Long-distance movement factor: a transport function of the potyvirus helper component proteinase

Transport of viruses from cell to cell in plants typically involves one or more viral proteins that supply dedicated movement functions. Transport from leaf to leaf through phloem, or long-distance transport, is a poorly understood process with requirements differing from those of cell-to-cell movement. Through genetic analysis of tobacco etch virus (TEV; potyvirus group), a novel long-distance movement factor was identified that facilitates vascular-associated movement in tobacco. A mutation in the central region of the helper component proteinase (HC-Pro), a TEV-encoded protein with previously described activities in aphid-mediated transmission and polyprotein processing, inactivated long-distance movement. This mutant virus exhibited only minor defects in genome amplification and cell-to-cell movement functions. In situ histochemical analysis revealed that the mutant was capable of infecting mesophyll, bundle sheath, and phloem cells within inoculated leaves, suggesting that the long-distance movement block was associated with entry into or exit from sieve elements. The long-distance movement defect was specifically complemented by HC-Pro supplied in trans by a transgenic host. The data indicate that HC-Pro functions in one or more steps unique to long-distance transport.     

Transgenic plants expressing potato virus X ORF2 protein (p24) are resistant to tobacco mosaic virus and Ob tobamoviruses

The p24 protein, one of the three proteins implicated in local movement of potato virus X (PVX), was expressed in transgenic tobacco plants (Nicotiana tabacum Xanthi D8 NN). Plants with the highest level of p24 accumulation exhibited a stunted and slightly chlorotic phenotype. These transgenic plants facilitate the cell-to-cell movement of a mutant of PVX that contained a frameshift mutation in p24. Upon inoculation with tobacco mosaic virus (TMV), the size of necrotic local lesions was significantly smaller in p24+ plants than in nontransgenic, control plants. Systemic resistance to tobamoviruses was also evidenced after inoculation of p24+ plants with Ob, a virus that evades the hypersensitive response provided by the N gene. In the latter case, no systemic symptoms were observed, and virus accumulation remained low or undetectable by Western immunoblot analysis and back-inoculation assays. In contrast, no differences were observed in virus accumulation after inoculation with PVX, although more severe symptoms were evident on p24-expressing plants than on control plants. Similarly, infection assays conducted with potato virus Y showed no differences between control and transgenic plants. On the other hand, a considerable delay in virus accumulation and symptom development was observed when transgenic tobacco plants containing the movement protein (MP) of TMV were inoculated with PVX. Finally, a movement defective mutant of TMV was inoculated on p24+ plants or in mixed infections with PVX on nontransgenic plants. Both types of assays failed to produce TMV infections, implying that TMV MP is not interchangeable with the PVX MPs.     

Host and viral factors determine the dispensability of coat protein for bipartite geminivirus systemic movement

Geminiviruses have unique, twinned icosahedral particles which encapsidate circular single-stranded DNA. Their genomes are composed of either one or two DNA segments. Monopartite geminiviruses absolutely require a functional coat protein (CP) for infectivity, whereas bipartite geminivirus CP null mutants can infect plants systemically. However, we show here that a CP mutant of the bipartite tomato golden mosaic virus (TGMV), which can infect Nicotiana benthamiana systemically, is confined to the inoculated leaves of Nicotiana tabacum or Datura stramonium. We also show that a CP mutant of the related bean golden mosaic virus (BGMV), which can infect beans systemically, is confined to the inoculated leaves of N. benthamiana. In each case, the extent of viral DNA accumulation in inoculated leaves was unaffected by the absence of CP, which suggests that CP is required specifically for systemic movement. The dispensability of CP is correlated with the degree of virus-host adaptation. TGMV is well adapted to N. benthamiana and does not require CP to infect this host systemically, whereas BGMV is poorly adapted to N. benthamiana and requires CP. Analysis of TGMV-BGMV hybrid viruses revealed that the viral genetic background can also affect the dispensability of CP for systemic movement in N. benthamiana. Thus, bipartite geminivirus movement in planta can be resolved genetically into three components: (i) local, cell-to-cell movement, which does not require CP; (ii) CP-dependent systemic movement, which occurs in all hosts tested; and (iii) CP-independent systemic movement, which occurs in hosts to which a given virus is well adapted.     

Selectable viruses and altered susceptibility mutants in Arabidopsis thaliana

The genetic basis for susceptibility or nonsusceptibility of plants to viruses is understood poorly. Two selectable tobacco etch virus (TEV) strains were developed for identification of Arabidopsis thaliana mutants with either gain-of-susceptibility or loss-of-susceptibility phenotypes. These strains conferred a conditional-survival phenotype to Arabidopsis based on systemic expression of herbicide resistance or proherbicide sensitivity genes, thereby facilitating mass selections and screens for Arabidopsis mutants that enhance or suppress TEV replication, cell-to-cell movement, or long-distance movement. A multicomponent mechanism that restricts systemic invasion of TEV was identified through isolation of gain-of-susceptibility mutants with alterations at two loci.     

Isolation of an Arabidopsis thaliana mutant in which the multiplication of both cucumber mosaic virus and turnip crinkle virus is affected

During the systemic infection of plants by viruses, host factors play an important role in supporting virus multiplication. To identify and characterize the host factors involved in this process, we isolated an Arabidopsis thaliana mutant named RB663, in which accumulation of the coat protein (CP) of cucumber mosaic virus (CMV) in upper uninoculated leaves was delayed. Genetic analyses suggested that the phenotype of delayed accumulation of CMV CP in RB663 plants was controlled by a monogenic, recessive mutation designated cum2-1, which is located on chromosome III and is distinct from the previously characterized cum1 mutation. Multiplication of CMV was delayed in inoculated leaves of RB663 plants, whereas the multiplication in RB663 protoplasts was similar to that in wild-type protoplasts. This suggests that the cum2-1 mutation affects the cell-to-cell movement of CMV rather than CMV replication within a single cell. In RB663 plants, the multiplication of turnip crinkle virus (TCV) was also delayed but that of tobacco mosaic virus was not affected. As observed with CMV, the multiplication of TCV was normal in protoplasts and delayed in inoculated leaves of RB663 plants compared to that in wild-type plants. Furthermore, the phenotype of delayed TCV multiplication cosegregated with the cum2-1 mutation as far as we examined. Therefore, the cum2-1 mutation is likely to affect the cell-to-cell movement of both CMV and TCV, implying a common aspect to the mechanisms of cell-to-cell movement in these two distinct viruses.     

Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene

The RNA genome of tobacco etch potyvirus (TEV) was engineered to express bacterial beta-glucuronidase (GUS) fused to the virus helper component proteinase (HC-Pro). It was shown previously that prolonged periods (approximately 1 month) of TEV-GUS propagation in plants resulted in the appearance of spontaneous deletion variants. Nine deletion mutants were identified by nucleotide sequence analysis of 40 cDNA clones obtained after polymerase chain reaction amplification. The mutants were missing between 1,741 and 2,074 nucleotides from TEV-GUS, including the sequences coding for most of GUS and the N-terminal region of HC-Pro. This region of HC-Pro contains determinants involved in helper component activity during aphid transmission, as well as a highly conserved series of cysteine residues. The deletion variants were shown to replicate and move systemically without the aid of a helper virus. Infectious viruses harboring the two largest HC-Pro deletions (termed TEV-2del and TEV-7del) were reconstructed by subcloning the corresponding mutated regions into full-length DNA copies of the TEV genome. Characterization of these and additional variants derived by site-directed mutagenesis demonstrated that deletion of sequences coding for the HC-Pro N-terminal domain had a negative effect on accumulation of viral RNA and coat protein. The TEV-2del variant possessed an aphid-nontransmissible phenotype that could be rescued partially by prefeeding of aphids on active HC-Pro from another potyvirus. These data suggest that the N-terminal domain of HC-Pro or its coding sequence enhances virus replication or genome expression but does not provide an activity essential for these processes. The function of this domain, as well as a proposed deletion mechanism involving nonhomologous recombination, is discussed.     

PACAP-27 causes negative and positive dromotropic effects in anesthetized dogs

Coinfection of tobacco plants with potato virus X (PVX) and any of several members of the potyvirus group causes a synergistic disease characterized by a dramatic increase in symptom severity correlated with a 3- to 10-fold increase in the accumulation of PVX in the first systemically infected leaves. We have recently shown that PVX/potyviral synergistic disease is mediated by expression of potyviral 5'-proximal sequences encoding P1, helper component-proteinase (HC-Pro), and a fraction of P3 (termed P1/HC-Pro sequence). Here we report the effect of mutations in this potyviral sequence on the induction of synergistic disease. Three transgenic tobacco lines expressing the tobacco etch potyvirus (TEV) P1/HC-Pro sequence with mutations within the P1 coding region were not impaired in their ability to mediate synergism when infected with PVX. In contrast, two of three transgenic lines with mutations in the HC-Pro coding region were unable to induce the synergistic increases in either symptom severity or PVX accumulation. Loss of synergistic function was associated with mutations within the region encoding the central domain of HC-Pro, while the ability to induce synergism was retained in a transgenic line expressing HC-Pro with an alteration in the amino-terminal "zinc-finger domain." In coinoculation experiments, a TEV mutant lacking the sequence encoding the zinc-linger domain of HC-Pro induced a typical synergistic response in interaction with PVX. The results indicate that the zinc-finger domain comprising the first 66 amino acid residues of HC-Pro is dispensable for induction of synergistic disease and transactivation of PVX multiplication, while regions within the central domain of HC-Pro are essential for both of these responses.     

Isolation and stability of histidine-tagged proteins produced in plants via potyvirus gene vectors

A system for the expression and purification of histidine-tagged proteins from plants has been developed using a tobacco etch potyvirus (TEV)-derived gene vectors. The vectors offered a convenient polylinker and a choice of histidine tagging at the recombinant proteins' N or C termini. These vectors were utilized for expression of proteins encoded by beet yellows closterovirus (BYV). Approximately 4 micrograms/g of 20-kDa BYV protein was readily isolated from plants systemically infected by hybrid TEV. In contrast, only minute quantities of 22-kDa BYV capsid protein (CP) histidine-tagged at its N or C terminus could be purified. Rapid degradation of the recombinant CP has been implicated in its failure to accumulate in infected plants. Fusion with TEV HC-Pro stabilized the histidine-tagged BYV CP and facilitated purification of the fusion product from infected plants. This same fusion approach was successfully used with the 24-kDa minor BYV CP. The recombinant proteins were recognized by histidine-tag-specific monoclonal antibody in immunoblot analysis. These results demonstrate the utility of a designed series of TEV vectors for expression, detection, and purification of the recombinant proteins and suggest that intrinsic protein stability is a major factor in a recovery of recombinant proteins from plants.     

Restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene

Nicotiana benthamiana plants transformed with the coat protein gene of tomato bushy stunt virus (TBSV) failed to elicit effective virus resistance when inoculated with wildtype virus. Subsequently, R1 and R2 progeny from 13 transgenic lines were inoculated with a TBSV mutant containing a defective coat protein gene. Mild symptoms typical of those elicited in nontransformed plants infected with the TBSV mutant initially appeared. However, within 2 to 4 weeks, up to 20% of the transgenic plants sporadically began to develop the lethal syndrome characteristic of wild-type virus infections. RNA hybridization and immunoblot analyses of these plants and nontransformed N. benthamiana inoculated with virus from the transgenic lines indicated that wild-type virus had been regenerated by a double recombination event between the defective virus and the coat protein transgene. Similar results were obtained with a TBSV deletion mutant containing a nucleotide sequence marker, and with a chimeric cucumber necrosis virus (CNV) containing the defective TBSV coat protein gene. In both cases, purified virions contained wild-type TBSV RNA or CNV chimeric RNA derived by recombination with the transgenic coat protein mRNA. These results thus demonstrate that recombinant tombus-viruses can arise frequently from viral genes expressed in transgenic plants.     

Plants transformed with a mutant alfalfa mosaic virus coat protein gene are resistant to the mutant but not to wild-type virus

Transgenic tobacco plants expressing the wild-type (wt) coat protein (CP) gene of alfalfa mosaic virus (AIMV) have been shown to be resistant to infection with viral particles and RNAs or to infection with viral particles only. The difference in resistance of these plants to RNA inocula was found to correlate with a difference in the expression level of the transgene. Plants expressing a mutant AIMV CP with the N-terminal serine residue changed to glycine have been shown to be susceptible to infection with wt viral particles or RNAs. By site-directed mutagenesis of AIMV cDNA a viable mutant virus encoding CP with the same N-terminal mutation was obtained. Plants expressing wt or mutant CP were resistant to the mutant virus, demonstrating that a single amino acid substitution in CP did not permit the virus to overcome CP-mediated resistance. Although the mutant CP did not confer resistance to wt virus when expressed in transgenic plants, it was still effective in classical cross-protection: plants infected with the mutant virus were resistant to severe strain of AIMV. A model to explain the data is discussed.     

Genetic engineering of potyvirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gene

Three versions of the zucchini yellow mosaic virus (ZYMV) coat protein gene were engineered for expression in plants: the full-length coat protein sequence, the conserved core portion of the gene, and an antisense version. These constructs were introduced into muskmelon (Cucumis melo) and tobacco plants (Nicotiana tabacum) via Agrobacterium tumefaciens-mediated transformation; gene expression was verified by Northern and Western analysis. Transgenic R0 and R1 muskmelon plants expressing the full-length coat protein gene exhibited apparent immunity to ZYMV infection: There was a lack of symptom development during a 3-mo observation period and no measurable virus accumulation as determined by ELISA. Melon plants expressing the core or antisense constructs showed a several-day delay of systemic symptom development and reduction in virus titer. Furthermore, transgenic R1 tobacco plants expressing the full-length coat protein, core, or antisense constructs of ZYMV, a nonpathogen of tobacco, showed a short delay in symptom development and reduced virus titer when inoculated with the heterologous potyviruses, potato virus Y, and tobacco etch virus. The transgenic tobacco plants were not protected against the non-potyvirus, tobacco mosaic virus.     

Molecular studies on bromovirus capsid protein. III. Analysis of cell-to-cell movement competence of coat protein defective variants of cowpea chlorotic mottle virus

To determine whether the role of coat protein (CP) in cell-to-cell movement of dicot-adapted cowpea chlorotic mottle bromovirus (CCMV) is distinct from that of monocot-adapted brome mosaic bromovirus (BMV), two reporter genes, beta-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP), were substituted for the CP in a biologically active clone of CCMV RNA3 (C3). Primary leaves of Nicotiana benthamiana, Chenopodium quinoa, and cowpea were co-inoculated with wild-type (wt) CCMV RNA 1 and -2 and either C3/delta CP-GUS or C3/delta CP-EGFP and analyzed for GUS activity or the presence of green fluorescence. The visual appearance of infections caused by GUS or EGFP variants indicated that, in CCMV, epidermal cell-to-cell movement can occur without a functional CP. By contrast, inoculation of MP defective variants of C3/delta CP-GUS or C3/delta CP-EGFP resulted in subliminal infections. Additional experiments examining the infectivity of wt BMV RNA 1 and -2 and a BMV RNA3 variant bearing the EGFP in the place of CP (B3/delta CP-EGFP) confirmed previous observations that, unlike CCMV, epidermal cell-to-cell movement of BMV is dependent on the expression of a functional CP. Taken together, the results demonstrate that BMV and CCMV use different mechanisms for initial epidermal cell-to-cell spread, and the individual role played by the respective CP genes in this active process is discussed.     

Probing the roles of active site residues in phosphatidylinositol-specific phospholipase C from Bacillus cereus by site-directed mutagenesis

The role of amino acid residues located in the active site pocket of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus[Heinz, D. W., Ryan, M., Bullock, T., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863] was investigated by site-directed mutagenesis, kinetics, and crystal structure analysis. Twelve residues involved in catalysis and substrate binding (His32, Arg69, His82, Gly83, Lys115, Glu117, Arg163, Trp178, Asp180, Asp198, Tyr200, and Asp274) were individually replaced by 1-3 other amino acids, resulting in a total number of 21 mutants. Replacements in the mutants H32A, H32L, R69A, R69E, R69K, H82A, H82L, E117K, R163I, D198A, D198E, D198S, Y200S, and D274S caused essentially complete inactivation of the enzyme. The remaining mutants (G83S, K115E, R163K, W178Y, D180S, Y200F, and D274N) exhibited reduced activities up to 57% when compared with wild-type PI-PLC. Crystal structures determined at a resolution ranging from 2.0 to 2.7 A for six mutants (H32A, H32L, R163K, D198E, D274N, and D274S) showed that significant changes were confined to the site of the respective mutation without perturbation of the rest of the structure. Only in mutant D198E do the side chains of two neighboring arginine residues move across the inositol binding pocket toward the newly introduced glutamic acid. An analysis of these structure-function relationships provides new insight into the catalytic mechanism, and suggests a molecular explanation of some of the substrate stereospecificity and inhibitor binding data available for this enzyme.     

Virus-induced cell death in plants expressing the mammalian 2',5' oligoadenylate system

The major components of the 2-5A system, responsible for the mammalian interferon-induced antiviral response, are the 2',5' oligoadenylate synthetase (2-5Aase) and 2',5' oligoadenylate (2-5A) dependent ribonuclease (RNase L). Transgenic tobacco plants expressing these two enzyme activities were produced by crossing the transgenic plants expressing RNase L with those expressing 2-5Aase. The double transgenic plants showed complete resistance against cucumber mosaic virus (CMV), infection with necrotic spots only forming on the virus-inoculated leaf. On the other hand, although plants inoculated with potato virus Y (PVY) formed necrotic spots on the inoculated leaf and virus amplification could not be detected, all plants died within 20 days of inoculation. The transgenic tobacco plants expressing either 2-5Aase or RNase L activity showed typical disease symptoms with CMV- or PVY-inoculation. These results suggest that the introduced 2-5A system is activated in tobacco cells by dsRNA, the replicating intermediates of RNA viruses, leading to death of the host cells, which has not been observed in mammalian cells.     

Functions of the tobacco etch virus RNA polymerase (NIb): subcellular transport and protein-protein interaction with VPg/proteinase (NIa)

The NIb protein of tobacco etch potyvirus (TEV) possesses several functions, including RNA-dependent RNA polymerase and nuclear translocation activities. Using a reporter protein fusion strategy, NIb was shown to contain two independent nuclear localization signals (NLS I and NLS II). NLS I was mapped to a sequence within amino acid residues 1 to 17, and NLS II was identified between residues 292 and 316. Clustered point mutations resulting in substitutions of basic residues within the NLSs were shown previously to disrupt nuclear translocation activity. These mutations also abolished TEV RNA amplification when introduced into the viral genome. The amplification defects caused by each NLS mutation were complemented in trans within transgenic cells expressing functional NIb, although the level of complementation detected for each mutant differed significantly. Combined with previous results (X. H. Li and J. C. Carrington, Proc. Natl. Acad. Sci. USA 92:457-461, 1995), these data suggest that the NLSs overlap with essential regions necessary for NIb trans-active function(s). The fact that NIb functions in trans implies that it must interact with one or more other components of the genome replication apparatus. A yeast two-hybrid system was used to investigate physical interactions between NIb and several other TEV replication proteins, including the multifunctional VPg/proteinase NIa and the RNA helicase CI. A specific interaction was detected between NIa and NIb. Deletion of any of five regions spanning the NIb sequence resulted in NIb variants that were unable to interact with NIa. Clustered point mutations affecting the conserved GDD motif or NLS II within the central region of NIb, but not mutations affecting NLS I near the N terminus, reduced or eliminated the interaction. The C-terminal proteinase (Pro) domain of NIa, but not the N-terminal VPg domain, interacted with NIb. The effects of NIb mutations within NLS I, NLS II, and the GDD motif on the interaction between the Pro domain and NIb were identical to the effects of these mutations on the interaction between full-length NIa and NIb. These data are compatible with a model in which NIb is directed to replication complexes through an interaction with the Pro domain of NIa.