Replication, stability, and gene expression of tobacco mosaic virus mutants with a second 30K ORF

A series of tobacco mosaic virus (TMV)-hybrids containing a second 30K open reading frame (ORF) inserted into different positions of the genome 3' region were constructed. These insertional mutants were used to evaluate the effects of a modified viral genome organization on replication and gene expression. They were evaluated for stability upon systemic infection and subsequent host passage using RNase protection assays. A mutant with the second 30K ORF fused in frame to two-thirds of the coat protein reading frame replicated as a free-RNA virus and produced increased amounts of the hybrid protein compared to the wild-type 30K protein, but substantially reduced amounts compared to the wild-type coat protein. A mutant with the second 30K ORF inserted between the native 30K and coat protein ORFs produced reduced amounts of 30K protein but replicated efficiently and was maintained for weeks of systemic infection before the population gradually shifted to progeny wild-type TMV. Mutants with the second 30K ORF fused behind different lengths of the coat protein subgenomic RNA promoter/leader region and inserted between the coat protein gene and the 3' nontranslated sequences replicated poorly and the mutations were not maintained during continued replication in plants.     

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.     

The complete nucleotide sequence of the genomic RNA of the tobamovirus tobacco mild green mosaic virus

The complete nucleotide sequence of the genomic RNA of the tobamovirus tobacco mild green mosaic virus (TM-GMV) was determined. It shows 64.4% sequence homology with the genomic RNA of tobacco mosaic virus (TMV) and 66.0% with that of tomato mosaic virus (ToMV). Its genomic organization is similar to that of TMV and ToMV. The 5' proximal open reading frame (ORF) encodes a 126K polypeptide and a 183K readthrough product in which nucleotide-binding and polymerase-sequence motifs are found. The third ORF encodes a 28.5K protein homologous to TMV and ToMV movement proteins. A conserved core is found with four other tobamoviruses and two tobraviruses suggesting a common mechanism of cell-to-cell movement for tobamo- and tobraviruses. The fourth ORF encodes the 17.5K coat protein. Homology between the RNAs of TMGMV and its satellite virus STMV is limited to their 3' termini, and structural comparisons suggest that this region may determine the nature of the satellite/helper virus interaction.     

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.     

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     

A stable RNA virus-based vector for citrus trees

Virus-based vectors are important tools in plant molecular biology and plant genomics. A number of vectors based on viruses that infect herbaceous plants are in use for expression or silencing of genes in plants as well as screening unknown sequences for function. Yet there is a need for useful virus-based vectors for woody plants, which demand much greater stability because of the longer time required for systemic infection and analysis. We examined several strategies to develop a Citrus tristeza virus (CTV)-based vector for transient expression of foreign genes in citrus trees using a green fluorescent protein (GFP) as a reporter. These strategies included substitution of the p13 open reading frame (ORF) by the ORF of GFP, construction of a self-processing fusion of GFP in-frame with the major coat protein (CP), or expression of the GFP ORF as an extra gene from a subgenomic (sg) mRNA controlled either by a duplicated CTV CP sgRNA controller element (CE) or an introduced heterologous CE of Beet yellows virus. Engineered vector constructs were examined for replication, encapsidation, GFP expression during multiple passages in protoplasts, and for their ability to infect, move, express GFP, and be maintained in citrus plants. The most successful vectors based on the 'add-a-gene' strategy have been unusually stable, continuing to produce GFP fluorescence after more than 4 years in citrus trees.     

In vitro viral RNA synthesis by a subcellular fraction of TMV-inoculated tobacco protoplasts

A subcellular fraction which can synthesize viral RNA and subgenomic RNA in vitro was prepared from tobacco mosaic virus (TMV)-inoculated tobacco protoplasts. S(1)-Resistant fragment analysis with strand specific TMV cDNA showed that a large amount of plus-stranded and a small amount of minus-stranded, genome-size RNA was synthesized by this subcellular fraction. Plus-stranded subgenomic RNA of coat protein mRNA size was also synthesized. The time course of the appearance of viral RNA synthetic activity was consistent with that of the appearance of TMV infectivity in vivo.     

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.     

Efficient expression of foreign proteins in roots from tobravirus vectors

Viral vectors were constructed from infectious cDNA clones of each of the three tobraviruses, tobacco rattle virus (TRV), pea early-browning virus (PEBV), and pepper ringspot virus (PepRSV). RNA2 of each of the three viruses was modified to carry an additional coat protein subgenomic promoter and was used to express green fluorescent protein (GFP) when inoculated to plants. The tobravirus expression vectors have a wide host range and were able to express GFP in, for example, Nicotiana species, tomato, pea, arabidopsis, and sugar beet. The TRV vector was able to invade and express GFP very efficiently in roots, whereas the widely used PVX vector was not.     

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.     

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.     

Umbravirus-encoded proteins both stabilize heterologous viral RNA and mediate its systemic movement in some plant species

The proteins encoded by open reading frame 3 (ORF3) of the umbraviruses pea enation mosaic virus-2 and tobacco mottle virus, like that of groundnut rosette virus, mediated the movement of viral RNA through the phloem of infected Nicotiana benthamiana or N. clevelandii plants when they were expressed from chimeric tobacco mosaic virus in place of the coat protein. However, these chimeras did not move systemically in N. tabacum. In lysates of N. benthamiana or N. tabacum protoplasts, the chimeric RNAs were more stable than was RNA of tobacco mosaic virus lacking the coat protein gene. The chimeric viruses also protected the latter in trans, suggesting that the ORF3 proteins can increase the stability of heterologous viral RNA. Umbraviral ORF3 proteins contain a conserved arginine-rich domain, and the possible roles of this motif in the functions of the proteins are discussed.     

Inhibition of uncoating of tobacco mosaic virus particles in protoplasts from transgenic tobacco plants that express the viral coat protein gene

The uncoating of tobacco mosaic virus (TMV) particles in protoplasts isolated from leaves of transgenic tobacco plants that express the TMV coat protein gene was investigated. Extracts of these protoplasts collected up to 1 hr after inoculation with TMV contained fewer of the complexes ("striposomes") thought to be involved in cotranslational disassembly of virus particles than did extracts of protoplasts that do not express the viral coat protein gene. These results are consistent with the hypothesis that TMV coat protein-mediated resistance is at least in part the result of inhibition of the uncoating of the virus particles in the inoculum.     

Independent assembly of virions in tobacco doubly infected by potato virus X and potato virus Y or tobacco mosaic virus

Enhancement of potato virus X (PVX) synthesis in tobacco cells doubly infected by PVX and potato virus Y (PVY) or by PVX and tobacco mosaic virus (TMV) is not a consequence of encapsidation of PVX-RNA by a protein coat containing the capsid subunits of PVY or TMV. Serology, electrophoresis, sedimentation analysis, and infectivity tests did not detect heterologous encapsidation of PVX-RNA, PVY-RNA, or TMV-RNA in doubly infected leaves.     

Engineering of Alfalfa mosaic virus RNA 3 into an expression vector

Summary.  RNA 3 of alfalfa mosaic virus (AMV) encodes the 5′-proximal movement protein (MP) gene and the 3′-proximal coat protein (CP) gene which is expressed from a subgenomic RNA. Several strategies were explored to use this RNA as a vector for expression of the green fluorescent protein (GFP) in Nicotiana tabaccum plants expressing the viral polymerase proteins P1 and P2 (P12 plants). Insertion of a subgenomic promoter (sgp)-GFP cassette between the CP gene and the 3′-untranslated region (UTR) interfered with RNA accumulation in protoplasts, indicating that cis-acting sequences required for replication were disrupted. When GFP was fused to the N-terminus of MP or CP, the chimeric RNAs accumulated in protoplasts but cell-to-cell movement in plants was blocked. Insertion of a GFP-sgp cassette immediately upstream of the CP gene caused a hypersensitive host response. However, insertion of a GFP-sgp cassette upstream of the MP gene did not affect symptom formation and yielded a vector that expressed GFP in inoculated but not in the systemic leaves of both P12 tobacco and non-transgenic N. benthamina plants. When the size of the GFP gene was reduced from 700 to 300 nucleotides, virus infection was observed in the non-inoculated leaves. Analysis of the progeny of some chimera revealed novel data on replication, encapsidation and recombination of AMV RNA 3. Received August 7, 2000 Accepted December 18, 2000