S. Yu. Morozov

Conserved and variable elements in RNA genomes of potexviruses

The nucleotide sequences of genomic RNAs and predicted amino acid sequences of two strains of potato virus X and white clover mosaic potexvirus were compared to each other, and the proteins of different plus‐RNA‐containing plant viruses. The predicted non‐virion proteins of potexviruses have direct sequence homology and common structural peculiarities with those of several ‘Sindbis‐like’ plant viruses. The most conserved amino acid sequences were found to be located in the polypeptide encoded by the long 5′‐proximal open reading frame (ORF1). The putative polypeptide encoded by the ORF2 starting beyond the ORF1 stop codon is clearly related to the presumptive NTP‐binding domain of the ORF1‐coded polypeptide. These results suggest possible functions for all of the potexvirus proteins and also indicate that potexviruses have a genome organization which is considerably different from that of other plant viruses.

Cell-to-cell movement of potato virus X involves distinct functions of the coat protein.

Complementation of movement-deficient potato virus X (PVX) coat protein (CP) mutants, namely PVX.CP-Xho lacking the 18 C-terminal amino acid residues and PVX.DeltaCP lacking the entire CP gene, was studied by transient co-expression with heterologous proteins. These data demonstrated that the potyvirus CPs and both the major and minor CPs of beet yellows closterovirus could complement cell-to-cell movement of PVX.CP-Xho but not PVX.DeltaCP. These data also indicated that the C-terminally truncated PVX CP lacked a movement function which could be provided in trans by the CPs of other filamentous viruses, whereas another movement determinant specified by some region outside the most C-terminal part of the PVX CP could not be complemented either by potyvirus or closterovirus CPs. Surprisingly, the CP of spherical cocksfoot mottle sobemovirus rescued all of the PVX CP movement functions, complementing the spread of PVX.CP-Xho and, to a lesser extent, PVX.DeltaCP. Both these mutants were also rescued by the tobacco mosaic virus (TMV) movement protein (MP). To shed light on the movement function of PVX CP, attempts were made to complement PVX.CP-Xho by a series of TMV MP mutants. An internal deletion abolished complementation, suggesting that the internal region of TMV MP, which includes a number of overlapping functional domains important for cell-to-cell transport, provides an activity complementing movement determinant(s) specified by the C-terminal region of PVX CP.

Complete nucleotide sequence and genome organization of a tobamovirus infecting cruciferae plants

Genomic RNA sequence of a tobamovirus infecting cruciferae plants (cr‐TMV) was determined. The RNA is composed of 6312 nucleotides and contains four ORFs encoding the proteins of 122K (ORF1), 178K (ORF2), 29K (ORF3) and 18K (capsid protein, ORF4). ORF4 overlaps ORF3 by 74 nucleotides and the overlapping region can be folded into a stable hairpin structure. The 3′‐terminal region of the cr‐TMV RNA preceding the tRNA‐like structure was shown to form six potentially stable pseudoknots.

Expression and biochemical analyses of the recombinant potato virus X 25K movement protein

The 25K movement protein (MP) of potato virus X (PVX) is encoded by the 5′‐proximal gene of three overlapping MP genes forming a ‘triple gene block’. The PVX 25K MP (putative NTPase‐helicase) has been synthesized in Escherichia coli as a recombinant containing a six‐histidine tag at the amino terminus. The His‐tagged 25K protein was purified in a one‐column Ni‐chelate affinity chromatography procedure. In the absence of any other viral factors, this protein had obvious Mg2+‐dependent ATPase activity, which was stimulated slightly (1.7–1.9‐fold) by various polynucleotides. Like other viral proteins possessing ATPase‐helicase motifs and many plant viral movement proteins, the PVX 25K MP was able to bind nucleic acids in vitro. The RNA binding activity of the 25K MP was pronounced only at very low salt concentrations and was independent of its ATPase activity.

Expression strategy of the potato virus X triple gene block.

The mode of expression of the overlapping genes of the triple block positioned internally in potato virus X (PVX) RNA was examined. The results of In vitro translation of synthetic RNA transcripts and natural PVX-specific methylmercuric hydroxide-denatured dsRNAs suggest that the 25K protein is expressed as a single translation product of the 2.1 kb subgenomic (sg) RNA and that both the 12K and 8K proteins are expressed from the same 1.4 kb sgRNA.

Dual-colour imaging of membrane protein targeting directed by poa semilatent virus movement protein TGBp3 in plant and mammalian cells.

The movement function of poa semilatent hordeivirus (PSLV) is mediated by the triple gene block (TGB) proteins, of which two, TGBp2 and TGBp3, are membrane proteins. TGBp3 is localized to peripheral bodies in the vicinity of the plasma membrane and is able to re-direct TGBp2 from the endoplasmic reticulum (ER) to the peripheral bodies. For imaging of TGBp3-mediated protein targeting, PSLV TGBp3 tagged with a red fluorescent protein (DsRed) was used. Coexpression of DsRed-TGBp3 with GFP targeted to the ER lumen (ER-GFP) demonstrated that ER-GFP was contained in typical ER structures and peripheral bodies formed by TGBp3 protein, suggesting an ER origin for these bodies. In transient coexpression with viral membrane proteins tagged with GFP, DsRed-TGBp3 directed to the peripheral bodies the homologous TGBp2 protein and two unrelated membrane proteins, the 6 kDa movement protein of beet yellows closterovirus and the putative movement protein encoded by the genome component 4 of faba bean necrotic yellows nanovirus. However, coexpression of TGBp3 with GFP derivatives targeted to the ER membranes by artificial hydrophobic tail sequences suggested that targeting to the ER membranes per se was not sufficient for TGBp3-directed protein trafficking to peripheral bodies. TGBp3-induced targeting of TGBp2 also occurred in mammalian cells, indicating the universal nature of the protein trafficking signals and the cotargeting mechanism.

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.

A study of TMV ts mutant Ni2519. I. Complementation experiments.

Two distinct virus-specific functions, i.e., virus assembly and spreading of infection from cell to cell (transport function), are temperature-sensitive (ts) in TMV mutant Ni2519. Assembly of Ni2519 cannot be complemented by the temperature-resistant TMV strains used: A14 (a wild type strain from which Ni2519 was derived) and dolichos enation mosaic virus (DEMV, or cowpea strain of TMV), a thermophilic strain. On the other hand, Ni2519 can serve as a donor of the coat protein to complement is strain Ni118, which has a mutation in the coat protein gene. The genomic RNA can be produced by Ni2519 at a nonpermissive temperature; functionally active Ni2519 coat protein (capable of coating Ni118 RNA upon mixed infection) is produced at a nonpermissive temperature as well. The is phenotype of Ni2519 upon virus assembly probably results not from the ts behavior of any virus-coded protein(s) but is due to the ts properties of the genomic RNA molecule itself, so the possibility of the complementation of assembly of Ni2519 is ruled out. Thus, Ni2519 appears to represent a novel class of virus mutants with is virion RNA. The second is function of Ni2519 (transport of infection) can be complemented by a helper virus. The experimental system used for complementation of the transport function allowed Ni2519 to spread from cell to cell at a nonpermissive temperature. Obviously, Ni2519 infection spreads under these conditions in a form different from that in the mature virions, since its assembly cannot be complemented by the helper virus. Some aspects of the transport function are discussed.

HOST-CONTROLLED CELL-TO-CELL MOVEMENT OF A HYBRID BARLEY STRIPE MOSAIC VIRUS EXPRESSING A DIANTHOVIRUS MOVEMENT PROTEIN

The triple gene block (TGB) of barley stripe mosaic virus (BSMV), coding for viral movement proteins (MPs), was replaced by the single MP gene of red clover necrotic mosaic virus (RCNMV). Accumulation of the hybrid virus in barley plants (the selective host for BSMV) was reduced compared to BSMV. The hybrid virus induced small necrotic local lesions on Chenopodium amaranticolor leaves and did not infect Nicotiana clevelandii (the selective host for RCNMV). The hybrid virus accumulated in the inoculated leaves of Nicotiana benthamiana, but not in the upper noninoculated leaves. Thus the RCNMV MP gene substituted for the BSMV TGB in cell-to-cell movement, but not in systemic spread. Hybrid virus movement was efficient only in N. benthamiana, the common host for BSMV and RCNMV. These data point to the involvement of host-specific factors in the function of virus-coded transport determinants.