[Translational regulation of potato virus X RNA-coat protein complexes: the key role of a coat protein N-terminal peptide].

The efficiency of in vitro translation of potato virus X (PVX) RNA within vRNP complexes assembled from genomic RNA and viral CP was examined. The vRNP particles contain the 5'-proximal RNA segments encapsidated by helically arranged CP head-like portions heterogeneous in length and the CP-free RNA tail. Translation of RNA is completely repressed upon incubation with PVX CP and is accompanied by vRNP particles production. By contrast, translation is activated in vRNPs in vitro assembled using two CP forms, differing in the principals of their N-terminal peptides modification. The N-terminal peptide of PVX CP represents the major phosphorylation site(s) for Thr/Ser-specific protein kinases. It was shown that: (i) CP phosphorylation results in a translational activation of vRNP; (ii) removal of N-terminal peptide from CP abolished activation and CP retains the translation repressing ability. It was suggested that substitution of Ser/Thr residues by non-phosphorylated Ala/Gly in N-terminal peptide of the mutant CP will led to a complete inhibition of vRNP translation. However, opposite results were obtained in our experiments: (i) RNA of such mutant virus (PVX-ST) was efficiently translated within the virions; (ii) RNA of a wild-type (wt) PVX also efficiently translated in mixedly assembled vRNP "wt PVX RNA + PVX-ST CP"; (iii) opposite result (repression of translation) was obtained with "mixed" vRNP (PVX-ST RNA + wtPVX CP). Therefore, the N-terminal peptide located at the surface of the particle and of the particles plays a key role in translation activation of the RNA encapsidated in vRNP and native virions.

The 3'-untranslated region of brome mosaic virus RNA does not enhance translation of capped mRNAs in vitro.

The translation enhancing ability of cis-acting 3'-terminal untranslated region (3'-UTR) of brome mosaic virus (BMV) was examined. Two chimeric mRNA constructs translated in rabbit reticulocyte lysates contained the BMV coat protein (CP) gene and NPTI gene, respectively. It was shown that the 3'-UTR of BMV RNA enhanced the translational efficiency of uncapped but not capped messages.

Translation arrest of potato virus X RNA in Krebs-2 cell-free system: RNase H cleavage promoted by complementary oligodeoxynucleotides.

Translation arrest of genomic potato virus X (PVX) RNA promoted by complementary oligodeoxynucleotides in Krebs-2 cell-free system is described. 14-15 mer oligodeoxynucleotides complementary to the 5'-proximal cistron of PVX RNA were shown to induce specific truncation of the major non-structural polypeptide coded by PVX RNA. Evidence is presented that effective translational arrest of PVX RNA in the presence of complementary oligonucleotides results from the site-specific cleavage of RNA by endogenous RNase H intrinsic to the Krebs-2 extract. No similar translational arrest was found in the rabbit reticulocyte lysate cell-free system.

Translation enhancing properties of the 5'-leader of potato virus X genomic RNA.

The double-stranded DNA copy corresponding to the 5'-nontranslated alpha beta-leader of potato virus X (PVX) genomic RNA (positions -3 to-85 according to AUG initiator) was chemically synthesized and fused to the transcription plasmids containing three different reporter genes: neomycinphosphotransferase type II (NPT II) gene, Bacillus thuringiensis coleopteran-specific toxic protein gene and beta-glucuronidase (GUS) gene. Expression of the reporter genes in vitro and in plant protoplasts (in the case of GUS gene) reveals that the alpha beta-leader of PVX RNA acts as a translation enhancer despite the presence of the upstream vector-derived sequence and irrespective of the length of the spacer sequence preceding the reporter genes.

Site-specific enzymatic cleavage of TMV RNA directed by deoxyribo- and chimeric (deoxyribo-ribo)oligonucleotides.

The TMV RNA molecule can be cleaved at a single site by RNase H directed by chimeric oligo(deoxyribo-ribo)nucleotide with an internucleotide pyrophosphate bond.

Translational efficiency and competitive ability of mRNAs with 5'-untranslated alpha beta-leader of potato virus X RNA.

The 5'-untranslated leader sequence of potato virus X (PVX) RNA (63 nucleotides apart from cap-structure) consists of two sub-sequences referred to as alpha-sequence (41 nucleotides with no G) and beta-sequence (42 nucleotides upstream from the first AUG). Computer-based folding predictions suggest that the 5'-proximal region of alpha beta-leader is unstructured. The second structural feature of alpha beta-leader is the presence of the sequences apparently complementary to the 3'-terminal region of 18S rRNA. The alpha beta-leader has been shown to strongly enhance the translation of the contiguous foreign gene (NPT1) transcripts in cell-free translation systems from rabbit reticulocytes (RRL), wheat germ (WG) and Krebs-2 ascite cell extract (KA). In competitive translation PVX, RNA strongly inhibited tobacco mosaic virus (TMV) RNA, in RRL and WG systems. No competition occurred between PVX and TMV RNAs in KA system. There was no correlation between the translational efficiency and competitive ability of PVX RNA in different cell-free translation systems. The competitive ability did not solely depend on the presence of alpha beta-leader in mRNA. We present evidence to suggest that alpha beta-leader together with about 150 bases of the coding sequence is responsible for the translation competitive ability of PVX RNA.

[Directed fragmentation of tobacco mosaic virus RNA: excision of 3' terminal sections, containing coat protein genes of tobacco mosaic virus]. / Napravlennaia fragmentatsiia RNK virusa tabachnoi mozaiki: vyrezanie 3'-kontsevogo uchastka, soderzhashchego gen belka obolochki virusa tabachnoi mozaiki.

The present work demonstrated that RNA of different Tobacco mosaic virus strains (common; A14; Ni118, U2; wheat strain) are selectively cleaved by RNase H from E. coli in the presence of oligodeoxyribonucleotide d(TTTTTTTCCGG) complementary to the stretch of TMV RNA from the 842-nd to the 852-nd nucleotide from 3'-end, e. g. corresponding to the region adjacent to the 3'-terminus of origin of assemble of TMV. Such cleavage yields two main products. One of them--the short S-fragment--is the 3'-terminal part of TMV RNA. This conclusion is based on its ability to accept histidine and its molecular weight is about 0.28 X 10(6). The other main product of TMV RNA cleavage by RNase H is the L-fragment. L-fragment is the 5'-terminal moiety of TMV RNA, as follows from its ability to direct the in vitro synthesis of p110-130, which is known to be encoded in the 5'-terminal TMV RNA region.

[Addressed enzymatic fragmentation of RNA molecules]. / Napravlennyi fermentativnyi gidroliz RNK.

RNase H has been used for selective cleavage of RNA of MS2 and R17 bacteriophages and 16S RNA from E. coli ribosomes in the region of formation of heteroduplex composed of RNA and an oligodeoxyribonucleotide complementary to a certain part of it. The oligonucleotides used--d(C-T-C-A-T-G-T-T-), d(C-C-A-T-C-T-T-T-T) and d(T-T-T-C-C-A-T-C-T-T-T-T)--were synthesized by chemical methods. The molecular weight of the fragments produced on cleavage of the RNA of MS2 and R17 were estimated with the use of gel electrophoresis under denaturating conditions. The dependence of the enzyme activity on Mg2+ and Na+ concentration and of RNA cleavage on the RNA: oligodeoxyribonucleotide ratio was investigated.

[Translation of the genomic and subgenomic RNAs of cucumber virus 3 in a cell free system from wheat embryos and in Xenopus laevis oocytes]. / Transliatsiia genomnoi i subgenomnoi RNK ogurechnogo virusa 3 v beskletochnoi sisteme iz zarodyshei pshenitsy i v ootsitakh Xenopus laevis.

The purified preparation of cucumber virus 3 (CV3) (one of the member of tobamovirus group) contains, besides full-length (2.0 X 10(6)) genomic RNA, a short (0.24 X 10(6)) subgenomic RNA coding for coat protein in Xenopus laevis oocytes as well as in the cell-free protein synthesizing system from wheat embryos. The similarity of the polypeptide coded by short CV3 RNA in vitro to natural CV3 coat protein is proved by the similarity of their molecular weights, antigenic specificity and by the similarity of the peptide maps. Individual CV3 genomic RNA coded in vitro for polypeptide 130 X 10(3), and was incapable of coding for the coat protein. The results of translation of an artificial mixture of short and full-length RNAs suggest that it is the short RNA that is preferentially translated.

[Increased selectivity of directed RNA hydrolysis mediated by RNAse H from E. coli using mixed oligo(deoxyribo-ribo)nucleotides]. / Povyshenie izbiratel'nosti napravlennogo gidroliza RNK RNKazoi H iz Ecoli s pomoshch'iu smeshannykh oligo(dezoksiribo-ribo)-nukleotidov.

Chimeric oligo(deoxyribo-ribo)nucleotides appeared to be a valuable tool to achieve the high selectivity of RNA cleavage as shown by RNA-ase H-mediated hydrolysis of TMV RNA directed by d(TGTGTATGCC), d(TGTGTAT), d(TGTGTAT)GCCAU and d(TGTGTAT)ppGCCAU.

Characteristics of Artificial Virus-like Particles Assembled in vitro from Potato Virus X Coat Protein and Foreign Viral RNAs.

Potato virus X (PVX) and some other potexviruses can be reconstitutedin vitrofrom viral coat protein (CP) and RNA. PVX CP is capable of forming viral ribonucleoprotein complexes (vRNP) not only with homologous, but also with foreign RNAs. This paper presents the structure and properties of vRNP assembledin vitroupon incubation of PVX CP and RNAs of various plant and animal viruses belonging to different taxonomic groups. We have shown that the morphology and translational properties of vRNPs containing foreign (heterologous) RNA are identical to those of homological vRNP (PVX RNA - PVX CP). Our data suggest that the assembly of the "mixed" vRNPin vitrocould be started at the 5'-proximal region of the RNA, producing a helical structure of vRNPs with foreign nucleic acids. The formation of heterologous vRNPin vitrowith PVX CP appears not to require a specific 5' end RNA nucleotide sequence, and the PVX CP seems to be able to pack foreign genetic material of various sizes and compositions into artificial virus-like particles.

Potato virus X RNA-mediated assembly of single-tailed ternary 'coat protein-RNA-movement protein' complexes.

Different models have been proposed for the nature of the potexvirus transport form that moves from cell to cell over the infected plant: (i) genomic RNA moves as native virions; or (ii) in vitro-assembled non-virion ribonucleoprotein (RNP) complexes consisting of viral RNA, coat protein (CP) and movement protein (MP), termed TGBp1, serve as the transport form in vivo. As the structure of these RNPs has not been elucidated, the products assembled in vitro from potato virus X (PVX) RNA, CP and TGBp1 were characterized. The complexes appeared as single-tailed particles (STPs) with a helical, head-like structure composed of CP subunits located at the 5'-proximal region of PVX RNA; the TGBp1 was bound to the terminal CP molecules of the head. Remarkably, no particular non-virion RNP complexes were observed. These data suggest that the CP-RNA interactions resulting in head formation prevailed over TGBp1-RNA binding upon STP assembly from RNA, CP and TGBp1. STPs could be assembled from the 5' end of PVX RNA and CP in the absence of TGBp1. The translational ability of STPs was characterized in a cell-free translation system. STPs lacking TGBp1 were entirely non-translatable; however, they were rendered translatable by binding of TGBp1 to the end of the head. It is suggested that the RNA-mediated assembly of STPs proceeds via two steps. Firstly, non-translatable CP-RNA STPs are produced, due to encapsidation of the 5'-terminal region. Secondly, the TGBp1 molecules bind to the end of a polar head, resulting in conversion of the STPs into a translatable form.