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.

[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.

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.

Translational activation of encapsidated potato virus X RNA by coat protein phosphorylation.

Previously we showed that encapsidated potato virus X (PVX) RNA is nontranslatable in vitro, but can be converted into a translatable form after binding to PVX particles of PVX-coded movement protein, the product of the first gene of triple gene block (TGBp1). Here we report that a similar effect occurs via in situ phosphorylation of the PVX coat protein (CP) by Ser/Thr protein kinase (PK) C, the mixture of casein kinases I and II or by cytoplasmic PK(s) from Nicotiana glutinosa leaves. Immunochemical analyses indicated that phosphorylation induced conformational changes in PVX CP. The N-terminal region of the PVX CP, rich in Ser and Thr residues, is exposed at the virion surface and can be removed by treatment with trypsin. We showed that (i) trypsin treatment removed the bulk of (32)P-radioactivity from in situ phosphorylated PVX CP, (ii) PVX containing N-terminally truncated CP (PVX-Ptd) failed to be translationally activated by phosphorylation, and (iii) the specific infectivity of PVX-Ptd was reduced. However, the PVX-Ptd RNA remained intact and PVX-Ptd could be translationally activated by the PVX MP TGBp1. We hypothesize that phosphorylation of the parental PVX by cytoplasmic PK(s) in vivo renders PVX RNA translatable in primary inoculated cells, whereas translational activation of the progeny virions destined for plasmodesmata trafficking is triggered by TGBp1.

The movement protein-triggered in situ conversion of potato virus X virion RNA from a nontranslatable into a translatable form.

Plant virus-encoded movement protein(s) (MP), and for many viruses the coat protein (CP), is required to mediate viral spread between plant cells via plasmodesmata (PD). Most probably, the genomic RNA of potexviruses moves through PD as assembled virions and/or as ribonucleoprotein complexes containing the CP and 25-kDa MP. Here we report that encapsidated potato virus X (PVX) virion RNA, which is nontranslatable in a cell-free protein synthesizing system, can be converted into a fully translatable form after interaction of intact PVX particles with the PVX 25-kDa MP. The 25-kDa MP molecules bind selectively to only one extremity of the viral particle (that presumably contains the 5' end of the genomic RNA). The process of complex formation is ATP-independent; i.e., the ATPase activity of the 25-kDa MP is not involved in the binding of the MP to PVX virion.

Internal initiation of translation directed by the 5'-untranslated region of the tobamovirus subgenomic RNA I(2).

Previously we reported that, unlike RNA of typical tobamoviruses, the translation of the coat protein (CP) gene of a crucifer-infecting tobamovirus (crTMV) in vitro occurred by an internal ribosome entry mechanism mediated by the 148-nt region that contained an internal ribosome entry site (IRES(CP,148)(CR)). The equivalent 148-nt sequence from TMV U1 RNA (U1(CP,148)(SP)) was incapable of promoting internal initiation. In the present work, we have found that the 228-nt region upstream of the movement protein (MP) gene of crTMV RNA (IRES(MP,228)(CR)) contained an IRES element that directed in vitro translation of the 3'-proximal reporter genes from chimeric dicistronic transcripts. Surprisingly, the equivalent 228-nt sequence upstream from the MP gene of TMV U1 directed translation of the downstream gene of a dicistronic transcripts as well. Consequently this sequence was termed IRES(MP,228)(U1). It was shown that IRES(MP,228)(CR), IRES(MP,228)(U1), and IRES(CP,148)(CR) could mediate expression of the 3'-proximal GUS gene from dicistronic 35S promoter-based constructs in vivo in experiments on transfection of tobacco protoplasts and particle bombardment of Nicotiana benthamiana leaves. The results indicated that an IRES element was located within the 75-nt region upstream of MP gene (IRES(MP,75)), which corresponded closely to the length of the 5'UTR of TMV subgenomic RNA (sgRNA) I(2). The RNA transcripts structurally equivalent to I(2) sgRNAs of TMV U1 and crTMV, but containing a hairpin structure (H) immediately upstream of IRES(MP,75) (HIRES(MP), (75)(CR)-MP-CP-3'UTR; HIRES(MP,75)(U1)-MP-CP-3'UTR), were able to express the MP gene in vitro. The capacity of HIRES(MP,75)(CR) sequence for mediating internal translation of the 3'-proximal GUS gene in vivo, in tobacco protoplasts, was demonstrated. We suggested that expression of the MP gene from I(2) sgRNAs might proceed via internal ribosome entry pathway mediated by IRES(MP) element contained in the 75-nt 5'UTR. Our results admit that a ribosome scanning mechanism of the MP gene expression from I(2) sgRNA operates concurrently.

Phosphorylation of tobacco mosaic virus movement protein abolishes its translation repressing ability.

Previously we showed that the ribonucleoprotein complexes (RNPs) of the TMV 30-kDa movement protein (MP) with TMV RNA are nontranslatable in vitro and noninfectious to protoplasts, but are infectious to intact plants. It has been suggested that MP-TMV RNA complexes could be converted into the translatable and replicatable form in planta in the course of passage through plasmodesmata (Karpova et al., 1997, Virology 230, 11-21). The role of TMV MP phosphorylation was investigated in terms of its capacity to modulate the translation-repressing ability of the MP. Phosphorylation of the TMV MP, either before or after RNP complex formation, caused a conversion of nontranslatable MP-RNA complexes into a form that was translatable in vitro and infectious to protoplasts and plants.

A tobamovirus genome that contains an internal ribosome entry site functional in vitro.

Most eukaryotic mRNAs are translated by a "scanning ribosome" mechanism. We have found that unlike the type member of the genus Tobamovirus, translation of the 3'-proximal coat protein (CP) gene of a crucifer infecting tobamovirus (crTMV) (Dorokhov et al., 1993; 1994) occurred in vitro by an internal ribosome entry mechanism. Three types of synthetic dicistronic RNA transcripts were constructed and translated in 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 tobamoviruses in vivo. (ii) "deltaNPT-CP" transcripts contained partially truncated neomycin phosphotransferase I gene and CP gene. (iii) "CP-GUS" transcripts contained the first CP gene and the gene of Escherichia coli beta-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 (IRES(CP)) promoting internal initiation of translation in vitro. Dicistronic IRES(CP), containing chimeric mRNAs with the 5'-terminal stem-loop structure preventing translation of the first gene (MP, deltaNPT, or CP), expressed the CP or GUS genes despite their 3'-proximal localization. The capacity of crTMV IRES(CP) for 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 IRES(CP). It was concluded that integrity of IRES(CP) was 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.

Nontranslatability and dissimilar behavior in plants and protoplasts of viral RNA and movement protein complexes formed in vitro.

It was found that the fusion (His)6-movement proteins (MPs) of two tobamoviruses (TMV UI and a crucifer-infecting tobamovirus, crTMV) were efficient nonspecific translational repressors. The in vitro translation of viral RNAs was blocked by incomplete 30K MP-RNA complexes formed at the MP:RNA molar ratios of 100-150:1. Similar results were obtained with the barley stripe mosaic hordeivirus (BSMV)-encoded 58K MP; however, the translation inhibiting activity of the 58K MP was manifested only in the presence of magnesium. By contrast, the 25K MP of potato virus X (PVX) was incapable of forming MP-RNA complexes under experimental conditions used and did not inhibit in vitro translation. The translation repressing ability correlated with the level of MP affinity to RNA. The complexes of the 30K MP and 58K MP with TMV RNA were not infectious in isolated protoplasts; however, they were infectious in indicator plants. Reduction of MP affinity to RNA resulted in translatability of MP-TMV RNA complexes that apparently was due to their destabilization. Thus, the deletion mutant DEL4 MP formed MP-TMV RNA complexes that were translatable in vitro, infectious to protoplasts and plants. In contrast to this, the complexes of TMV RNA with the mammalian RNA-binding protein p50 were nontranslatable and noninfectious to either protoplasts or intact plants. These results implied that nontranslatable MP-RNA complexes which could not replicate in the primary infected cells were converted into a translatable and replicatable form in the course of passage through plasmodesmata in planta.

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.

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

The 5' non-translated alpha beta-leader sequence of potato virus X RNA consists of two regions: the alpha sequence (41 nucleotides with no G) and the beta sequence (42 nucleotides upstream from AUG). The alpha beta-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 alpha region of the CCACC pentanucleotide complementary to the 3'-terminal conserved structure of 18S rRNA. Different derivatives of alpha beta-leader have been constructed for use in determining the contribution of separate elements of the alpha beta sequence to translational enhancement. It was found that deletion of the alpha sequence large fragment which was supposed to be unfolded did not reduce the delta alpha beta-leader enhancement activity. Moreover, translational enhancement was greater for this derivative. Deletion of the beta sequence resulted in a considerable increase in activity of the alpha-leader showing that the beta region was dispensable for translation. Disruption or 'masking' of CCACC led to inactivation of the alpha beta-leader as a translational enhancer. Thus, we identified the CCACC pentanucleotide as the primary motif responsible for the translation enhancing ability of alpha beta-leader.

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.