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Tomato spotted wilt virus (TSWV) particles are spherical and enveloped, an uncommon feature among plant infecting viruses. Previous studies have shown that virus particle formation involves the enwrapment of ribonucleoproteins with viral glycoprotein containing Golgi stacks. In this study, the localization and behaviour of the viral glycoproteins Gn and Gc were analysed, upon transient expression in plant protoplasts. When separately expressed, Gc was solely observed in the endoplasmic reticulum (ER), whereas Gn was found both within the ER and Golgi membranes. Upon co-expression, both glycoproteins were found at ER-export sites and ultimately at the Golgi complex, confirming the ability of Gn to rescue Gc from the ER, possibly due to heterodimerization. Interestingly, both Gc and Gn were shown to induce the deformation of ER and Golgi membranes, respectively, also observed upon co-expression of the two glycoproteins. The behaviour of both glycoproteins within the plant cell and the phenomenon of membrane deformation are discussed in light of the natural process of viral infection.

Isolates of two distinct begomovirus species, the severe strain of the species Tomato leaf curl New Delhi virus (tomato leaf curl New Delhi virus-[India:New Delhi:Severe:1992]; ToLCNDV-[IN:ND:Svr:92], bipartite) and the Varanasi strain of the species Tomato leaf curl Gujarat virus (tomato leaf curl Gujarat virus-[India:Varanasi:2001]; ToLCGV-[IN:Var:01], mono/bipartite) infect tomato (Lycopersicon esculentum) and cause severe yield losses in northern India. This study investigated the infectivity properties of genomic components of these two species. Both pseudorecombinants were infectious in Nicotiana benthamiana, Nicotiana tabacum and L. esculentum. Enhanced pathogenicity was observed when DNA-A of ToLCNDV-[IN:ND:Svr:92] was trans-complemented with ToLCGV-[IN:Var:01] DNA-B, and was consistently associated with an increase in accumulation of ToLCGV-[IN:Var:01] DNA-B. Mixed infection of ToLCNDV-[IN:ND:Svr:92] and ToLCGV-[IN:Var:01] always showed extremely severe symptoms, suggesting a synergistic interaction between these two viruses. Southern blot analysis of viral DNAs from infected plants showed a significantly higher level of accumulation of both ToLCNDV components and DNA-B of ToLCGV-[IN:Var:01] with no alteration to levels of DNA-A of ToLCGV-[IN:Var:01]. Symptom development and/or higher infectivity of the supervirulent pseudorecombinants correlated with the increased levels of DNA-B accumulation. Protoplast replication assays revealed that enhanced infectivity by the pseudorecombinant occurred at the level of replication, as DNA-A of ToLCNDV-[IN:ND:Svr:92] enhanced ToLCGV-[IN:Var:01] DNA-B replication, whose accumulation was in turn increased by ToLCGV-[IN:Var:01] DNA-A. This is the first report demonstrating a virulent pseudorecombinant between two distinct species of begomoviruses that infect tomato, and is the second report on synergism between begomoviruses. The results revealed that ToLCGV-[IN:Var:01] DNA-B is capable of associating with different DNA-A components, despite having different iteron sequences.

Plum pox virus (PPV) populations from peaches are able to adapt consistently to herbaceous hosts, characterized by a reduction in time to symptom development, increases in inoculation efficiency and increased titres. PPV adaptation was studied by using pea (Pisum sativum) as an alternative host. Two isolates of PPV from peaches were inoculated and passaged in peas ten times using either aphid or mechanical inoculation, generating four independent passage lines. Mechanical-transmission efficiency from peach to pea improved from 3 % at passage 1 to 100 % by serial passage 4 on peas. Inoculation using aphid vectors required six to ten serial passages in pea to reach a peak of 50-60 % transmission efficiency. Sequence analyses of all four PPV population lines inoculated sequentially to pea identified a specific mutation occurring consistently in the NIb gene when compared with the same PPV isolates passaged in parallel in peach. The mutation allowed PPV to replicate up to 20 times faster in the new host. Pea-adapted strains of PPV at every passage were also tested for their ability to infect the original host, peach. Regardless of the number of previous passages, all pea-adapted PPV strains consistently infected peach at low levels using aphid inoculation.

The 3' untranslated region (UTR) of turnip crinkle virus (TCV) RNA is 253 nt long (nt 3798-4050) with a 27 nt hairpin structure near its 3' terminus. In this study, the roles of the 3' UTR in virus accumulation were investigated in protoplasts of Hibiscus cannabinus L. and Arabidopsis thaliana (L.) Heynh. Our results showed that, in Hibiscus protoplasts, the minimal 3' UTR essential for TCV accumulation extends from nt 3922 to 4050, but that maintenance of virus accumulation at wild-type (wt) levels requires the full-length 3' UTR. However, in Arabidopsis protoplasts, only 33 nt (nt 4018-4050) at the 3' extremity of the UTR is required for wt levels of accumulation, whereas other parts of the 3' UTR are dispensable. The 27 nt hairpin within the 33 nt region is essential for virus accumulation in both Hibiscus and Arabidopsis protoplasts. However, transposition of nucleotides in base pairs within the upper or lower stems has no effect on virus accumulation in either Hibiscus or Arabidopsis protoplasts, and alterations of the loop sequence also fail to affect replication. Disruption of the upper or lower stems and deletion of the loop sequence reduce viral accumulation in Arabidopsis protoplasts, but abolish virus accumulation in Hibiscus protoplasts completely. These results indicate that strict conservation of the hairpin structure is more important for replication in Hibiscus than in Arabidopsis protoplasts. In conclusion, both the 3' UTR primary sequence and the 3'-terminal hairpin structure influence TCV accumulation in a host-dependent manner.

A series of deletion mutants of tobacco mosaic virus movement protein (TMV-MP) was used to identify domains of the protein necessary for membrane association. A membrane fraction was isolated from tobacco BY-2 protoplasts infected with wild-type and mutant TMV that produce MP carrying a 3 aa deletion. Deletions that affected membrane association were clustered around the two major hydrophobic regions of MP that are predicted to be transmembrane. Deletions in other hydrophobic regions also reduced membrane association. In addition, a non-functional mutant of MP, in which one of the known phosphorylation sites was eliminated, was not associated with cellular membranes, while a functional second site revertant restored membrane association. This indicates that MP function requires interaction with membrane; however, membrane association was not sufficient for function. Results are consistent with the hypothesis that TMV-MP is an integral or tightly associated membrane protein that includes two hydrophobic transmembrane domains.

The subcellular distribution of the movement proteins (MPs) of nine alanine-scanning mutants of Cucumber mosaic virus (CMV), fused to the green fluorescent protein (GFP) and expressed from CMV, was determined by confocal microscopy of infected epidermal cells of Nicotiana tabacum and Nicotiana benthamiana, as well as infected N. benthamiana protoplasts. Only those mutant MPs that were functional for movement in all host species tested localized to plasmodesmata of infected epidermal cells and to tubules extending from the surface of infected protoplasts, as for wild-type CMV 3a MP. Various mutant MPs that were either conditionally functional for movement or dysfunctional for movement did not localize to plasmodesmata and did not form tubules on the surface of infected protoplasts. Rather, they showed distribution to different extents throughout the infected cells, including the cytoplasm, nucleus or the plasma membrane. The CMV 3a MP also did not associate with microtubules.

In this study, quantitative parameters affecting in vitro acquisition and whitefly (Bemisia tabaci) transmission of Lettuce infectious yellows virus (LIYV) were examined and B. tabaci transmission of an engineered defective RNA (D-RNA) was demonstrated. Virions purified from virus- and virion RNA-inoculated Chenopodium murale plants and protoplasts of Nicotiana tabacum, respectively, were consistently transmitted to plants by B. tabaci when virion concentrations were 0.1 ng microl(-1) or greater. Transmission efficiency increased with increasing virion concentration and number of whiteflies used for inoculation. When in vitro-derived transcripts of the M5gfp D-RNA (engineered to express the green fluorescent protein, GFP) were co-inoculated to protoplasts with wild-type LIYV virion RNAs, the resulting virions were transmissible to plants. LIYV and the M5gfp D-RNA systemically invaded inoculated plants; however, GFP expression was not detected in these plants. Unlike LIYV, the M5gfp D-RNA was not subsequently transmitted by B. tabaci from the initially infected plants, but, when high concentrations of virions from plants infected by LIYV and the M5gfp D-RNA were used for in vitro acquisition by whiteflies, both were transmitted to plants. Quantitative and qualitative analyses showed that, although the M5gfp D-RNA replicated within and systemically invaded plants along with LIYV, compared with LIYV RNA 2 it was not as abundant in plants or in the resulting virions, and concentration of encapsidated RNAs is an important factor affecting transmission efficiency.

For the cell-to-cell movement of cucumoviruses both the movement protein (MP) and the coat protein (CP) are required. These are not reversibly exchangeable between Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV). The MP of CMV is able to function with the TAV CP (chimera RT), but TAV MP is unable to promote the cell-to-cell movement in the presence of CMV CP (chimera TR). To gain further insight into the non-infectious nature of the TR recombinant, RNA 3 chimeras were constructed with recombinant MPs and CPs. The chimeric MP and one of the CP recombinants were infectious. The other recombinant CP enabled virus movement only after the introduction of two point mutations (Glu-->Lys and Lys-->Arg at aa 62 and 65, respectively). The mutations served to correct the CP surface electrostatic potential that was altered by the recombination. The infectivity of the TR virus on different test plants was restored by replacing the sequence encoding the C-terminal 29 aa of the MP with the corresponding sequence of the CMV MP gene or by exchanging the sequence encoding the C-terminal 15 aa of the CP with the same region of TAV. The analysis of the recombinant clones suggests a requirement for compatibility between the C-terminal 29 aa of the MP and the C-terminal two-thirds of the CP for cell-to-cell movement of cucumoviruses.

Cucumber mosaic virus (CMV) and Peanut stunt virus (PSV) each have genomes consisting of three single-stranded RNA molecules: RNA 1, 2 and 3. RNAs 1 and 2 encode the 1a and 2a proteins, respectively, which are necessary for replication of the viral genome. Although RNA 3 is exchangeable between CMV and PSV, exchange of RNA 1 and 2 between the two viruses has been unsuccessful. In this study, reassortants containing PSV RNA 1 and CMV RNA 2 together with RNA 3 of CMV or PSV were shown to be able to replicate their genomic RNA, but not to transcribe subgenomic RNA 4 in tobacco protoplasts. Conversely, the reassortant consisting of CMV RNA 1 and PSV RNA 2 together with RNA 3 of CMV or PSV could not replicate. Subsequently, a yeast two-hybrid system was used to analyse the in vivo interaction between the 1a and 2a proteins. The C-terminal half of PSV-1a protein interacted with the N-terminal region of 2a protein of both PSV and CMV, but the C-terminal half of CMV-1a and the N-terminal region of PSV-2a did not interact. These results suggest that RNA replication in the interspecific reassortant between CMV and PSV requires compatibility between the C-terminal half of the 1a protein and the N-terminal region of the 2a protein, and this compatibility is insufficient for transcription of subgenomic RNA 4.

Spring beauty latent virus (SBLV) is a member of the genus Bromovirus, and is closely related to Brome mosaic virus (BMV) and Cowpea chlorotic mottle virus (CCMV). Compatible interactions between viral components are required for successful infection of plants by BMV and CCMV. To further our understanding of interactions between bromovirus components, we used SBLV to produce reassortants among the three bromoviruses. We found that SBLV RNA 2 functioned with heterologous bromovirus RNA 1 in infections of whole plants and protoplasts of Nicotiana benthamiana, although SBLV RNA 1 did not function with heterologous bromovirus RNA 2. A DNA-based transient assay for 1a and 2a proteins, which are encoded by RNAs 1 and 2, respectively further suggested that SBLV 2a protein may function in combination with heterologous bromovirus 1a protein. Moreover, analysis of the ability of reassortants to spread locally revealed that an RNA 2-mediated interaction between viral components may be required for efficient cell-to-cell movement of bromoviruses.

A full-length cDNA corresponding to the RNA genome of Potato leafroll virus (PLRV) was modified by inserting cDNA that encoded the jellyfish green fluorescent protein (GFP) into the P5 gene near its 3' end. Nicotiana benthamiana protoplasts electroporated with plasmid DNA containing this cDNA behind the 35S RNA promoter of Cauliflower mosaic virus became infected with the recombinant virus (PLRV-GFP). Up to 5% of transfected protoplasts showed GFP-specific fluorescence. Progeny virus particles were morphologically indistinguishable from those of wild-type PLRV but, unlike PLRV particles, they bound to grids coated with antibodies to GFP. Aphids fed on extracts of these protoplasts transmitted PLRV-GFP to test plants, as shown by specific fluorescence in some vascular tissue and epidermal cells and subsequent systemic infection. In plants agroinfected with PLRV-GFP cDNA in pBIN19, some cells became fluorescent and systemic infections developed. However, after either type of inoculation, fluorescence was mostly restricted to single cells and the only PLRV genome detected in systemically infected tissues lacked some or all of the inserted GFP cDNA, apparently because of naturally occurring deletions. Thus, intact PLRV-GFP was unable to move from cell to cell. Nevertheless, PLRV-GFP has novel potential for exploring the initial stages of PLRV infection.

A full-length cDNA copy of the genome of Potato leafroll virus (PLRV) was introduced into the genome of tobacco and potato plants by Agrobacterium tumefaciens-mediated transformation. Transgenic lines were obtained in which the transgene was readily detected by PCR with DNA extracted from T(1) tobacco seedlings and clonally multiplied potato plants. PLRV-specific genomic and sub- genomic RNAs, coat protein antigen and virus particles were detected in transgenic plants. Aphids fed on the transgenic tobacco plants readily transmitted PLRV to test plants. Infected transgenic tobacco plants, like non-transgenic (WT) PLRV-infected plants, displayed no symptoms of the infection but transgenic plants of potato were severely stunted. In parallel tests, the mean PLRV titres in WT tobacco plants and transgenic tobacco plants were 600 and 630 ng virus/g leaf, respectively, although differences in PLRV titres among transgenic plants were much greater than those among infected WT plants. In similar tests with potato, the mean PLRV titre of WT plants was 50 ng virus/g leaf whereas higher concentrations (up to 3400 ng virus/g leaf) accumulated in transgenic potato plants. In tissue prints of stems, PLRV was detected in similar proportions of phloem cells in transgenic and infected WT plants. In transgenic tobacco and potato plants, but not in infected WT plants, a few stem epidermal cells also contained virus. From tissue prints of transgenic tobacco leaves, it was estimated that about one in 40000 mesophyll cells contained virus, but in transgenic potato, a greater proportion of mesophyll cells was infected.

Capsicum annuum cv. Avelar plants resist systemic infection by the Florida isolate of pepper mottle potyvirus (PepMoV-FL). Immuno-tissue blot analysis for detection of PepMoV-FL infection in selected stem segments revealed that virus moved down the stem in external phloem, and, over time, accumulated to detectable levels throughout stem sections (appearing to accumulate in external and internal phloem) taken from below the inoculated leaf. At 21 days post-inoculation, PepMoV-FL was detected in stem segments one or two internodes above the inoculated leaf; however, no virus was observed in internal phloem in stem segments beyond these internodes. In contrast to these observations, PepMoV-FL was detected in the internal phloem of all internodes of the stem located above the inoculated leaf, with subsequent movement into non-inoculated leaves, in Avelar plants co-infected with PepMoV-FL and cucumber mosaic cucumovirus (CMV-KM). No apparent enhancement of PepMoV-FL accumulation occurred in protoplasts inoculated with PepMoV-FL alone versus a mixed inoculum of PepMoV-FL and CMV-KM. These findings confirm earlier observations that potyvirus movement up the stem of Capsicum species occurs via internal phloem. It is also shown that PepMoV-FL does not accumulate to detectable levels in internal phloem in the stem of Avelar plants, thereby limiting its movement to within the inoculated leaf and lower portions of the stem; however, co-infection of Avelar plants with CMV-KM alleviates this restricted movement, allowing PepMoV-FL to invade young tissues systemically.

Nicotiana benthamiana mesophyll protoplasts, either mock-inoculated or inoculated using in vitro transcripts derived from lettuce infectious yellows virus (LIYV) RNA 1- and/or RNA 2-cloned cDNAs were analysed by transmission electron microscopy (TEM) and, in some cases, also by immunogold labelling. TEM revealed the main cytopathological effects of LIYV infections in N. benthamiana protoplasts infected with RNAs 1 and 2: (a) typical closterovirus-induced (beet yellows virus-type) accumulations of vesiculated cytoplasmic membranes as inclusion bodies, sometimes with associated virions; (b) scattered aggregations of virions within the cytoplasm; and (c) electron-dense plasmalemma deposits. These were not seen in mock-inoculated protoplasts. Protoplasts inoculated only with LIYV RNA 1 contained vesiculated cytoplasmic inclusion bodies, but not virions or plasmalemma deposits. Thus, infection by only LIYV RNA 1 is sufficient to induce characteristic closterovirus vesiculated cytoplasmic inclusion bodies. However, both LIYV RNAs 1 and 2 are needed for production of virions and plasmalemma deposits.

Bean yellow dwarf virus (BeYDV) is an atypical member of the geminivirus genus Mastrevirus that infects dicotyledonous plants. BeYDV DNA contains six open reading frames (ORFs) with the capacity to encode proteins in excess of 10 kDa. Two virion-sense ORFs (V1 and V2) and two complementary-sense ORFs (C1 and C2) have homologues in all mastreviruses, while ORFs C3 and C4 are not conserved. To investigate their functions, each of the ORFs has been truncated by either frame-shifting or the introduction of a stop codon. We demonstrate that an ORF V1 mutant replicated efficiently in Nicotiana tabacum protoplasts but was unable to systemically infect Phaseolus vulgaris and Datura stramonium, consistent with a role for V1 protein in virus movement. However, the mutant was able to systemically infect Nicotiana benthamiana although the onset of symptoms was appreciably delayed in comparison with wild-type virus. Disruption of ORF V2, encoding the coat protein, prevented systemic infection of all three hosts but the mutant replicated in protoplasts. Both ORF C1 and ORF C2 were essential for replication in protoplasts. Modification of the complementary-sense splice donor and acceptor sequences also prevented replication. Removal of the intron prevented systemic infection, although the intronless mutant was able to produce functional replication-associated protein (Rep) and replicated efficiently in protoplasts. ORFs C3 and C4 were not required for systemic infection. Our results indicate that four ORFs are spatially and functionally conserved in mastreviruses that infect both monocotyledonous and dicotyledonous plants.

Mutagenesis of the artichoke mottled crinkle virus (AMCV) genome and complementation studies between replication-defective mutants were undertaken to identify viral protein(s) essential for AMCV replication. Inoculation of Nicotiana benthamiana protoplasts with mutant transcripts revealed that null mutations in ORFs 1 [tA33(-)], 2 [tA92(-)] and 6 [tA7(-)], as well as an ORF 2 mutation [tA92GED] in the GDD motif of the 92 kDa protein, the putative replicase, prevented accumulation of detectable levels of progeny RNA. Conversely, mutations of ORFs 3 [tA41(-)], 4 [tA21(-)] and 5 [tA19(-)] did not substantially affect the accumulation of AMCV genomic and subgenomic RNAs of both positive and negative polarity. Inoculation of N. benthamiana plants with transcripts impaired in replication revealed that tA92(-) and tA7(-) mutants lead to replicating pseudorevertants. Functional analysis of these pseudorevertants showed that: (i) the double stop codon introduced at the end of ORF 1 to prevent the translational readthrough of the 92 kDa protein reverted to a single amber, ochre or opal codon, giving rise to viable genomes; (ii) the putative 7 kDa protein is not essential for genome viability, although the RNA region spanning ORF 6 plays a role in cis in replication. Finally, the two replication-defective mutants tA33(-) and tA92(-) complemented when co-inoculated to N. benthamiana protoplasts, definitively proving that the 33 kDa protein is essential for tombusvirus genome replication. Analysis of viral RNAs from the coinfection experiments showed that tA92(-) was preferentially amplified over tA33(-).

The structural phenotype of the movement proteins (MPs) of two representatives of the Bromoviridae, alfalfa mosaic virus (AMV) and brome mosaic virus (BMV), was studied in protoplasts. Immunofluorescence microscopy showed that the MPs of these viruses, for which there has been no evidence of a tubule-guided mechanism, assemble into long tubular structures at the surface of the infected protoplast. Electron microscopy and immunogold analysis confirmed the presence of both MP and virus particles in the tubules induced by AMV and BMV. The significance of the tubule-forming properties of these viral MPs is discussed.

A plant protoplast system for studying tomato spotted wilt tospovirus (TSWV) infection was established and tested. Using polyethylene glycol-mediated inoculation with highly infectious TSWV particles, generally 50% or more of Nicotiana rustica protoplasts were infected. In these cells viral RNA and viral protein synthesis became detectable at 16 h post-inoculation (p.i.) and continued at least until 90 h p.i. Both the structural viral proteins [nucleoprotein (N) and the envelope glycoproteins G1 and G2] and the nonstructural viral proteins NSs and NSm accumulated to amounts sufficient for detection and immunocytological analysis. Local lesion tests on petunia leaves and electron microscopical analysis confirmed the production of mature, infectious virus particles, underlining the conclusion that a full infection cycle was completed in this system. Upon inoculation of Vigna unguiculata (cowpea) protoplasts with TSWV particles, comparable proportions of infected cells and amounts of NSs, NSm and N protein were obtained, but much lower amounts of viral glycoproteins were detected than in N. rustica protoplasts, and progeny virus particles were less abundant. With the N. rustica-based protoplast system, a powerful synchronized single-cell infection system has now become available for more precise in vivo studies of the processes occurring during tospovirus infection.

RNA transcribed from cloned satellite tobacco mosaic virus (STMV) cDNA replicated in Nicotiana benthamiana protoplasts when co-inoculated with tobacco mild green mosaic virus (TMGMV) genomic RNA, but degraded when inoculated alone. STMV genomic RNA extracted from wild-type virions replicated in protoplasts when co-inoculated with TMGMV, tobacco mosaic virus (TMV) or tomato mosaic virus (ToMV). Transcripts from clones of two STMV coat protein (CP) mutants accumulated to the same level as wild-type transcripts in protoplasts when co-inoculated with TMGMV, whereas a third mutant accumulated to detectable levels in some, but not all, experiments. These results confirm that STMV RNA requires helper virus for replication, and that the helper specificity exhibited by cloned STMV reflects a specific requirement for the TMGMV replicase. It also demonstrates that the low accumulation of STMV CP mutants observed previously in whole plants cannot be attributed to inefficient RNA replication.

Transgenic tobacco plants (V123 plants) expressing a set of full-length brome mosaic virus (BMV) genomic RNAs from the cauliflower mosaic virus 35S promoter were produced. The accumulation level of BMV RNAs in V123 plant cells was approximately 1% of that in nontransgenic tobacco protoplasts inoculated with BMV RNAs. The level of BMV RNA in V123 protoplasts did not increase after inoculating the protoplasts with BMV RNAs, whereas V123 protoplasts supported the accumulation of cucumber mosaic virus (CMV) RNAs to a level similar to that in non-transgenic tobacco protoplasts after inoculation with CMV RNA. Such BMV-specific resistance was also observed in protoplasts from V12 plants expressing full-length BMV RNA1 and RNA2, both of which are required and sufficient for BMV RNA replication. On the other hand, protoplasts from M12 plants, expressing truncated BMV RNA1 and RNA2 in which the 3' 200 nucleotides required for BMV RNA replication were deleted, exhibited weaker resistance to infection with BMV RNA than V12 protoplasts, although the accumulation level of truncated BMV RNA1 and RNA2 in M12 protoplasts was higher than that of BMV RNA1 and RNA2 in V12 protoplasts. These results suggest that expression of BMV RNA replicons is involved in the induction of resistance, rather than high-level accumulation of BMV RNAs and/or their encoded proteins.

We have assessed the functional importance of the NTP-binding motif (NTBM) in the cowpea mosaic virus (CPMV) B-RNA-encoded 58K domain by changing two conserved amino acids within the consensus A and B sites (GKSRTGK500S and MDD545, respectively). Both Lys-500 to Thr and Asp-545 to Pro substitutions are lethal as mutant B-RNAs were no longer replicated in cowpea protoplasts. Transiently produced mutant proteins were not able to support trans-replication of CPMV M-RNA in cowpea protoplasts in contrast to transiently produced wild-type B proteins. Therefore loss of viral RNA synthesis was a result of a protein defect rather than an RNA template defect. Mutant B polyproteins were correctly processed in vitro and in vivo and the regulatory function of the 32K protein on processing of B proteins was not affected by these mutations. Since regulation of processing by the 32K protein depends on interaction with the 58K domain, the mutations in the NTBM apparently do not interfere with this interaction. The Asp-545 to Pro substitution left intact the binding properties of the 84K precursor of the 58K protein, with respect to ATP-agarose, whereas the Lys-500 to Thr substitution decreased the binding capacity of the 84K protein, suggesting that the Lys-500 residue is directly involved in ATP binding. The Lys-500 to Thr substitution in the 58K domain resulted in an altered distribution of viral proteins, which failed to aggregate into large cytopathic structures as observed in protoplasts infected with wild-type B-RNA. However viral proteins containing the Asp-545 to Pro substitution showed a normal distribution in protoplasts.