The detection and characterization of viral-related double-stranded RNAs in tobacco mosaic virus-infected plants

The 2 M LiCl-soluble RNA fraction extracted from tobacco mosaic virus (TMV)-infected tobacco plants contains, in addition to the viral replicative form of 4 x 10(6) MW, three smaller double-stranded (ds) RNA species with apparent molecular weights (estimated by polyacrylamide gel electrophoresis, using ds RNAs as markers) of 2.25, 1.1, and 0.23 x 10(6). The synthesis of all four ds RNAs is insensitive to actinomycin D. They are completely RNase insensitive at high salt concentrations and are found both in directly inoculated and in apical tissues. In tissues incubated in the presence of 3H-uridine and actinomycin D, the three small ds RNAs accounted for 6 to 11.5% of the total radioactivity incorporated into viral ds RNA. On a molar basis, however, in apical leaves the smallest ds RNA was synthesized to almost the same level as the replicative form. By molecular hybridization, the three small ds RNAs have been shown to be of viral origin, and each contains sequences represented in the 5' end of complementary (negative strand) TMV RNA. Based on molecular weight data, none of the ds RNAs can be considered to be a ds form of the subgenomic TMV coat protein mRNA (the LMC), suggesting that it is not replicated independently. None of the small ds RNAs was found to be an endogenous product of the bound TMV RNA replicase.     

Isolation and analysis of virus-specific ribonucleoprotein of tobacco mosaic virus-infected tobacco

A ribonucleoprotein fraction (vRNP) of a characteristic buoyant density greater than the buoyant density of tobacco mosaic virus (TMV) particles has been isolated from infected tissue by Cs2SO4 density gradient centrifugation. The vRNP particles appear to be TMV specific because they are synthesized in the presence of actinomycin D and have RNAs identified as genomic and I-class subgenomic (apparent Mr 1.1-1.3 x 106 and 0.60.8 x 10(6)) RNAs by their electrophoretic mobility and hybridization to plasmid-bearing RNA sequences. Polypeptides of apparent molecular weights 17,500 (TMV coat), 31,000, 37,000, and 39,000 were major constituents of vRNP. Of the minor polypeptides, those of apparent molecular weights 70,000, 68,000, 55,000, and 25,000 had electrophoretic mobilities similar to mobilities of polypeptides found in a ribonucleoprotein preparation from uninoculated plants. vRNP from common TMV-infected plants, but not from plants infected with a mutant that did not form native coat protein, reacted with immunoglobins against TMV and TMV coat protein. Common TMV and its vRNP differed in the extent of reactivity toward the two immunoglobins, in electron microscopic appearance, and in the higher sensitivity of vRNP to ribonuclease.     

A tobacco mosaic virus-hybrid expresses and loses an added gene

An additional open reading frame from the chloramphenicol acetyltransferase (CAT) gene was fused behind a tobacco mosaic virus (TMV) subgenomic RNA promoter and inserted into different positions in the complete TMV genome to examine how much this viral genome can be altered with continued replication. One hybrid virus, CAT-CP, with the insertion between the 30K and coat protein genes, replicated efficiently, produced an additional subgenomic RNA and CAT activity, and assembled into 350-nm virions, compared to 300-nm virions of wild-type TMV. However, during systemic infection of plants, the inserted sequences were deleted. This deletion was exact, resulting in progeny wild-type TMV. Another hybrid virus examined was CP-CAT, which had the insertion between the coat protein gene and the nontranslated 3' region. This virus replicated poorly, produced only minimal levels of CAT activity, and did not systemically invade infected plants. These data show that some extensive modifications of the TMV genome still allow efficient virus replication.     

Replication of tobacco mosaic virus. IV. Further characterization of viral related RNAs

Experiments were performed to characterize the viral related RNA species which appear in extracts of tobacco mosaic virus (TMV)-infected tissue and, in particular, the low molecular weight (ca. 350,000 daltons) component, LMC. It was determined that LMC is probably not a component of the virus rod but is a fragment of unincapsidated TMV RNA. Synthesis of LMC in diseased tissue is not inhibited by the presence of actinomycin D. Because LMC would not reconstitute into a rod with TMV protein, it was considered not to contain a detectable amount of the 5′ terminus of TMV RNA. Polyadenylic acid sequences could not be detected by three analytical methods in any RNA component. In addition to LMC, which is homogeneous in size, TMV RNA fragments polydisperse in size are also present in leaf tissue extracts. In contrast, RNA complementary to TMV RNA was present in extracts only as a component of the double-stranded TMV replicative form and was not found free in the infected cell. Neither fragments of replicative form nor single-stranded TMV complementary RNA could be detected. In addition, the only RNA fraction of uniform size found to contain both an RNA species and its complement was the TMV replicative form.     

Subcellular localization of tobacco mosaic virus minus strand RNA in infected protoplasts

Radioactive RNA probes were prepared which specifically hybridize with sequences complementary to 5' and 3' regions of tobacco mosaic virus (TMV) RNA. These probes were used in Northern hybridization to locate TMV-RNA minus strands in the subcellular fractions of infected tobacco protoplasts. When the protoplasts were lysed with Triton X-100, full-length minus strands were present in the cytoplasmic but not in the nuclear fraction. With mechanically broken protoplasts, the crude nuclear fraction (250 g pellet) contained small amount of minus strands which appeared to derive from unbroken protoplasts, but most of minus strands were recovered in a fraction sedimented between 250 and 2500 g, little if any being found in lighter fractions. The results indicate that TMV-RNA replicates in association with an extranuclear structure.     

Properties of the tobacco mosaic virus intermediate length RNA-2 and its translation

The existence of subgenomic RNAs is well established in the case of plant viruses such as tobacco mosaic virus (TMV). However, except for the subgenomic coat protein mRNA, it is not known whether the other subgenomic RNAs have a function in the life cycle of the virus. In search of more information about one of the major subgenomic RNAs-intermediate length RNA-2 or I2 RNA-of TMV, in vitro and in vivo translational studies were performed. The I2 RNA, which codes in vitro for the synthesis of a 30K (K = kilodalton) protein, appears to be uncapped as judged by the need of different in vitro translation conditions for the synthesis of this protein, compared to the conditions required for the synthesis of the 126K and 183K proteins coded by the capped genomic RNA. In vivo a protein migrating in the same position as the 30K protein synthesized in vitro can be detected in infected tobacco leaves. Since this protein occurs transiently early upon infection, whether it is virus-coded or virus-induced, it could have an early function during infection.     

Replication of tobacco mosaic virus. VII. Further characterization of single- and double-stranded virus-related RNAs from TMV-infected plants

The single-stranded (ss) and double-stranded (ds) RNAs produced in tobacco tissue as a result of infection by tobacco mosaic virus (TMV) have been reinvestigated. 32P-labeled probes consisting of either cDNA or viral RNA, complementary to specific regions of either the viral RNA or its negative strand, respectively, were used in "Northern" hybridization experiments. Of the 10 ssRNA bands observed, all but four appeared to be artifacts of electrophoresis. These four RNAs were found on polyribosomes and are presumed to be true mRNAs; three were identified as the well-known genomic RNA, the I2-mRNA and the coat protein mRNA, or LMC. The fourth RNA species of MW approximately 1.2 x 10(6) had not previously been specifically identified as a subgenomic RNA of TMV. The viral RNA which gave rise to the six artifactual ssRNA bands was heterogeneous in size and was shown to be encapsidated in vivo. Upon electrophoresis, these heterogeneous RNA fragments comigrated approximately with plant rRNAs also present in the extracts, generating the observed artifactual bands. Four dsRNAs were also identified. From molecular weight and hybridization analyses, they appeared to be double-stranded forms of the above four polyribosome-associated ssRNAs. Attempts to translate proteins from the denatured dsRNAs in vitro were unsuccessful. A population of low-molecular-weight, TMV-specific ssRNAs, (+) and (-) in sequence, was generated during infection; however these RNAs were believed to be breakdown products.     

Cotranslational disassembly of a cowpea strain (Cc) of TMV: evidence that viral RNA-protein interactions at the assembly origin block ribosome translocation in vitro

Cotranslational disassembly of mixed rod-length populations of tobacco mosaic virus (TMV; vulgare, common or, U1 strain), reveals a reproducible, significant reduction in the level of expression of the intermediate-sized, subgenomic 12-RNA when compared with conventional total viral RNA preparations. I2-RNA encodes a 30-kDa protein, and recent evidence suggests that I2-RNA is unusual in that it lacks a 5'-cap structure. In TMV vulgare, the assembly origin is located within the 30-kDa protein-coding region. To resolve which of these structural features might be responsible for the decline in 30-kDa gene expression from packaged 12-RNAs, the products encoded in vitro by packaged or naked genomic and subgenomic RNAs from two strains of TMV, vulgare and a cowpea strain (Cc or Sunn-hemp mosaic virus), were compared. The results indicate that strong coat protein-RNA interactions, presumed to occur at the assembly origin, dictate the site at which translocation of 80 S ribosomes is inhibited. The implications of this conclusion for virus infection in vivo are discussed.     

Co-expression of multiple target proteins in plants from a tobacco mosaic virus vector using a combination of homologous and heterologous subgenomic promoters

To co-express multiple target proteins, we engineered a single-component chimeric tobacco mosaic virus (TMV)-based vector containing homologous and heterologous capsid protein subgenomic RNA promoters. Delivery of this vector into Nicotiana benthamiana plants via agroinfiltration resulted in co-expression of two reporter genes within a single cell. Furthermore, co-expression of a host-specific antisense RNA or a silencing suppressor protein from this vector augmented the accumulation of green fluorescent protein or a vaccine antigen, hemagglutinin from avian influenza virus A/Vietnam/1194/04. These findings suggest that this chimeric vector utilizing the homologous and heterologous subgenomic TMV promoters has a potential for high-level production of multiple therapeutic proteins including monoclonal antibodies.     

Trans complementation of virus-encoded replicase components of tobacco mosaic virus

We examined whether the 130K and 180K proteins of tobacco mosaic virus (TMV), the putative virus-encoded replicase components, produced by a replication-competent TMV mutant could complement a replication-defective mutant in a single cell. The replication-competent mutant (LDCS29) had a deletion in the coat protein gene and the replication-defective mutant (LDR28) had a large deletion in the gene encoding the 130K and 180K proteins. Neither the replication of LDR28 nor the production of the coat protein from LDR28 or LDCS29 was detected when the mutants were inoculated separately into tobacco protoplasts. However, when the two mutants were co-inoculated, the production of the LDR28 genomic RNA and the subgenomic RNA for the coat protein and accumulation of the coat protein were observed. These results show that the virus-encoded replicase components of TMV complemented the replication-defective mutant in trans.     

Stimulation by aurintricarboxylic acid of tobacco mosaic virus-specific RNA synthesis and production of informosome-like infection-specific ribonucleoprotein

It was shown that aurintricarboxylic acid (ATA), a well-known inhibitor of protein synthesis, markedly stimulates the synthesis of tobacco mosaic virus (TMV)-specific RNA species of the intermediate (I) class (apparent molecular weights 1.1-1.3 x 10(6) and 0.6-0.8 x 10(6)). No stimulation by ATA of full-length genomic TMV RNA or the subgenomic TMV RNA coding for TMV coat protein was detected. Informosome-like infection-specific ribonucleoprotein (vRNP) particles different from mature TMV particles were found in the TMV-infected cells (Yu. L. Dorokhov, N. M. Alexandrova, N. A. Miroshnichenko, and J. G. Atabekov, 1983, Virology 127, 237-252). It is shown here that [3H]uridine incorporation into vRNP RNAs was markedly stimulated in the presence of ATA. vRNP can be released from the TMV-specific polyribosomes by EDTA treatment, which suggests that it is involved in the translation process. The results of the pulse-chase experiments (including those in which TMV RNA synthesis is blocked by 2-thiouracil) suggest that vRNP does not serve as a precursor for mature virion.     

The tubule-forming NSm protein from Tomato spotted wilt virus complements cell-to-cell and long-distance movement of Tobacco mosaic virus hybrids

A Florida isolate of Tomato spotted wilt virus (TSWV) was able to complement cell-to-cell movement of a movement-defective Tobacco mosaic virus (TMV) vector expressing the jellyfish green fluorescent protein (GFP). To test for complementation of movement in the absence of other TSWV proteins, the open reading frame for the NSm protein was expressed from TMV constructs encoding only the TMV replicase proteins. NSm was expressed from either the coat protein or movement protein subgenomic promoter, creating virus hybrids that moved cell to cell in inoculated leaves of tobacco, providing the first functional demonstration that NSm is the TSWV movement protein. Furthermore, these CP-deficient hybrids moved into upper leaves of Nicotiana benthamiana, demonstrating that NSm can support long-distance movement of viral RNAs. Tubules, characteristic of the NSm protein, were also formed in tobacco protoplasts infected with the TMV-TSWV hybrids. The C-terminus of the NSm protein was shown to be required for movement. TMV-TSWV hybrids expressing NSm and GFP moved within inoculated leaves. Our combination of single-cell and intact plant experiments to examine multiple functions of a heterologous viral protein provides a generalized strategy with wider application to other viruses also lacking a reverse genetic system.     

Replication of tobacco mosaic virus RNA in tobacco mesophyll protoplasts inoculated in vitro

Synthesis of tobacco mosaic virus (TMV)-specific RNAs was investigated using tobacco mesophyll protoplasts inoculated in vitro. Three species of TMV-specific RNA were separated by polyacrylamide gel electrophoresis, and were identified as single-stranded viral RNA, its replicative form (RF), and replicative intermediate (RI) by examining their chromatographic behavior on cellulose, solubility in 1 M NaCl, and susceptibility to denaturation by dimethylsulfoxide. The molecular weight of RF was shown to be about 3.8 × 10⁶ by coelectrophoresis with rice dwarf virus RNA. Time course of the synthesis of RF and RI as well as the results of pulse-chase experiments were consistent with the possible precursor role of these structures in the synthesis of TMV-RNA. No other forms of TMV-specific RNA were detected in infected protoplasts. Kinetics of the replication of TMV-RNA in synchronously infected protoplasts showed the presence of three successive phases in virus replication. During the initial phase, viral RNA replicated exponentially and was encapsidated 4–5 hr later, so that most of the viral RNA synthesized remained free or only partially coated. The rate of viral RNA replication became linear at the end of the initial phase and remained so throughout the subsequent phases. Active formation of virus particles continued during the intermediate phase to encapsidate the bulk of viral RNA synthesized by this time. In the final phase, the synthesis of viral RNA was closely followed by encapsidation.     

Replication of tobacco mosaic virus : III. Viral RNA metabolism in separated leaf cells

The metabolism of RNA has been studied in cell suspensions prepared from tobacco mosaic virus (TMV)-infected tobacco leaves. The cell suspensions incorporated [³H]-uridine into encapsidated viral RNA at a constant rate for as long as 44 hr. Gel electrophoretic analyses of RNA extracted from infected and uninfected cells which had been exposed to [³H]uridine, showed that both incorporated label into ribosomal RNA species. However, RNA preparations from infected cells contained five additional species of RNA not observed in preparations from healthy cells; in addition to TMV RNA, label was detected in two species of double-stranded RNA identified as replicative form (RF) and replicative intermediate (RI), a single-stranded RNA component of low molecular weight (LMC) and one with a molecular weight greater than that of TMV RNA. Synthesis of all five species of virus-specific RNA was insensitive to actinomycin D indicating their independence of cellular DNA.     

Replication of TMV-L and Lta1 RNAs and their recombinants in TMV-resistant Tm-1 tomato protoplasts

Tm-1 is a gene that provides resistance to tomato plants against tobacco mosaic virus (TMV) infection. In tomato cells carrying the Tm-1 gene, multiplication of TMV is inhibited. From previous analysis of resistance-breaking mutants, the involvement of the 130- and 180-kDa proteins, putative viral replicases, in the resistance conferred by the Tm-1 gene was suggested. When wild-type TMV RNA was co-inoculated with a resistance-breaking mutant RNA, replication of the wild-type TMV genomic RNA could not be rescued by the 130- and 180-kDa proteins of a resistance-breaking strain, Lta1. To investigate how the putative resistance factor interacts with the 130- and 180-kDa proteins, we expressed the wild-type TMV protein sequence that is associated with the resistance-breaking phenomenon as part of a recombinant virus derived from Lta1 in Tm-1/Tm-1 protoplasts. No specific degradation of wild-type TMV protein sequences was observed, suggesting that the mechanism of the resistance does not involve the instability of a viral protein.     

Recombination between a 3-kilobase tobacco mosaic virus transgene and a homologous viral construct in the restoration of viral and nonviral genes

Summary.  Transgenic plants harboring various plant virus sequences have shown resistance to viral infections. An environmental risk associated with the use of these plants is the possibility of forming a novel virus by recombination between challenging viruses and transgenic viral mRNA. Two experiments were designed using tobacco mosaic virus (TMV) vectors and transgenic Nicotiana benthamiana to determine if recombinant viral RNA would be detectable. N. benthamiana was transformed with a nontranslatable portion of a TMV viral vector including part of the replicase gene, the movement protein gene, a gene for green fluorescent protein (GFP), and the coat protein gene. When transformed plants were inoculated with a TMV vector coat protein mutant which could not move efficiently through the host, recombinant RNA was detected in 32% of the infected plants, although virions were not detected. When transformed plants were infected with a TMV vector with a normal coat sequence but three base changes in the GFP sequence, no recombinant RNA or virions were detected. Thus, recombinant RNA between TMV RNA and host mRNA did not accumulate to detectable levels under nonselective conditions, and though recombinant RNA did accumulate in the presence of selective pressure, an encapsidated recombinant viral population did not develop. Received September 2, 1999/Accepted March 13, 2000     

Complementation of coat protein-defective TMV mutants in transgenic tobacco plants expressing TMV coat protein

Transgenic tobacco plants (Nicotiana tabacum cv. Xanthi) which express tobacco mosaic virus (TMV) U1 strain coat protein (CP) can complement both the assembly and the long-distance spread of CP-defective (DT1) or coat proteinless (DT1G) mutants of TMV. Both mutants arose spontaneously from PM2 and exist only as unencapsidated RNA in the inoculated leaves of control tobacco plants, where they are unable to form virus particles or to spread systemically. TMV CP expressed in transgenic tobacco plants [CP+ line 3404; P. Powell Abel, R. S. Nelson, B. De, N. Hoffman, S. G. Rogers, R. T. Fraley, and R. N. Beachy, 1986, Science 232, 738-743] was able to package some of either mutant viral RNA into TMV-like particles in vivo and resulted in the long-range spread of infection. In vivo encapsidated DT1 RNA was recovered and reinoculated onto control or new CP+ transgenic tobacco plants. Localized infection of control plants confirmed that no RNA recombination or reversion of the mutant RNA to wild-type had occurred during passage in the first CP+ plant. In contrast, encapsidated DT1 RNA was unable to produce even local infection in CP+ transgenic plants confirming that CP-mediated protection operates during the early stages of virus infection, including particle uncoating. By positive complementation, these results also confirm that TMV CP is required for the long-distance spread of infection.