Virus (BMV) RNA 3 by site-specific cleavage with RNase H from E.coli: the 5′-proximal (L)fragment encoding the 32 kDa protein and the 3′-proximal (Sh) fragment carrying the coat protein gene. The translational efficiency of the L- and Sh-fragments was compared with those of the native BMV RNA 3 and RNA 4, encoding the 32 kDa and coat proteins, respectively. The Sh-fragment template activity was similar to that of RNA 4, although it was uncapped and contained 20–22 additional 5′-terminal nucleotides in comparison with BMV RNA 4

A Tobamovirus Genome That Contains an Internal Ribosome Entry Site Functionalin Vitro

AbstractMost eukaryotic mRNAs are translated by a “scanning ribosome” mechanism. We have found that unlike the type member of the genusTobamovirus,translation of the 3′-proximal coat protein (CP) gene of a crucifer infecting tobamovirus (crTMV) (Dorokhovet al.,1993; 1994) occurredin vitroby an internal ribosome entry mechanism. Three types of synthetic dicistronic RNA transcripts were constructed and translatedin vitro:(i) “MP-CP-3′NTR” transcripts contained movement protein (MP) gene, CP gene and the 3′-nontranslated region of crTMV RNA. These constructs were structurally equivalent to dicistronic subgenomic RNAs produced by tobamovirusesin vivo.(ii) “ΔNPT-CP” transcripts contained partially truncated neomycin phosphotransferase I gene and CP gene. (iii) “CP-GUS” transcripts contained the first CP gene and the gene ofEscherichia coliβ-glucuronidase (GUS) at the 3′-proximal position. The results indicated that the 148-nt region upstream of the CP gene of crTMV RNA contained an internal ribosome entry site (IRESCP) promoting internal initiation of translationin vitro.Dicistronic IRESCP, containing chimeric mRNAs with the 5′-terminal stem–loop structure preventing translation of the first gene (MP, ΔNPT, or CP), expressed the CP or GUS genes despite their 3′-proximal localization. The capacity of crTMV IRESCPfor mediating internal translation distinguishes this CP tobamovirus from the well-known-type member of the genus, TMV UI. The equivalent 148-nt sequence from TMV RNA was incapable of mediating internal translation. Two mutants were used to study structural elements of IRESCP. It was concluded that integrity of IRESCPwas essential for internal initiation. The crTMV provides a new example of internal initiation of translation, which is markedly distinct from IRESs shown for picornaviruses and other viral and eukaryotic mRNAs

Spherical particles derived from TMV virions enhance the protective properties of the rabies vaccine

In this study the ability of spherical particles (SPs) obtained from the tobacco mosaic virus (TMV) virions to enhance the immunogenic potential of the vaccine was evaluated. TMV SPs were shown to increase the protective properties of the widely used effective Russian adjuvant-free rabies vaccine, composed of killed rabies virions. The results of the NIH potency test showed enhancement of protectivity, that is comparable with the effect of the incomplete Freund׳s adjuvant on the same vaccine. Keywords: Rabies vaccine, Plant virus, Spherical particles, Adjuvan

Data in support of toxicity studies of structurally modified plant virus to safety assessment

This data article is related to the research article entitled “Assessment of structurally modified plant virus as a novel adjuvant in toxicity studies” (Nikitin et al., 2018), devoted to the safety study of structurally modified plant virus - spherical particles (SPs). SPs are generated by thermally denatured tobacco mosaic virus (TMV) coat protein and act as effective adjuvant for development of new vaccine candidates. This article reports the additional results on the toxicity studies of TMV SPs. The weight coefficients of laboratory animals internal organs complements the data of the subchronic toxicity studies. Also plaque-forming cell assay, delayed-type hypersensitivity test and peritoneal macrophage assay as a part of immunotoxicity studies of TMV SPs are presented. Keywords: Plant virus, Tobacco mosaic virus, Spherical particles, Toxicit