Foreign complementary sequences facilitate genetic RNA recombination in brome mosaic virus

We have demonstrated that local antisense sequences can mediate genetic recombination within the 3' noncoding region among brome mosaic virus (BMV) RNAs (P. Nagy and J. J. Bujarski, 1993, Proc. Natl. Acad. Sci. USA 90, 6390-6394). Here we show that foreign complementary inserts can direct crossovers between BMV RNA3 components within an internal region. A 170-nt polynucleotide derived from the cowpea chlorotic mottle virus (CCMV) RNA3 was inserted just upstream of the initiation codon of the BMV coat protein open reading frame in either sense or antisense orientations. The resulting respective mutants, BCC+ and BCC-, maintained unchanged CCMV inserts when inoculated separately on leaves of a local lesion host for BMV. In contrast, when a mixture containing both mutated RNAs3 was inoculated, a significant fraction of lesions accumulated the BMV RNA3 lacking the CCMV insert. The presence of a 3' marker mutation confirmed that the BMV RNA3 progeny arose due to crossovers between BCC+ and BCC- within the complementary sequences. The highest frequency of recombinant appearance was observed when the RNA mixtures were annealed prior to inoculation on the host plants. Our results confirm a concept predicting the general nature of the heteroduplex-mediated recombination functioning in RNA viruses. Examples of possible applications of this approach in recombinant RNA technology are discussed.     

Application of GFP-tagged Plum pox virus to study Prunus-PPV interactions at the whole plant and cellular levels

The Sharka disease caused by the potyvirus Plum pox virus (PPV) is one of the most serious viral diseases affecting stone fruit trees. The study of PPV/Prunus interaction under greenhouse controlled conditions is space, time, labor consuming. While the PPV/Prunus interactions are now quite well known at the whole plant level, few data however are available on the interactions between the virus and the Prunus host plants at the cellular level. Using a green fluorescent protein (GFP)-tagged M type PPV strain, combined to an in vitro inoculation procedure, we developed a novel tool to track PPV invasion in Prunus persica (peach) cv. GF305 and Prunus armeniaca (apricot) cv. Screara susceptible hosts. Different graft combinations were performed using in vitro-maintained healthy or GFP-tagged PPV infected 'GF305' and 'Screara'. Contact for 30 days in grafts between the inoculum and the genotype to be tested were found sufficient to allow the systemic spread of the recombinant virus: fluorescence from GFP-tagged PPV could easily be detected in the entire plant under a binocular microscope allowing quick and reliable sorting of infected plants. Using a fluorescence stereomicroscopy or confocal microscopy, GFP could also be observed in stem cross-sections especially in epidermis and pith cells. In vitro grafting inoculation with GFP-tagged PPV provides a new and powerful tool to facilitate mid-term virus maintenance. Moreover, this tool will be of special importance in the study of PPV infection dynamics in Prunus, allowing as well precise observations of cellular events related to PPV/Prunus interactions.     

Tobacco mosaic virus replicase-mediated cross-protection: contributions of RNA and protein-derived mechanisms

Specific sequences of the tobacco mosaic virus (TMV) RNA-dependent RNA-polymerase (RdRp) gene were investigated for their ability to confer cross-protection. Nine overlapping segments ranging from 713 to 1070 nucleotides in length and covering the methyltransferase, helicase, and polymerase (POL) domains of the TMV RdRp open reading frame were systemically expressed in Nicotiana benthamiana using a potato X virus (PVX) vector [Chapman, S., Kavanagh, T., and Baulcombe, D. C. (1992). Plant J., 1, 549-557]. PVX-infected plants were subsequently challenge inoculated with 10 microg of wild-type TMV and monitored for TMV accumulation. Mock inoculated plants and plants preinfected with the unmodified PVX vector rapidly accumulated high levels of challenge virus. In contrast, plants preinfected with PVX vectors expressing segments of the TMV RdRp open reading frame displayed either high or low levels of protection. High protection levels were observed for PVX constructs expressing segments of the TMV POL domain, whereas low protection levels were observed for PVX constructs expressing segments covering the methyltransferase and helicase domains. Frameshift mutations that blocked protein expression from RdRp segments disrupted only the high levels of protection derived from POL segments and not the low levels derived from the other segments. However, all RdRp segments conferred similarly high levels of protection against a TMV construct with restricted local movement. Thus both RNA and protein sequences in conjunction with the speed of the infecting challenge virus can affect the protection derived from the TMV RdRp gene.     

Inhibition of replication of HIV-1 by retroviral vectors expressing tat-antisense and anti-tat ribozyme RNA.

A ribozyme was constructed that specifically cleaves RNA that contains the first coding exon of the tat gene of HIV-1. This anti-tat ribozyme was incorporated into a Moloney murine leukemia virus vector. A sequence containing only the 48-nucleotide antisense region of the ribozyme was also inserted into the retroviral vector. Human T-cell lines constitutively producing the tat-antisense and the anti-tat ribozyme RNA were created by transduction into Jurkat cells. When challenged with HIV-1, both the tat-antisense and anti-tat ribozyme-producing cells inhibited the replication of HIV-1. The antisense vector conferred a greater resistance to HIV-1 replication than did the anti-tat ribozyme vector.     

Enhancement by potato virus Y of potato virus X synthesis in doubly infected tobacco depends on the timing of invasion by the viruses

The time required for movement of potato virus Y (PVY) and two strains of potato virus X (PVX) from an inoculated lower leaf of small tobacco plants to the fourth leaf above, where maximal enhancement of PVX synthesis occurs, was determined. PVY required 12 hr longer than one PVX strain and 6 hr longer than the other; timing was from the time of inoculation of lower leaves. The rates of movement of the two viruses were the same in singly or doubly inoculated plants. Lower leaves were then inoculated with PVX and PVY at times so that invasion of fourth leaves by the two viruses occurred at predicted intervals. Maximum enhancement of one PVX strain occurred when PVY invaded fourth leaves 12 hr before PVX; enhancement of the other PVX strain occurred when PVY invaded 6 hr after PVX. Enhancement was always less than maximal when invasion by either virus preceded invasion by the other by more than 24 hr. These results were interpreted as indicating that enhancement occurs only in cells invaded by the two viruses within a relatively short period of time and that maximum enhancement results when critical stages in the replication cycle of each virus coincide; in such cells replication of both viruses was probably occurring simultaneously.     

Genetic stability of recombinant potato virus X virus vectors presenting foreign epitopes

Summary We investigated the genetic stability of recombinant potato virus X vectors presenting beet necrotic yellow vein virus (BNYVV) epitopes. Following N-terminal PVX coat protein (CP) fusion of the BNYVV epitopes, we inoculated Nicotiana benthamiana plants with recombinant (r)PVX and carried out five serial passages through systemically-infected plants. RT-PCR investigation of the BNYVV epitope sequences revealed the accumulation of several point mutations and deletions, predominantly affecting positively-charged residues. A comparison of the isoelectric point (pI) values and charges of the wild type and rCPs showed that the initial high rCP pI values had changed to values closer to that of the wild-type CP.     

Antiphytoviral activity of 2,4 dioxo hexahydro triazine.

Three treatments with 2,4 dioxo hexahydro triazine (DHT) significantly reduced the concentration of potato virus X (PVX) in systemically infected tobacco plants. In hypersensitive plants DHT caused a reduction in the number of local lesions produced by PVX. In systemic and hypersensitive hosts, treatment with DHT resulted in a more or less marked reduction in the concentration of, and in the number of local lesions caused by, potato virus Y (PVY), potato virus A (PVA), tobacco mosaic virus (TMV), and cucumber mosaic virus (CMV). Three days after treatment with DHT the incorporation of 32P into TMV RNA was significantly reduced, while that into host RNA was less affected.     

Tobravirus 2b protein acts in trans to facilitate transmission by nematodes

Analysis of RNA2 of TRV PaY4 showed it to be recombinant, carrying 3'-terminal sequences derived from RNA1. Virus produced using an infectious cDNA clone of PaY4 RNA2 was nematode transmissible, demonstrating that natural TRV recombinant isolates are not necessarily defective. Mutations introduced into PaY4 RNA2 showed that the 2b gene, but not the 2c gene, is required for transmission by both Paratrichodorus pachydermus and P. anemones nematodes. Experiments examined whether infection of plants with two different virus clones would impact upon nematode transmission of either virus. Simultaneous inoculation with TRV clones expressing green or red fluorescent proteins revealed that mixing of the two virus populations did not occur, although, in roots, adjacent cells were found containing green- or red-tagged viruses. Subsequently, in similar experiments it was found that a TRV PaY4 2b mutant was transmitted when combined with wild-type TRV PaY4. Also, transmission of a 2b mutant of an in vitro TRV/PEBV recombinant virus (TRV-C1) occurred after coinfection with wild-type virus. Thus, the tobravirus 2b transmission protein is trans-acting. Although TRV PaY4 and TRV PpK20 are both transmitted by P. pachydermus, a 2b mutant of TRV PaY4 was not transmitted when coinoculated to plants with TRV PpK20.     

Complementation of the movement-deficient mutations in potato virus X: potyvirus coat protein mediates cell-to-cell trafficking of C-terminal truncation but not deletion mutant of potexvirus coat protein

The cell-to-cell movement of the GUS-tagged potato virus X (PVX) coat protein (CP) movement-deficient mutant was restored by potyviral CPs of potato virus A (PVA) and potato virus Y (PVY) in Nicotiana benthamiana leaves in transient cobombardment experiments. Viral cell-to-cell movement of PVX CP mutant was complemented in Nicotiana tabacum cv. SR1 transgenic plants expressing PVY CP: PVX RNA and polymerase were detected in the PVX CP mutant-inoculated leaves of transgenic plants. These findings demonstrated the ability of the PVX CP-deficient mutant to move from cell to cell but not long distances in the transgenic plants and suggest that CPs of potex- and potyviruses display complementary activities in the movement process. Potyviral CP alone is not able to carry out these activities, since the mutated PVX CP is indispensable for restored movement. No trans-encapsidation between potyviral CP and PVX RNA was observed. Therefore, potyviral CP facilitates the PVX CP mutant movement by the mechanism that cannot be explained by coat protein substitution. Our data also suggest that CP functioning in cell-to-cell movement is not restricted to a simple passive role in forming virions.     

Replication of potato virus X RNA is altered in coinfections with potato virus Y

Potato virus X (PVX) and potato virus Y (PVY) may coinfect tobacco to cause a classic synergistic disease. In the acute stage the disease is characterized by a dramatic increase in the accumulation of infectious PVX particles, with no corresponding increase or decrease in the accumulation of PVY. The accumulation of PVX genomic RNA and coat protein has been examined in doubly versus singly infected tobacco leaves. These experiments indicate that the levels of both viral components increase in doubly infected plants to about the same extent as the level of infectious PVX particles. The level of PVX subgenomic coat protein mRNA found associated with polyribosomes of synergistically infected plants is also increased to a similar extent. Pulse labelling experiments suggest that the increase in PVX coat protein is due to an increased rate of synthesis. The level of PVX (-) strand RNA template increases disproportionately in doubly infected tissue, to a level three times higher than that of the virion or its component parts. This result suggests that PVX/PVY synergism involves an alteration in the normal regulation of the relative levels of PVX (+) and (-) strand RNAs during viral replication.     

Expression of multiple foreign epitopes presented as synthetic antigens on the surface of Potato virus X particles

Summary. We describe the construction of recombinant Potato virus X (PVX) vectors expressing two different epitopes, ep4 and ep6, from Beet necrotic yellow vein virus (BNYVV). The seven-amino-acid epitopes were expressed as N-terminal coat protein fusions and were displayed on the surface of PVX particles. Particle assembly into full virions was successful even though no wild type coat protein subunits were present, and the epitopes could be detected in crude extracts and purified virus preparations with appropriate antibodies. A construct containing both epitope sequences in tandem was also prepared. The resulting PVX particles could be detected by antibodies against ep4 and ep6, either individually or simultaneously, showing that both epitopes were accessible. In addition mixed infections with PVX vectors containing the individual ep4 and ep6 sequences were carried out. This resulted in the formation of PVX particles displaying ep4 alone, ep6 alone, or both epitopes. These experiments demonstrate for the first time that PVX can be utilized to present multiple epitopes, either tandemly on every coat protein subunit or as heteromultimeric assemblies, both of which could be useful vaccination strategies. The production of epitope-presenting viruses in which every coat protein subunit contains a foreign epitope allows the high-level expression of defined numbers of foreign antigen sites, making such viruses useful standards for immune detection.     

Cellular protein binds to sequences near the 5' terminus of potato virus X RNA that are important for virus replication

The sequences in the 5'-nontranslated region (NTR) of Potato virus X (PVX) genomic RNA were previously reported to contain several regulatory elements that are required for genomic and subgenomic RNA accumulation. To investigate whether cellular proteins bind to these elements, we conducted electrophoretic mobility shift assays (EMSA) with protoplast protein extracts and RNA sequences from the PVX 5'-NTR. These analyses showed that the 5' region of PVX positive-strand RNA formed complexes with cellular proteins. UV cross-linking studies of complexes formed with various deletions of the PVX RNA indicated that a 54-kDa cellular protein (p54) was bound to nt 1-46 at the 5' terminus of PVX RNA. Site-directed mutations introduced within this 46-nt region further indicated that an ACCA sequence element located at nt 10-13 was important for optimal binding. In addition, mutations that decreased the affinity of the template RNA for the cellular factor decreased PVX plus-strand RNA accumulation in protoplasts. These studies suggest that the p54 may function in PVX RNA replication by binding to the 5' terminus of the viral genomic RNA.