Replication In Protoplasts Research Articles

Host Adaptation and Replication Properties of Two Bipartite Geminiviruses and Their Pseudorecombinants

To investigate factors involved in host adaptation and specificity of bipartite geminiviruses, the infectivity of bean dwarf mosaic (BDMV) and tomato mottle (ToMoV) geminiviruses and the BDMV/ToMoV pseudorecombinants [BDMV DNA-A + ToMoV DNA-B (BA+TB) and ToMoV DNA-A + BDMV DNA-B (TA+BB)] in Phaseolus vulgaris, Lycopersicon esculentum, Nicotiana benthamiana, and N. tabacum plants was determined. Additionally, replication of these viruses was examined in protoplasts prepared from N. tabacum BY2 and Xanthi-nc cells. In adapted hosts and the permissive experimental host, N. benthamiana, BDMV and ToMoV infected nearly 100% of inoculated plants, induced severe symptoms, and had high levels of both DNA components. In nonadapted hosts, BDMV and ToMoV infected approximately 40% of inoculated plants, induced no symptoms, and had reduced levels of both DNA components. For the pseudorecombinants, symptoms were observed only in TA+BB-infected N. benthamiana and P. vulgaris plants. In the other pseudorecombinant/host combinations, symptomless infections were detected and some plants were infected with the DNA-A component only. Symptom development and/or higher infection rates for the pseudorecombinants were correlated with the host-adapted DNA-B component, and pseudorecombinant-infected plants had reduced levels of DNA-B. Protoplast replication assays revealed inefficient DNA-B replication for the pseudorecombinants, and differences in viral replication properties in the two N. tabacum cell lines.

Replication of alfalfa mosaic virus RNA 3 with movement and coat protein genes replaced by corresponding genes of Prunus necrotic ringspot ilarvirus.

Alfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) are tripartite positive-strand RNA plant viruses that encode functionally similar translation products. Although the two viruses are phylogenetically closely related, they infect a very different range of natural hosts. The coat protein (CP) gene, the movement protein (MP) gene or both genes in AMV RNA 3 were replaced by the corresponding genes of PNRSV. The chimeric viruses were tested for heterologous encapsidation, replication in protoplasts from plants transformed with AMV replicase genes P1 and P2 (P12 plants) and for cell-to-cell transport in P12 plants. The chimeric viruses exhibited basic competence for encapsidation and replication in P12 protoplasts and for a low level of cell-to-cell movement in P12 plants. The potential involvement of the MP gene in determining host specificity in ilarviruses is discussed.

Complete nucleotide sequence of tobacco necrosis virus strain DH and genes required for RNA replication and virus movement.

The complete genome sequence of tobacco necrosis virus strain D (Hungarian isolate, TNV-DH) was determined. The genome (3762 nt) has an organization identical to that reported for TNV-D. Highly infectious synthetic transcripts from a full-length TNV-DH cDNA clone were prepared, the first infectious necrovirus transcript reported. This clone was used for reverse genetic studies to map the viral genes required for replication and movement. Protoplast inoculation with delta 22 and delta 82 mutants revealed that both the 22 kDa and 82 kDa gene products are required for RNA replication. Although the products of three small central genes (p7(1), p7a and p7b) were not essential for RNA replication in protoplasts, mutations in these ORFs prevented infection of plants. In contrast, viral RNA accumulation and cell-to-cell movement were observed in the inoculated, but not the systemically infected, leaves of Nicotiana benthamiana challenged with RNA lacking the intact coat protein (CP) gene. These results strongly suggest that p7(1), p7a, p7b and CP are involved in TNV-DH cell-to-cell and long-distance movement, respectively.

Independent Expression of the First Two Triple Gene Block Proteins of Beet Necrotic Yellow Vein Virus Complements Virus Defective in the Corresponding Gene but Expression of the Third Protein Inhibits Viral Cell-to-Cell Movement

Cell-to-cell movement of beet necrotic yellow vein furovirus is controlled by three slightly overlapping genes on RNA 2 called the triple gene block (TGB) encoding, in order, P42, P13, and P15. Synthesis of P42 is directed by subgenomic RNA 2suba while synthesis of both P13 and P15 is probably directed by a dicistronic subgenomic RNA, 2subb. For complementation experiments, each TGB protein gene was inserted into a “replicon” derived from viral RNA 3. In mixed infections, the replicons expressing P42 and P13 complemented RNA 2 mutants defective in the corresponding gene. A P15-containing replicon did not complement a P15-defective RNA 2 but complementation was observed with a dicistronic replicon containing the P15 gene placed behind the P13 gene. In mixed infections with wild-type viral RNAs, the P15-containing replicon did not inhibit viral RNA replication in protoplasts but blocked local lesion formation on leaves. Infection of leaves was also inhibited by an RNA3-derived replicon containing the third TGB gene from another furovirus, peanut clump virus. The results are consistent with a model in which viral cell-to-cell movement requires production of appropriate relative amounts of P13 and P15, and their expression from a dicistronic subgenomic RNA provides a mechanism for coordinating their synthesis.

Replication of Citrus Tristeza Closterovirus in Citrus Protoplasts

The study of citrus tristeza closterovirus (CTV) has been hindered by particle fragility, the inherently low virus titers, and its woody citrus hosts. We report the development of an in vitro Citrus sinensis cv. Hamlin protoplast system useful for the study of CTV replication. Full-length CTV genomic RNA from purified virions was inoculated into 'Hamlin' protoplasts in the presence of polyethylene glycol. After 24 h, the protoplasts were assayed for virus replication. Positive- and negative-strand CTV genomic RNA were detected by Northern hybridization using digoxigenin-labeled RNA probes. Three viral-encoded proteins of 20, 25 (capsid protein), and 27 kDa were detected by Western blotting, demonstrating that the viral RNA was translated in vivo. CTV virus particles also were isolated and observed in the electron microscope. This was the first demonstration of the infectivity of CTV RNA and the first report of CTV replication in protoplasts. This system will enable a more detailed study of the replication strategy of this economically important and complex virus.

The Tomato Bushy Stunt Virus Replicase Proteins Are Coordinately Expressed and Membrane Associated

Two open reading frames at the 5′-end of the tomato bushy stunt virus genomic RNA are predicted to encode a 33-kDa (p33) protein and its 92-kDa (p92) readthrough product. From amino acid sequence comparisons with other small singlestranded RNA viruses, these proteins resemble viral components of the replicase—transcriptase complex. To investigate the accumulation of these proteins in the infected cell, two chimeric proteins were produced that expressed either a portion of p33 or the carboxy-terminal "half" of p92 fused with glutathione S-transferase, and polyclonal ascites fluids specific to p33 or p92 were elicited in mice. As expected, the anti-p33 antibody recognized p33 and the p92 readthrough protein, but the anti-p92 antibody was specific for p92. Immunoblot analyses revealed that at an early stage of infection both proteins were associated with the membrane fractions isolated from virus-infected plants, but later in the infection, prior to collapse of the tissues, these proteins were also associated with the cytoplasmic fraction. At all time points in plants and protoplasts p33 was about 20-fold more abundant than p92. A series of mutations derived from an infectious cDNA clone demonstrated that both the p33 and the p92 proteins were required for replication in protoplasts and the ratio of the two proteins was maintained in the replication-competent mutants. The wild-type amber (UAG) and in vitro-generated ochre (UAA) readthrough codon derivatives replicated in protoplasts. However, the tyrosine mutants (UAC or UAU) that were predicted to express only p92 were not viable in protoplasts.

Genetic Analysis of Beet Curly Top Virus: Examination of the Roles of L2 and L3 Genes in Viral Pathogenesis

The monopartite DNA genome of beet curly top geminivirus (BCTV, strain Logan) contains four leftward (complementary sense) open reading frames (ORFs) designated L1, L2, L3, and L4. We investigated the functions of the L2 and L3 ORFs by mutational analysis We found that in Nicotiana benthamiana and sugarbeet plants, neither a functional L2 nor a functional L3 gene is required for infectivity. Double mutants were also infectious, and no evidence for a synergistic effect of these genes was evident. However, while sugarbeet plants inoculated with L2 or L3 mutants showed symptoms that were indistinguishable from those elicited by wild type virus, mutant-inoculated N. benthamiana plants displayed a novel phenotype in which recovery of the plant from initially severe disease symptoms was greatly enhanced. Enhanced recovery was associated with a large reduction in viral DNA levels. Our studies did not provide evidence for functional homology between the BCTV L2 gene and its presumed homologue (AL2) in the bipartite geminiviruses. In contrast, mutants with lesions in the L3 ORF accumulated three- to five-fold less DNA than wild type virus in a protoplast replication assay, consistent with the interpretation that the BCTV L3 gene is a homologue of the bipartite geminivirus AL3 gene which is known to function as a replication enhancer. Functional homology was directly confirmed in experiments which demonstrated that the BCTV L3 gene can complement a tomato golden mosaic virus AL3 mutant, and vice versa.

Asp → Asn Substitutions in the Putative Calcium-Binding Site of the Turnip Crinkle Virus Coat Protein Affect Virus Movement in Plants

Many plant virus coat proteins have binding sites for divalent cations, particularly calcium. It has been speculated that the purpose of such sites is to ensure that plant viruses release their RNA only in the host cytoplasm which has a low calcium concentration. By comparison to tomato bushy stunt virus, two of the amino acids that probably interact directly with calcium ions in turnip crinkle virus (TCV) capsids, Asp155 and Asp157, were substituted with Asn residues by oligonucleotide-directed mutagenesis to create the mutant TCV-M18. TCV-M18 coat protein, and virions accumulated to wild-type levels in isolated protoplasts. Mutant virions were able to uncoat and to initiate de novo replication in protoplasts although no symptoms were observed in plants inoculated with TCV-M18. Mutant RNA accumulated to much lower levels than wild-type RNA in inoculated leaves and was not detected in upper, uninoculated leaves. The lower infectivity of TCV-M18 in plants may be due to a decreased capacity to move efficiently from cell to cell, and we suggest the possibility that TCV coat protein plays an active, pivotal role in cell-to-cell movement interactions.

Enhanced tobacco mosaic virus production and suppressed synthesis of the inhibitor of virus replication in protoplasts and plants of local lesion responding cultivars exposed to 35 °C

A 5-20 fold increase in tobacco mosaic virus (TMV) titres was observed in protoplasts of Samsun NN, a cultivar in which the infection in the intact plant is localized, when incubated at 35 °C, in comparison with parallel protoplasts incubated at 25 °C. No increase was observed when TMV-infected protoplasts of Samsun, a systemic-responding cultivar, were incubated at 35 °C, compared to incubation at 25 °C. Concomitant with the increase in virus replication in protoplasts of Samsun NN, production of the inhibitor of virus replication (IVR) from these protoplasts, as determined by immunoblotting and biological assay, was suppressed completely. In Samsun NN and the amphidiploid Nicotiana glutinosa × N. debneyi hybrid (ADH) plants kept at 35 °C, no necrotic lesions were observed in the inoculated leaves, but high titres of TMV were recovered from the upper non-inoculated leaves; in the inoculated leaves, TMV titres increased 8-9 times compared to those in plants maintained at 25 °C. No increase in TMV titres was observed in inoculated leaves of Samsun plants kept at 35 °C compared to those at 25 °C. Parallel to the increase in TMV titres in Samsun NN and ADH plants kept at 35 °C, production of IVR, as determined by immunoblotting, was suppressed almost completely. These results support the suggestion that IVR is associated with the localizing mechanism by suppressing virus replication.

Cis-preferential replication of the turnip yellow mosaic virus RNA genome.

The largest open reading frame of the turnip yellow mosaic virus RNA genome encodes a 206-kDa protein that is cleaved to yield N-terminal 150-kDa (p150) and C-terminal 70-kDa (p70) proteins. Using a genomic cDNA clone capable of generating infectious transcripts in vitro, we have introduced substitution, frameshift, and in-frame deletion mutations into the regions encoding both proteins. None of the mutant RNAs was able to replicate independently in turnip protoplasts, indicating that p150 and p70 are both essential. The replication in protoplasts of most of these defective RNAs was poorly supported in trans by a coinoculated helper genome with a deletion in the coat protein gene; replication could also be supported in trans by certain defective RNAs, but this complementation was likewise inefficient in most cases. The replication in trans was more efficient for defective RNAs encoding wild-type p150 and defective p70 than for those encoding defective p150 and wild-type p70. One defective RNA with a large deletion in the p70 coding region was able to replicate efficiently, both when inoculated with the helper genome and when inoculated with a second complementing defective RNA that supplied a wild-type p70. Thus, the cis preference of replication can be overcome in some cases. A model in which p150 and p70 form a complex with the 3' end of the RNA is proposed to explain the cis-preferential replication of turnip yellow mosaic virus RNA.

Bromovirus movement protein genes play a crucial role in host specificity.

Monocot-adapted brome mosaic virus (BMV) and dicot-adapted cowpea chlorotic mottle virus (CCMV) are closely related bromoviruses with tripartite RNA genomes. Although RNAs 1 and 2 together are sufficient for RNA replication in protoplasts, systemic infection also requires RNA3, which encodes the coat protein and the nonstructural 3a movement protein. We have previously shown with bromoviral reassortants that host specificity determinants in both viruses are encoded by RNA3 as well as by RNA1 and/or RNA2. Here, to test their possible role in host specificity, the 3a movement protein genes were precisely exchanged between BMV and CCMV. The hybrid viruses, but not 3a deletion mutants, systemically infected Nicotiana benthamiana, a permissive host for both parental viruses. The hybrids thus retain basic competence for replication, packaging, cell-to-cell spread, and long-distance (vascular) spread. However, the hybrids failed to systemically infect either barley or cowpea, selective hosts for parental viruses. Thus, the 3a gene and/or its encoded 3a protein contributes to host specificity of both monocot- and dicot-adapted bromoviruses. Tests of inoculated cowpea leaves showed that the spread of the CCMV hybrid containing the BMV 3a gene was blocked at a very early stage of infection. Moreover, the BMV hybrid containing the CCMV 3a gene appeared to spread farther than wt BMV in inoculated cowpea leaves. Several pseudorevertants directing systemic infection in cowpea leaves were obtained from plants inoculated with the CCMV(BMV 3a) hybrid, suggesting that the number of mutations required to adapt the hybrid to dicots is small.

Second-site suppressor mutations assist in studying the function of the 3' noncoding region of turnip yellow mosaic virus RNA.

The 3' noncoding region of turnip yellow mosaic virus RNA includes an 82-nucleotide-long tRNA-like structure domain and a short upstream region that includes a potential pseudoknot overlapping the coat protein termination codon. Genomic RNAs with point mutations in the 3' noncoding region that result in poor replication in protoplasts and no systemic symptoms in planta were inoculated onto Chinese cabbage plants in an effort to obtain second-site suppressor mutations. Putative second-site suppressor mutations were identified by RNase protection and sequencing and were then introduced into genomic cDNA clones to permit their characterization. A C-57----U mutation in the tRNA-like structure was a strong suppressor of the C-55----A mutation which prevented both systemic infection and in vitro valylation of the viral RNA. Both of these phenotypes were rescued in the double mutant. An A-107----C mutation was a strong second-site suppressor of the U-96----G mutation, permitting the double mutant to establish systemic infection. The C-107 and G-96 mutations are located on opposite strands of one helix of a potential pseudoknot, and the results support a functional role for the pseudoknot structure. A mutation near the 5' end of the genome (G + 92----A), at position -3 relative to the initiation codon of the essential open reading frame 206, was found to be a general potentiator of viral replication, probably as a result of enhanced expression of open reading frame 206. The A + 92 mutation enhanced the replication of mutant TYMC-G96 in protoplasts but was not a sufficiently potent suppressor to permit systemic spread of the A + 92/G-96 double mutant in plants.

Requirement for ICR-like sequences in the replication of brome mosaic virus genomic RNA

Previous studies using a brome mosaic virus (BMV) RNA-2 deletion mutant (pRNA-2 M/S) and additional derivatives as reporters established that viral sequences resembling internal control regions (ICRs) 1 and 2 of tRNA gene promoters are vital to (+)-strand replication in protoplasts. Transfer of these mutations to genomic RNA-2 and functional analysis in protoplast, local lesion, and systemic infections revealed a sequence-specific requirement for bases within the ICR2-like motif. Despite the low (generally <20% of wild-type) and sometimes undetectable levels of replication of these RNA-2 mutants, sufficient p2a protein was produced to support at least modest levels of RNA-1, −3 and −4 replication in protoplasts. However, only those RNA-2 ICR2 mutants supporting substantial replication of the viral genome in protoplasts were capable of establishing local lesions in Chenopodium hybridum and systemic infections in barley, further establishing the essential role of the ICR-like sequences in viral infectivity. Upon passage through a second set of barley plants, accumulation patterns for progeny from inocula containing certain RNA-2 mutants paralleled those from wild-type inocula, indicating repair of the introduced mutations. RNA stability and translatability were shown to be unaffected by the introduced mutations. BMV RNA-3 contains several ICR-like sequences, each of which was individually deleted. Whereas deletion of the 5′-terminal ICR2-like motif had little effect on RNA-3 accumulation in protoplasts or local lesion formation, it debilitated systemic spread in barley. Deletion of an internal ICR2-like motif at position 1100 decreased (+):(−) strand asymmetry from >100:1 to 14:1, reduced RNA-3 replication in protoplasts to less than 15% of wild-type, and abolished local lesion and systemic infectivity.

Resistance to tobacco mosaic virus induced by the 54-kDa gene sequence requires expression of the 54-kDa protein.

Tobacco plants transformed with the sequence encoding the 54-kDa putative replicase protein of tobacco mosaic virus were resistant to systemic virus disease (D. B. Golemboski, G. P. Lomonossoff, and M. Zaitlin, Proc. Natl. Acad. Sci. USA 87:6311-6315, 1990). Resistance was due to a marked suppression of virus replication at the site of inoculation (J. P. Carr and M. Zaitlin, Mol. Plant-Microbe Interact. 4:579-585, 1991). Although RNA transcripts encoding the 54-kDa protein were present in resistant plants, the 54-kDa protein itself was not observed in vivo. We wished to assess the relative importance of the 54-kDa protein versus its RNA in mediating resistance. Further attempts to detect the 54-kDa protein in plant tissues were unsuccessful; therefore, an indirect approach was taken using a protoplast-based transient gene expression system. Electroporation of protoplasts with plasmids capable of expressing the wild-type 54-kDa protein gene sequence or a mutant lacking the first AUG initiation codon of the 54-kDa open reading frame and encoding a slightly truncated protein reduced virus replication in protoplasts. In contrast, a frameshift mutant that was capable of directing synthesis of a protein only 20% the size of the 54-kDa protein, did not produce resistance in protoplasts. These results show that expression of the 54-kDa protein gene sequence at the RNA level alone is insufficient for resistance, and they implicate the 54-kDa protein itself in mediating this resistance phenomenon.

Deletion analysis of brome mosaic virus 2a protein: effects on RNA replication and systemic spread

Brome mosaic virus (BMV) genomic RNA2 encodes the 94-kDa 2a protein, which is one of two BMV nonstructural proteins required for RNA replication and subgenomic mRNA transcription. 2a contains a central polymeraselike region, which has extensive sequence similarity with the Sindbis virus nsP4 and tobacco mosaic virus (TMV) 183-kDa replication proteins, and also contains N- and C-terminal flanking segments without counterparts in the Sindbis virus and TMV nonstructural proteins. To further investigate the roles of the central and flanking segments in 2a, we have constructed a series of deletion and frameshift mutants in a biologically active BMV RNA2 cDNA clone and tested their ability to support viral RNA replication in barley protoplasts and systemic infection in whole barley plants. The entire 125-amino-acid C-terminal segment following the polymeraselike region was dispensable for RNA replication and transcription. Within the 200-amino-acid N-terminal flanking segment, deletion of the first 50 residues dramatically reduced genomic and subgenomic RNA accumulation, and deletion of 100 or more residues abolished detectable RNA synthesis. All mutations removing residues from the central polymeraselike domain also blocked RNA replication in trans. Sequences required in cis for RNA2 replication or stability were found to occur within the first 300 nucleotides of the 2a coding region. In whole barley plants, systemic infection was inhibited even by 2a deletions that supported strong RNA replication in protoplasts. Some replication-competent 2a variants failed to spread to uninoculated leaves, while other showed 10- to 500-fold-reduced virus yield in both inoculated and uninoculated leaves. These reductions were not due to any defects in RNA2 encapsidation.

Turnip yellow mosaic virus RNAs with anticodon loop substitutions that result in decreased valylation fail to replicate efficiently

Single and multiple nucleotide substitutions have been introduced into the anticodon loop of the tRNA-like structure of turnip yellow mosaic virus (TYMV) genomic RNA. We studied the effects of these mutations on in vitro valylation and on replication in Chinese cabbage protoplasts and plants. Only those mutants capable of efficient and complete valylation showed efficient replication in protoplasts and gave rise to systemic symptoms in whole plants. Mutants that accepted valine inefficiently (in some cases Vmax/Km values were less than 10(-3) relative to wild-type values) replicated to levels 200- to 500-fold below wild-type levels in protoplasts (estimated on the basis of coat protein and genomic RNA levels). These mutants could not support systemic spread in plants. In one plant inoculated with TYMC-A55 RNA, which replicates poorly in protoplasts, systemic symptoms developed after a delay. The reversion in replication was accompanied by improved valine acceptance and the appearance of a U57 second-site mutation. Our results indicate a correlation between valine acceptance activity and viral yield. Possible roles for valylation are discussed, and the results are compared with those of similar studies with brome mosaic virus which suggested that tyrosylation is not crucial for brome mosaic virus replication (T. W. Dreher, A. L. N. Rao, and T. C. Hall, J. Mol. Biol. 206:425-438, 1989).

Mutational analysis of complementary-sense genes of African cassava mosaic virus DNA A

We have investigated the ability of African cassava mosaic virus DNA A mutants, containing disrupted complementary-sense genes, to infect Nicotiana benthamiana and to replicate in Nicotiana tabacum protoplasts. Three overlapping open reading frames (ORFs) with the capacity to encode proteins with an Mr greater than 10K (AC1, AC2 and AC3) are highly conserved between geminiviruses that infect dicotyledonous plants and one (AC4) is less well conserved. Of these, only AC1 is a prerequisite for DNA replication; disruption of this ORF rendered the DNA noninfectious in plants and prevented DNA replication in protoplasts. Disruption of ORF AC2 prevented plant infection but mutants were capable of autonomous replication and replicated DNA B in trans in protoplasts to produce DNA forms that comigrated with wild-type virus DNAs. The AC2 mutant phenotype suggests that the product of this ORF is involved in virus spread within the plant. Mutants in which ORF AC3 had been disrupted retained the ability to replicate and to infect plants systemically although symptom development was delayed and attenuated, and mutant DNA accumulated to much lower levels (10 to 20%) in comparison with wild-type infection. Typical geminate virus particles were observed in extracts of plants infected with ORF AC3 mutants indicating that this gene is not essential for coat protein synthesis or virus assembly but possibly acts by modulating virus levels in infected tissues. Disruption of ORF AC4 had no effect on infectivity or symptom development suggesting that this ORF is maintained only because it overlaps the highly conserved ORF AC1.

Analysis of the role of brome mosaic virus 1a protein domains in RNA replication, using linker insertion mutagenesis

Brome mosaic virus (BMV) belongs to a "superfamily" of plant and animal positive-strand RNA viruses that share, among other features, three large domains of conserved sequence in nonstructural proteins involved in RNA replication. Two of these domains reside in the 109-kDa BMV 1a protein. To examine the role of 1a, we used biologically active cDNA clones of BMV RNA1 to construct a series of linker insertion mutants bearing two-codon insertions dispersed throughout the 1a gene. The majority of these mutations blocked BMV RNA replication in protoplasts, indicating that both intervirally conserved domains function in RNA replication. Coinoculation tests with a large number of mutant combinations failed to reveal detectable complementation between mutations in the N- and C-terminal conserved domains, implying that these two domains either function in some directly interdependent fashion or must be present in the same protein. Four widely spaced mutations with temperature-sensitive (ts) defects in RNA replication were identified, including a strongly ts insertion near the nucleotide-binding consensus of the helicaselike C-terminal domain. Temperature shift experiments with this mutant show that 1a protein is required for continued accumulation of all classes of viral RNA (positive strand, negative strand, and subgenomic) and is required for at least the first 10 h of infection. ts mutations were also identified in the 3' noncoding region of RNA1, 5' to conserved sequences previously implicated in cis for replication. Under nonpermissive conditions, the cis-acting partial inhibition of RNA1 accumulation caused by these noncoding mutations was also associated with reduced levels of the other BMV genomic RNAs. Comparison with previous BMV mutant results suggests that RNA replication is more sensitive to reductions in expression of 1a than of 2a, the other BMV-encoded protein involved in replication.

Regeneration of a functional RNA virus genome by recombination between deletion mutants and requirement for cowpea chlorotic mottle virus 3a and coat genes for systemic infection.

RNAs 1 and 2 of the tripartite cowpea chlorotic mottle virus (CCMV) genome are sufficient for RNA replication in protoplasts, whereas systemic infection of cowpea plants additionally requires RNA3, which encodes the 3a noncapsid protein and coat protein. By using biologically active CCMV cDNA clones, we find that deletions in either RNA3 gene block systemic infection. Thus, though some plant RNA viruses are able to spread systemically without encapsidation, both the coat and 3a genes are required for systemic infection of cowpeas by CCMV. When plants were coinoculated with CCMV RNAs 1 and 2 and both the 3a and coat deletion mutants of RNA3, 30-60% rapidly developed systemic infection. Progeny RNA recovered from systemically infected leaves in such infections contained neither of the starting deletion mutants but rather a single full-length RNA3 component with both genes intact. Nucleotide substitutions introduced into the coat protein deletion mutant as an artificial marker were recovered in the full-length progeny RNA, confirming its recombinant nature. Intermolecular RNA recombination in planta can, therefore, rescue a complete infectious genome from coinoculated mutants independently disabled for systemic spread. These results have implications for the repair of defective genomes produced by frequent natural replication errors, the possible emergence of newly adapted RNA viruses upon coinfection of new hosts, and further studies of RNA virus recombination.

Identification of the initiation codons for translation of cowpea mosaic virus middle component RNA using site-directed mutagenesis of an infectious cDNA clone

A full-length cDNA copy of CPMV M RNA has been cloned downstream of a phage λ promoter in the plasmid pPMl. Transcripts obtained from this clone can be translated in vitro and replicated in cowpea mesophyll protoplasts in the presence of viral B RNA. We have constructed a series of site-directed mutants of this clone to investigate the mechanism of translation of CPMV M RNA. The results obtained confirm that the AUG at position 161 is used to direct the synthesis of a 105K protein in vitro and the detection of a 58K protein in infected cowpea protoplasts suggests that it is also used in vivo. The synthesis of the 95K protein can be initiated from either of the AUGs at positions 512 and 524, though synthesis of this protein does not appear to be essential for CPMV replication in protoplasts.