The enigma of pX: A host-dependent cis-acting element with variable effects on tombusvirus RNA accumulation

Tomato bushy stunt virus (TBSV) is a small isometric virus that contains a single-stranded RNA genome with five major genes. In this study, we have analyzed the importance of an additional small sixth open reading frame (ORF) of 207 nucleotides, designated pX, which resides at the 3' end of the genome. Bioassays showed that deletions or additions of nucleotides at the 5' end of the pX gene that were designed to disrupt the ORF, or site-specific inactivation of its start codon, all gave rise to TBSV mutants which were unable to accumulate to detectable levels in cucumber or Nicotiana benthamiana protoplasts. Although these results suggested a role for the putative pX protein, introduction of a premature stop codon in the pX gene had no strong negative effect. However, a comparable mutation that affected the same nucleotides without changing the predicted amino acid sequence greatly reduced RNA accumulation. Therefore, we hypothesize that cis-acting RNA sequences within the pX gene, rather than the predicted protein influence genome accumulation. The requirement of the cis-acting pX ORF sequences appears to be host-dependent because comparisons revealed that subtle pX gene mutations that prohibited accumulation of TBSV RNA in cucumber or N. benthamiana, failed to interfere substantially with replication in Chenopodium quinoa protoplasts or plants. Irrespective of the host, the cis-acting pX gene sequences were dispensable on replicase-deficient RNAs that require helper TBSV for replication in trans. In addition, the pX gene was not essential for in vitro translation of replicase proteins from genomic RNA. These results suggest that neither translation nor polymerase activity of the replicase proteins require pX gene sequences. However, it is possible that very early in the replication cycle of genomic RNA in vivo, the pX gene cis-acting element is essential for some other unidentified function which involves interaction with one or more host components whose composition varies slightly between different plants.     

Assembly and movement of a plant virus carrying a green fluorescent protein overcoat

Potato virus X (PVX) is a filamentous plant virus infecting many members of the family Solanaceae. A modified form of PVX, PVX.GFP-CP which expressed a chimeric gene encoding a fusion between the 27-kDa Aequorea victoria green fluorescent protein and the amino terminus of the 25-kDa PVX coat protein, assembled into virions and moved both locally and systemically. The PVX.GFP-CP virions were over twice the diameter of wild-type PVX virions. Assembly of PVX.GFP-CP virions required the presence of free coat protein subunits in addition to the fusion protein subunits. PVX.GFP-CP virions accumulated as paracrystalline arrays in infected cells similar to those seen in cells infected with wild-type PVX The formation of virions carrying large superficial fusions illustrates a novel approach for production of high levels of foreign proteins in plants. Aggregates of PVX.GFP-CP particles were fluorescent, emitting green light when excited with ultraviolet light and could be imaged using confocal laser scanning microscopy. The detection of virus particles in infected tissue demonstrates the potential of fusions between the green fluorescent protein and virus coat protein for the non-invasive study of virus multiplication and spread.     

Stem-loop structure in the 5' region of potato virus X genome required for plus-strand RNA accumulation

Computer-generated thermodynamic predictions and solution structure probing indicated two stem-loop structures, stem-loop 1 (SL1; nt 32-106) and stem-loop 2 (SL2; nt 143-183), within the 5' 230 nt of potato virus X (PVX) RNA. Because the existence of SL1 was further supported by covariation analysis of several PVX strains, the functional significance of this structure was investigated by site-directed mutational analysis in a tobacco protoplast system. In general, mutations that reduced genomic plus-strand RNA accumulation similarly affected coat protein accumulation, indicating that subgenomic plus-strand RNA was also affected. In contrast, minus-strand RNA levels remained relatively unchanged. Mutational analysis of the stem C (SC) region of SL1 indicated that pairing was more important than sequence, which was consistent with the covariation analysis. Alterations that increased length and stability of either SC or stem D (SD) were deleterious to plus-strand RNA accumulation. The formation of internal loop C between SC and SD, as well as specific nucleotides within this loop, were also required. Several modifications were made to the terminal GAAA tetraloop, a motif known for enhanced RNA stability. Both GANA and GAAG motifs resulted in wild-type levels of RNA accumulation. However, a UUCG tetraloop was detrimental, indicating that the sequence of this element was important beyond just providing stabilization of the structure. These data indicate that multiple features of SL1 are critical for accumulation of PVX plus-strand RNA.     

Symptom attenuation by a normally virulent satellite RNA of turnip crinkle virus is associated with the coat protein open reading frame

Many satellite RNAs (sat-RNAs) can attenuate or intensify the symptoms produced by their helper virus. Sat-RNA C, associated with turnip crinkle virus (TCV), was previously found to intensify the symptoms of TCV on all plants in which TCV produced visible symptoms. However, when the coat protein open reading frame (ORF) of TCV was precisely exchanged with that of cardamine chlorotic fleck virus, sat-RNA C attenuated the moderate symptoms of the chimeric virus when Arabidopsis plants were coinoculated with the chimeric virus. Symptom attenuation was correlated with a reduction in viral RNA levels in inoculated and uninoculated leaves. In protoplasts, the presence of sat-RNA C resulted in a reduction of approximately 70% in the chimeric viral genomic RNA at 44 hr postinoculation, whereas the sat-RNA wa consistently amplified to higher levels by the chimeric virus than by wild-type TCV. TCV with a deletion of the coat protein ORF also resulted in a similar increase in sat-RNA C levels in protoplasts, indicating that the TVC coat protein, or its ORF, downregulates the synthesis of sat-RNA C. These results suggest that the coat protein or its ORF is a viral determinant for symptom modulation by sat-RNA C, and symptom attenuation is at least partly due to inhibition of virus accumulation.     

Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes

Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes.     

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.     

Genetic requirements for local and systemic movement of tomato golden mosaic virus in infected plants

Tomato golden mosaic geminivirus (TGMV) has two DNA components, A and B. Replication of DNA A can be detected in inoculated leaves, but DNA B is additionally required for virus movement in planta. Using viral DNA accumulation as an indication of the number of infected cells, we show here that both the BL1 and BR1 genes are necessary for local TGMV movement. We also demonstrate that transient expression of BL1 and BR1 together allows wild-type TGMV DNA A to move systemically. When the transient movement assay was used to analyze various A component mutants, all were found to move locally in inoculated leaves, and only an ar1 (coat protein) mutant was unable to move systemically. In addition, we confirm that a TGMV al2 (AR1 and BR1 trans-activator) mutant has a defect in local movement which can be rescued by provision of exogenous BR1, but not BL1. Finally, we show that the ability of TGMV coat protein mutants to accumulate single-stranded (ss) DNA is dependent on BR1. These results provide experimental evidence obtained in planta which supports three predictions of published models for bipartite geminivirus movement: (i) BL1 and BR1 have distinct and essential roles in cell-to-cell movement as well as systemic movement; (ii) BR1 may interact with viral ssDNA in vivo; and (iii) AL2 is indirectly required for movement through its effect on BR1 expression. In addition, our data suggest that specific models of bipartite geminivirus systemic movement should accommodate a role for the coat protein.     

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.     

Nucleotide sequence of the coat protein gene of pelargonium leaf curl virus and comparison of the deduced coat protein amino acid sequence with those of other tombusviruses

The sequence of 1,787 nucleotides (nts) in the genomic RNA of pelargonium leaf curl virus (PLCV) was determined. It included the entire coat protein (cp) gene (nts 585 to 1,754), 558 nts of the 3' end of the putative RNA polymerase gene, 26 nts of an intercistronic region between the two genes and 33 nts downstream of the stop codon of the cp gene. The cp gene was cloned into the expression vector pET8c and expressed in E. coli. The deduced cp amino acid sequence of PLCV was compared with those of five other tombusviruses. The closer the degree of serological relatedness between two viruses, the more similarity was found in their cp amino acid sequences not only in the protruding domains, but also in their random and shell domains and in the arm regions. Nucleic acid hybridization tests, cp amino acid comparisons and serological tests all suggest the same order of sequence for the relationships in the tombusvirus group.     

Nucleotide sequence of a resistance breaking mutant of southern bean mosaic virus

SBMV-S is a resistance-breaking mutant of an Arkansas isolate of the bean strain of southern bean mosaic virus (SBMV-BARK) that is able to move systemically in Phaseolus vulgaris cvs. Pinto and Great Northern, whereas the wild-type SBMV-BARK causes local necrotic lesions and is restricted to the inoculated leaves of these hosts. Sequence analysis of the 4136 nucleotide genomes of SBMV-BARK and SBMV-S revealed seven nucleotide differences, but only four deduced amino acid changes. A single amino acid change occurred in the C-terminal region of the putative RNA-dependent RNA polymerase and three differences were identified in the N-terminal portion of the virus coat protein. SBMV-BARK and SBMV-S were compared with other sobemoviruses and were found to contain a high level of nucleotide sequence identity (91.3%) to SBMV-B. Unlike SBMV-B however, SBMV-BARK and SBMV-S contained four putative overlapping open reading frames, making them more similar in genome organization to the cowpea strain, SBMV-C. The possibility exists that mutations or even errors, that resulted in mis-identification of open reading frames, occurred in previously published information on nucleotide sequence and genomic organization for SBMV-B.     

A phage single-stranded DNA (ssDNA) binding protein complements ssDNA accumulation of a geminivirus and interferes with viral movement

Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles. Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants. In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus. To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5). The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA. The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA. The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication. ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms. In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei. We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection.     

Evidence that a viral replicase protein is involved in the disassembly of tobacco mosaic virus particles in vivo

Tobacco mosaic virus (TMV) particles have been shown to undergo bidirectional disassembly when they are introduced into host cells. Approximately three-quarters of the genomic RNA (i.e., the 126-kDa and 183-kDa protein ORFs) is first uncoated in the 5'-to-3' direction and the process is then completed by removal of coat protein molecules in the 3'-to-5' direction. An effort was made to determine whether the 126-kDa protein or the 183-kDa protein, both of which are involved in replication of the viral RNA, is required for the second part of the disassembly reaction. It was shown that progeny negative-strand viral RNA begins to be produced in inoculated cells at about the same time that 3'-to-5' disassembly is initiated thus suggesting that the two processes may be coupled. Particles containing mutant forms of the viral RNA in which large sections of the 126-kDa and 183-kDa protein ORFs were missing were not disassembled in the 3'-to-5' direction when they were introduced into cells. However, they were disassembled when the inoculum contained purified TMV RNA from which, presumably, the two functional proteins could be translated Particles containing mutants of the RNA from which a few codons had been deleted in or near conserved regions in the 126-kDa protein ORF also did not undergo 3'-to-5' disassembly unless mixed with wild type viral RNA prior to inoculation. These results suggest that the 126-kDa and/or 183-kDa protein plays a role in the completion of disassembly of TMV particles at the onset of the infection process.     

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.