Identification of a major pathogenicity determinant and suppressors of RNA silencing encoded by a South Pacific isolate of <Emphasis Type="Italic">Banana bunchy top virus</Emphasis> originating from Pakistan

Five genes encoded by Banana bunchy top virus (BBTV) originating from Pakistan were expressed in Nicotiana benthamiana using a Potato virus X (PVX) vector. Expression of the master replication-associated protein (mRep) and movement protein (MP) resulted in necrotic cell death of inoculated tissues, as well as leaf curling and necrosis along the veins in newly emerging leaves. The systemic necrosis induced by the expression of MP was discolored (dark) in comparison to that induced by mRep. Expression of the cell-cycle link protein (Clink), the coat protein (CP), and the nuclear shuttle protein from the PVX vector induced somewhat milder symptoms, consisting of mild leaf curling and mosaic, although expression of the CP caused a necrotic response in inoculated leaf. The accumulation of viral RNA was enhanced by MP, Clink, and CP. Of the five BBTV-encoded gene products two, the MP and Clink, stabilized GFP-specific mRNA and reduced GFP-specific small interfering RNA in N. benthamiana line 16c when expressed under the control of the 35S promoter and co-inoculated with a construct for the expression of GFP hairpin RNA construct. These results identified MP and Clink as suppressors of RNA silencing. Taken together the ability of MP to induce severe symptoms in plants and suppress RNA silencing implicates this product as a major pathogenicity determinant of BBTV.     

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     

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     

Cell-to-cell movement of Alfalfa mosaic virus can be mediated by the movement proteins of Ilar-, bromo-, cucumo-, tobamo- and comoviruses and does not require virion formation

RNA 3 of Alfalfa mosaic virus (AMV) encodes the movement protein (MP) and coat protein (CP). Chimeric RNA 3 with the AMV MP gene replaced by the corresponding MP gene of Prunus necrotic ringspot virus, Brome mosaic virus, Cucumber mosaic virus or Cowpea mosaic virus efficiently moved from cell-to-cell only when the expressed MP was extended at its C-terminus with the C-terminal 44 amino acids of AMV MP. MP of Tobacco mosaic virus supported the movement of the chimeric RNA 3 whether or not the MP was extended with the C-terminal AMV MP sequence. The replacement of the CP gene in RNA 3 by a mutant gene encoding a CP defective in virion formation did not affect cell-to-cell transport of the chimera's with a functional MP. A GST pull-down technique was used to demonstrate for the first time that the C-terminal 44 amino acids of the MP of a virus belonging to the family Bromoviridae interact specifically with AMV virus particles. Together, these results demonstrate that AMV RNA 3 can be transported from cell-to-cell by both tubule-forming and non-tubule-forming MPs if a specific MP-CP interaction occurs.     

Geminivirus coat protein gene replacement alters insect specificity

Chimeric clones have been constructed in which the coat protein encoded by DNA A of the bipartite genome of the geminivirus African cassava mosaic virus (ACMV) has been replaced by that of beet curly top virus (BCTV). Constructs containing the coding region inserted in either orientation were infectious when co-inoculated with ACMV DNA B onto Nicotiana benthamiana, producing symptoms typical of ACMV infection. The onset of symptom production was delayed relative to plants inoculated with parental ACMV clones and remission of symptoms was observed. When inserted in the correct orientation for expression from the ACMV coat protein promoter, the BCTV gene was expressed in plants and the coat protein synthesized encapsidated ssDNA of both ACMV genomic components. The BCTV leafhopper vector, Circulifer tenellus (Baker), transmitted both BCTV and the chimeric virus but not ACMV when injected with virus preparations and transferred to N. benthamiana seedlings. The results show that the specificity of leafhopper transmission from insect to plant resides with the coat protein.     

Nucleic acid-binding properties of the alfalfa mosaic virus movement protein produced in yeast.

The movement protein of alfalfa mosaic virus (P3) was purified from yeasts transformed with an expression vector containing the P3 gene. Its nucleic acid-binding properties were tested by electrophoretic retardation, nitrocellulose retention, and RNA-protein cross-linking. The recombinant protein had a higher affinity for single-stranded RNA and DNA than for double-stranded nucleic acids. Each nucleic acid molecule bound several protein molecules without sequence specificity. The binding was 80% inhibited by 0.2 M NaCl. These properties are qualitatively similar, but not strictly identical, to those of two other viral movement proteins, the 30-kDa protein of tobacco mosaic virus and the gene I product of cauliflower mosaic virus.     

Molecular characterization and coat protein serology of watermelon leaf mottle virus (Potyvirus)

Summary.  A cDNA library was generated from purified RNA of watermelon leaf mottle virus (WLMV) (Genus Potyvirus). Two overlapping clones totaling 2,316 nucleotides at the 3′terminus of the virus were identified by immunoscreening with coat protein antiserum. The sequence analyses of the clones indicated an open reading frame (ORF) of 2,050 nucleotides which encoded part of the replicase and the coat protein, a 243-nucleotide non-coding region (3′UTR), and 23 adenine residues of the poly (A) tail. The taxonomic status of WLMV was determined by comparisons of the sequence of the cloned coat protein gene and 3′UTR with potyvirus sequences obtained from GenBank. The nucleotide sequence identities of WLMV compared with 17 other potyviruses ranged from 55.6 to 63.5% for the coat protein, and from 37.2 to 48.3% for the 3′UTR. Phylogenetic analyses of the coat protein region and the 3′UTR indicated that WLMV did not cluster with other potyviruses in a clade with high bootstrap support. The coat protein gene was expressed in Escherichia coli and a polyclonal antiserum was prepared to the expressed coat protein. In immunodiffusion tests, WLMV was found to be serologically distinct from papaya ringspot virus type W, watermelon mosaic virus 2, zucchini yellow mosaic virus, and Moroccan watermelon mosaic virus. In Western blots and ELISA, serological cross-reactivity with other cucurbit potyviruses was observed. Serological and sequence comparisons indicated that watermelon leaf mottle virus is a distinct member of the Potyvirus genus. Accepted September 23, 1999     

Inhibition of African cassava mosaic virus systemic infection by a movement protein from the related geminivirus tomato golden mosaic virus.

Plant viruses encode proteins that mediate their movement through the host plant leading to the establishment of a systemic infection. We have analyzed the effect of tomato golden mosaic virus (TGMV) genes BL1 and BR1, which are thought to be involved in the process of virus movement, on the infectivity of African cassava mosaic virus (ACMV) in Nicotiana benthamiana. Recombinant genomes were constructed by replacing the ACMV coat protein coding sequence with those of either BL1 or BR1. Replication of recombinants containing BL1 and BR1 coding sequences in the sense orientation with respect to the coat protein promoter was detected in the inoculated leaves only when the constructs were co-inoculated, suggesting that both genes are being expressed and act in a cooperative manner. Co-inoculated recombinants induced localized symptoms on inoculated leaves but did not spread systematically, either because of a defect in BL1 and/or BR1 expression or due to the inability of the TGMV gene products to functionally complement their ACMV counterparts. Systemic spread of ACMV was inhibited when the recombinant containing the BL1 coding sequence in the sense, but not in the antisense, orientation was co-inoculated with ACMV DNA B. Disruption of the BL1 coding sequence by a frameshift mutation restored the ability of the recombinant to spread systemically, suggesting that the gene product is responsible for the inhibitory effect. The inhibitory phenotype was mimicked by a chimera containing amino-terminal sequences of TGMV BL1 and carboxy-terminal sequences of its ACMV homologue, BC1. The chimera has characteristics of a dominant negative mutant. We suggest that dominant negative mutants of virus movement genes may provide a novel source for virus resistance genes.     

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.     

Engineering the genome of Grapevine virus A into a vector for expression of proteins in herbaceous plants

Grapevine virus A (GVA), a species of the genus Vitivirus, consists of a approximately 7.4 kb single-stranded RNA genome of positive polarity, organized into five open reading frames (ORFs). In addition to grape varieties, GVA infects Nicotiana benthamiana plants and protoplasts. We engineered the genome of GVA as a vector that includes duplication of homologous sequences that contain the promoter of the movement protein (MP) sgRNA, supplemented by enzymatic restriction sites to be used as a convenient tool for transient expression of foreign genes from an individual sgRNA. The resulting vector was able to infect and to move in N. benthamiana plants in a manner similar to the wild-type GVA, but it was not stable and the inserted sequence was lost from the genome. Replacing the duplicated promoter with a GVA-MP promoter derived from a distantly related isolate of GVA improved the stability of the inserted sequence. The resulting vector was successfully used to express the reporter gene beta-glucuronidase (GUS) and the coat protein gene of Citrus tristeza virus in inoculated N. benthamiana plants. Development of a useful GVA vector is expected to find a use as a biotechnological tool for improvement of grapevines and it may enable vine breeders to bypass obstacles involved in genetic manipulation of perennial and fruiting plants.     

Long-distance movement and viral assembly of tobacco mosaic virus mutants

Spreading of tobacco mosaic virus in infected plants is of two modes: cell-to-cell movement (to adjacent cells) and long-distance movement (to distant parts of the plant). Viral coat protein has been suggested to be involved in long-distance movement. To analyze the function of coat protein in the movement, we used mutants with modifications in the coat protein gene or in the assembly origin on the genomic RNA. A mutant which has the coding region for the C-terminal 5 amino acids of the protein deleted and mutants with 1 amino acid inserted after residue 101 or 152 of the protein retained both the abilities of long-distance movement and assembly into virus particles. Other mutants in the coat protein gene eliminated the two abilities. A mutant with modifications in the assembly origin displayed greatly reduced abilities of both the movement and assembly. These results suggest that both the coat protein with its ability to assemble into virus particles and the assembly origin are involved in long-distance movement, and that virus particles may play a pivotal role in the movement.     

Assembly of trans-encapsidated recombinant viral vectors engineered from Tobacco mosaic virus and Semliki Forest virus and their evaluation as immunogens

RNA virus vectors are attractive vaccine delivery agents capable of directing high-level gene expression without integration into host cell DNA. However, delivery of non-encapsidated RNA viral vectors into animal cells is relatively inefficient. By introducing the tobacco mosaic virus (TMV) origin of assembly into the RNA genome of Semliki Forest virus (SFV), we generated an SFV expression vector that could be efficiently packaged (trans-encapsidated) in vitro by purified TMV coat protein (CP). Using cellular assays, pseudovirus disassembly, RNA replication and reporter gene expression were demonstrated. We also evaluated the immune response to trans-encapsidated recombinant SFV carrying a model antigen gene (beta-galactosidase) in C57/B6 mice. Relative to RNA alone, vector encapsidation significantly improved the humoral and cellular immune responses. Furthermore, reassembly with recombinant TMV CPs permitted the display of peptide epitopes on the capsid surface as either genetic fusions or through chemical conjugation, to complement the immunoreactivity of the encapsidated RNA genetic payload. The SFV vector/TMV CP system described provides an alternative nucleic acid delivery mechanism that is safe, easy to manufacture in vitro and that also facilitates the generation of unique nucleic acid/protein antigen compositions.