N.P. Rodionova

Mutagenic analysis of Potato Virus X movement protein (TGBp1) and the coat protein (CP): in vitro TGBp1–CP binding and viral RNA translation activation

Previously, we have shown that encapsidated Potato virus X (PVX) RNA was non-translatable in vitro, but could be converted into a translatable form by binding of the PVX movement protein TGBp1 to one end of the virion or by coat protein (CP) phosphorylation. Here, a mutagenic analysis of PVX CP and TGBp1 was used to identify the regions involved in TGBp1-CP binding and translational activation of PVX RNA by TGBp1. It was found that the C-terminal (C-ter) 10/18 amino acids region was not essential for virus-like particle (VP) assembly from CP and RNA. However, the VPs assembled from the CP lacking C-ter 10/18 amino acids were incapable of TGBp1 binding and being translationally activated. It was suggested that the 10-amino-acid C-ter regions of protein subunits located at one end of a polar helical PVX particle contain a domain accessible to TGBp1 binding and PVX remodelling. The non-translatable particles assembled from the C-ter mutant CP could be converted into a translatable form by CP phosphorylation. The TGBp1-CP binding activity was preserved unless a conservative motif IV was removed from TGBp1. By contrast, TGBp1-dependent activation of PVX RNA translation was abolished by deletions of various NTPase/helicase conservative motifs and their combinations. The motif IV might be essential for TGBp1-CP binding, but insufficient for PVX RNA translation activation. The evidence to discriminate between these two events, i.e. TGBp1 binding to the CP-helix and TGBp1-dependent RNA translation activation, is discussed.

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

The double‐stranded DNA copy corresponding to the 5′‐nontranslated αβ‐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 β‐glucuronidase (GUS) gene. Expression of the reporter genes in vitro and in plant protoplasts (in the case of GUS gene) reveals that the αβ‐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.

Effects of sequence elements in the potato virus X RNA 5' non-translated αβ-leader on its translation enhancing activity

The 5′ non-translated αβ-leader sequence of potato virus X RNA consists of two regions: the α sequence (41 nucleotides with no G) and the β sequence (42 nucleotides upstream from AUG). The αβ-leader has been shown to enhance strongly the expression of adjacent genes in chimeric mRNAs. This phenomenon has been postulated to be due to the unpaired conformation of the 5′-terminal 30 nucleotides and/or to the presence within the α region of the CCACC pentanucleotide complementary to the 3′-terminal conserved structure of 18S rRNA. Different derivatives of αβ-leader have been constructed for use in determining the contribution of separate elements of the αβ sequence to translational enhancement. It was found that deletion of the α sequence large fragment which was supposed to be unfolded did not reduce the Δαβ-leader enhancement activity. Moreover, translational enhancement was greater for this derivative. Deletion of the β sequence resulted in a considerable increase in activity of the α-leader showing that the β region was dispensable for translation. Disruption or ‘masking’ of CCACC led to inactivation of the αβ-leader as a translational enhancer. Thus, we identified the CCACC pentanucleotide as the primary motif responsible for the translation enhancing ability of αβ-leader.

Tritium planigraphy study of structural alterations in the coat protein of Potato virus X induced by binding of its triple gene block 1 protein to virions

Alterations in Potato virus X (PVX) coat protein structure after binding of the protein, encoded by the first gene of PVX triple gene block (triple gene block 1 protein, TGBp1), to the virions were studied using tritium planigraphy. Previously, it has been shown that TGBp1 molecules interact with the PVX particle end, containing the 5′‐terminus of PVX RNA, and that this interaction results in a strong decrease in virion stability and its transformation to a translationally active state. In this work, it has been shown that the interaction of TGBp1 with PVX virions leads to an increase of ∼ 50% in tritium label incorporation into the 176–198 segment of the 236‐residue‐long PVX coat protein subunit, with some decrease in label incorporation into the N‐terminal coat protein region. According to the new ‘sandwich’ variant of our recently proposed model of the three‐dimensional structure of the intravirus PVX coat protein, the 176–198 segment is assigned to the β‐sheet region located at the subunit surface, presumably participating in coat protein interactions with the intravirus RNA and/or in protein–protein interactions, whereas the N‐terminal coat protein region corresponds to the other part of the same β‐sheet. For the remaining segments of the PVX coat protein subunit, no significant difference between tritium incorporation into untreated and TGBp1‐treated PVX was observed. A detailed description of the ‘sandwich’ version of the intravirus PVX coat protein model is presented.

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 restults 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.

Addressed fragmentation of RNA molecules

Many problems of molecular biology require specific fragmentation of nucleic acids. For DNAs this can be done with the help of specific endonucleases (restriction enzymes). However, no reliable and efflcient methods are known for strictly specific cleavage of RNA molecules. This paper describes an experimental approach to addressed fragmentation of polyribonucleotides. It is known that RNase H splits RNA in RNA-DNA heteroduplexes [l] or in the heteroduplexes made of monotonous synthetic polyribonucleotides and

Regulation of RNA Translation in Potato Virus X RNA-Coat Protein Complexes: The Key Role of the N-Terminal Segment of the Protein

The efficiency of in vitro translation of the potato virus X (PVX) RNA was studied for viral ribonucleoprotein complexes (vRNP) assembled from the genomic RNA and the viral coat protein (CP). In vRNP particles the 5′-proximal RNA segments were encapsidated into the CP, which formed helical headlike structures differing in length. Translation of the PVX RNA was completely suppressed upon incubation with PVX CP and was activated within vRNPs assembled in vitro with two CP forms, differing in the modification of the N-terminal peptide containing the main phosphorylation site(s) for Thr/Ser protein kinases. It was shown that CP phosphorylation activates RNA translation within vRNPs and that the removal of the N-terminal peptide of CP suppresses activation, but CP still acts as a translational suppressor. This fact made it possible to suppose that the replacement of Ser/Thr by amino acid residues that are not subject to phosphorylation in the N-terminal peptide of CP of the mutant PVX (PVX-ST) completely inhibits RNA translation within vRNP. However, experiments disproved this assumption: PVX-ST RNA was efficiently translated within native virions, RNA of the wild-type (wt) PVX was efficiently translated in heterogeneous vRNP (wtRNA + PVX-ST CP), and the opposite result (repression of translation) was obtained for another heterogeneous vRNP (PVX-ST RNA + wtCP). Therefore, the N-terminal CP peptide located on the surface of the PVX virion or vRNP particles plays a key role in the activation of viral RNA translation.

Deletion of the Intercistronic Poly(A) Tract from Brome Mosaic Virus RNA 3 by Ribonuclease H and Its Restoration in Progeny of the Religated RNA 3

Summary The dicistronic genomic RNA 3 of brome mosaic virus (BMV) was used in experiments on site-specific cleavage by RNase H and subsequent religation of large BMV RNA 3 fragments with T4 RNA ligase. BMV RNA 3 was cleaved at the intercistronic poly(A) tract into two fragments: a long (L-BMV 3) 5′-terminal fragment (M r 0.40 × 106) containing the 3a gene, and a short (Sh-BMV 3) fragment (M r 0.28 × 106) containing the coat protein gene and the 3′-terminal tRNA-like tyrosine-accepting structure. Two or three adenylate residues were present at the 5′ end of Sh-BMV 3 and one adenylate at the 3′ end of L-BMV 3. After religation of these RNA fragments BMV RNA 3 was constructed with a deletion including the entire intercistronic poly(A) tract but not the flanking sequences. The religated RNA 3 replicated in wheat plants co-inoculated with BMV RNA 1 and RNA 2. The normal poly(A) tract was restored in progeny BMV RNA 3 during the course of replication.

Analysis of the role of the coat protein N-terminal segment in Potato virus X virion stability and functional activity

Previously, we have reported that intact Potato virus X (PVX) virions cannot be translated in cell-free systems, but acquire this capacity by the binding of PVX-specific triple gene block protein 1 (TGBp1) or after phosphorylation of the exposed N-terminal segment of intravirus coat protein (CP) by protein kinases. With the help of in vitro mutagenesis, a nonphosphorylatable PVX mutant (denoted ST PVX) was prepared in which all 12 S and T residues in the 20-residue-long N-terminal CP segment were substituted by A or G. Contrary to expectations, ST PVX was infectious, produced normal progeny and was translated in vitro in the absence of any additional factors. We suggest that the N-terminal PVX CP segment somehow participates in virion assembly in vivo and that CP subunits in ST virions may differ in structure from those in the wild-type (UK3 strain). In the present work, to test this suggestion, we performed a comparative tritium planigraphy study of CP structure in UK3 and ST virions. It was found that the profile of tritium incorporation into ST mutant virions in some CP segments differed from that of normal UK3 virions and from UK3 complexed with the PVX movement protein TGBp1. It is proposed that amino acid substitutions in ST CP and the TGBp1-driven remodelling of UK3 virions induce structural alterations in intravirus CPs. These alterations affect the predicted RNA recognition motif of PVX CP, but in different ways: for ST PVX, labelling is increased in α-helices 6 and 7, whereas, in remodelled UK3, labelling is increased in the β-sheet strands β3, β4 and β5.

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