Tomato spotted wilt virus glycoproteins exhibit trafficking and localization signals that are functional in mammalian cells

The glycoprotein precursor (G1/G2) gene of tomato spotted wilt virus (TSWV) was expressed in BHK cells using the Semliki Forest virus expression system. The results reveal that in this cell system, the precursor is efficiently cleaved and the resulting G1 and G2 glycoproteins are transported from the endoplasmic reticulum (ER) to the Golgi complex, where they are retained, a process that could be blocked by tunicamycin. Expression of G2 alone resulted in transport to and retention in the Golgi complex, albeit less efficient, suggesting that G2 contains a Golgi retention signal. G1 alone was retained in the ER, irrespective of whether it contained the precursor's signal sequence or its own N-terminal hydrophobic sequence. Coexpression of G1 and G2 from separate gene constructs resulted in rescue of efficient G1 transport, as the proteins coaccumulated in the Golgi complex, indicating that their interaction is essential for proper targeting to this organelle. The results demonstrate that transport and targeting of the plant TSWV glycoproteins in mammalian BHK cells are strikingly similar to those of animal-infecting bunyavirus glycoproteins in mammalian cells. The observations are likely to reflect the dual tropism of TSWV, which replicates both in its plant host and in its animal (thrips) vector.     

Biolistic transformation of chrysanthemum with the nucleocapsid gene of tomato spotted wilt virus

In vitro regeneration and biolistic transformation procedures were developed for several commercial chrysanthemum Dendranthema grandiflora Tzvelev, syn. Chrysanthemum morifolium Ramat. cultivars using leaf and stem explants. Studies on the effect of several growth regulators and kanamycin on chrysanthemum regeneration were conducted, and a step-wise procedure to optimize kanamycin selection and recovery of transgenic plants was developed. A population of putative transformed chrysanthemum plants cvs. Blush, Dark Bronze Charm, Iridon, and Tara, was obtained after bombardment with tungsten microprojectiles coated with the binary plasmid pBIN19 containing the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) and the marker gene neomycin phosphotransferase (NPT II). PCR analysis of 82 putative transgenic plants selected on kanamycin indicated that the majority of the lines (89%) were transformed and contained both genes (71%). However, some transgenic lines contained only one of the genes: either the NPT II (15%) or the TSWV (N) gene (14%). Southern blot analysis on selected transgenic lines confirmed the integration of the TSWV (N) gene into the chrysanthemum genome. These results demonstrate the development of an efficient procedure to transfer genetic material into the chrysanthemum genome and selectively regenerate transgenic chrysanthemum plants at frequencies higher than previously reported.     

Analysis of Tomato spotted wilt virus NSs protein indicates the importance of the N‐terminal domain for avirulence and RNA silencing suppression

Recently, Tomato spotted wilt virus (TSWV) nonstructural protein NSs has been identified unambiguously as an avirulence (Avr) determinant for Tomato spotted wilt (Tsw)‐based resistance. The observation that NSs from two natural resistance‐breaking isolates had lost RNA silencing suppressor (RSS) activity and Avr suggested a link between the two functions. To test this, a large set of NSs mutants was generated by alanine substitutions in NSs from resistance‐inducing wild‐type strains (NSs^RI), amino acid reversions in NSs from resistance‐breaking strains (NSs^RB), domain deletions and swapping. Testing these mutants for their ability to suppress green fluorescent protein (GFP) silencing and to trigger a Tsw‐mediated hypersensitive response (HR) revealed that the two functions can be separated. Changes in the N‐terminal domain were found to be detrimental for both activities and indicated the importance of this domain, additionally supported by domain swapping between NSs^RI and NSs^RB. Swapping domains between the closely related Tospovirus Groundnut ringspot virus (GRSV) NSs and TSWV NSs^RI showed that Avr functionality could not simply be transferred between species. Although deletion of the C‐terminal domain rendered NSs completely dysfunctional, only a few single‐amino‐acid mutations in the C‐terminus affected both functions. Mutation of a GW/WG motif (position 17/18) rendered NSs completely dysfunctional for RSS and Avr activity, and indicated a putative interaction between NSs and Argonaute 1 (AGO1), and its importance in TSWV virulence and viral counter defence against RNA interference.     

Agrobacterium-mediated transformation of cultivated tomato with construct carrying the nucleoprotein (N) gene from tomato spotted wilt virus (TSWV)

Significant yield losses in commercial tomato production caused by tomato spotted wilt virus (TSWV) are the reason why we have undertaken studies on resistance to this pathogen. One of the possible sources of resistance can be the incorporation of the nucleoprotein N viral gene by Agrobacterium transformation. The N gene was introduced into three Lycopersicon esculentum forms. Out of the total of 3044 cotyledon explants 14.7% regenerated shoots, but only a few were rooted on medium containing kanamycin. The preliminary analysis indicated that 18 plants are putative transformants.     

Bean dwarf mosaic virus BV1 protein is a determinant of the hypersensitive response and avirulence in Phaseolus vulgaris

The capacities of the begomoviruses Bean dwarf mosaic virus (BDMV) and Bean golden yellow mosaic virus (BGYMV) to differeBean dwarf mosaic viru certain common bean (Phaseolus vulgaris) cultivars were used to identify viral determinants of the hypersensitive response (HR) and avirulence (avr) in BDMV. A series of hybrid DNA-B components, containing BDMV and BGYMV sequences, was constructed and coinoculated with BDMV DNA-A (BDMV-A) or BDMVA-green florescent protein into seedlings of cv. Topcrop (susceptible to BDMV and BGYMV) and the BDMV-resistant cvs. Othello and Black Turtle Soup T-39 (BTS). The BDMV avr determinant, in bean hypocotyl tissue, was mapped to the BDMV BV1 open reading frame and, most likely, to the BV1 protein. The BV1 also was identified as the determinant of the HR in Othello. However, the HR was not required for resistance in Othello nor was it associated with BDMV resistance in BTS. BDMV BV1, a nuclear shuttle protein that mediates viral DNA export from the nucleus, represents a new class of viral avr determinant. These results are discussed in terms of the relationship between the HR and resistance.     

The nucleoprotein of Tomato spotted wilt virus as protein tag for easy purification and enhanced production of recombinant proteins in plants

Upon infection, Tomato spotted wilt virus (TSWV) forms ribonucleoprotein particles (RNPs) that consist of nucleoprotein (N) and viral RNA. These aggregates result from the homopolymerization of the N protein, and are highly stable in plant cells. These properties feature the N protein as a potentially useful protein fusion partner. To evaluate this potential, the N protein was fused to the Aequorea victoria green fluorescent protein (GFP), either at the amino or carboxy terminus, and expressed in plants from binary vectors in Nicotiana benthamiana leaves were infiltrated with Agrobacterium tumefaciens and evaluated after 4 days, revealing an intense GFP fluorescence under UV light. Microscopic analysis revealed that upon expression of the GFP:N fusion a small number of large aggregates were formed, whereas N:GFP expression led to a large number of smaller aggregates scattered throughout the cytoplasm. A simple purification method was tested, based on centrifugation and filtration, yielding a gross extract that contained large amounts of N:GFP aggregates, as confirmed by GFP fluorescence and Western blot analysis. These results show that the homopolymerization properties of the N protein can be used as a fast and simple way to purify large amounts of proteins from plants.     

Transgenic peanut plants containing a nucleocapsid protein gene of tomato spotted wilt virus show divergent levels of gene expression

The nucleocapsid protein (N) gene of the lettuce isolate of tomato spotted wilt virus (TSWV) was inserted into peanut (Arachis hypogaea L.) via microprojectile bombardment. Constructs containing the hph gene for resistance to the antibiotic hygromycin and the TSWV N gene were used for bombardment of peanut somatic embryos. High frequencies of transformation and regeneration of plants containing the N gene were obtained. Southern blot analysis of independent transgenic lines revealed that one to several copies of the N gene were integrated into the peanut genome. Northern blot, RT-PCR and ELISA analyses indicated that a gene silencing mechanism may be operating in primary transgenic lines containing multiple copy insertions of the N transgene. One transgenic plant which contained a single copy of the transgene expressed the N protein in the primary transformant, and the progeny segregated in a 3 :1 ratio based upon ELISA determination. Received: 24 October 1997 / Revision received: 9 February 1998 / Accepted: 21 February 1998     

A CAPS marker to assist selection of tomato spotted wilt virus (TSWV) resistance in pepper.

The hypersensitive resistance to tomato spotted wilt virus (TSWV) in pepper is determined by a single dominant gene (resistant allele: Tsw) in several Capsicum chinense genotypes. In order to facilitate the selection for this resistance, four RAPD (among 250 10-mer primers tested) were found linked to the Tsw locus using the bulked segregant analysis and 153 F2 individuals. A close RAPD marker was converted into a codominant cleaved amplified polymorphic sequence (CAPS) using specific PCR primers and restriction enzymes. This CAPS marker is tightly linked to Tsw (0.9 +/- 0.6 cM) and is helpful for marker-assisted selection in a wide range of genetic intercrosses.     

The nuclear shuttle protein of Tomato leaf curl New Delhi virus is a pathogenicity determinant

The role of the movement protein (MP) and nuclear shuttle protein (NSP) in the pathogenicity of Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, was studied. Both genes were expressed in Nicotiana benthamiana, Nicotiana tabacum, and Lycopersicon esculentum plants with the Potato virus X (PVX) expression vector or by stable transformation of gene constructs under the control of the 35S promoter in N. tabacum. No phenotypic changes were observed in any of the three species when the MP was expressed from the PVX vector or constitutively expressed in transgenic plants. Expression of the ToLCNDV NSP from the PVX vector in N. benthamiana resulted in leaf curling that is typical of the disease symptoms caused by ToLCNDV in this species. Expression of NSP from PVX in N. tabacum and L. esculentum resulted in a hypersensitive response (HR), demonstrating that the ToLCVDV NSP is a target of host defense responses in these hosts. The NSP, when expressed as a transgene under the control of the 35S promoter, resulted in necrotic lesions in expanded leaves that initiated from a point and then spread across the leaf. The necrotic response was systemic in all the transgenic plants. Deletion of 100 amino acids from the C terminus did not compromise the HR response, suggesting that this region has no role in HR. Deletion of 60 or 100 amino acids from the N terminus of NSP abolished the HR response, suggesting that these sequences are required for the HR response. These findings demonstrate that the ToLCNDV NSP is a pathogenicity determinant as well as a target of host defense responses.     

Overcoming host- and pathogen-mediated resistance in tomato and tobacco maps to the M RNA of Tomato spotted wilt virus

A viral genetic system was used to map the determinants of the ability of Tomato spotted wilt virus (TSWV) to overcome the R gene (Sw-5) in tomato and the resistance conferred by the nucleocapsid gene of TSWV (N gene) in tobacco. A complete set of reassortant genotypes was generated from TSWV isolates A and D. TSWV-A was able to overcome the Sw-5 gene in tomato and the TSWV N gene in tobacco, whereas TSWV-D was repressed by both forms of resistance. The ability to overcome both forms of resistance was associated with the M RNA segment of TSWV-A (M(A)). Overcoming the Sw-5 gene was linked solely to the presence of M(A), and the ability of M(A) to overcome the TSWV N gene was modified by the L RNA and the S RNA of TSWV-A, which is consistent with previous reports that suggest that the nucleocapsid gene is not the primary determinant for overcoming the nucleocapsid-mediated resistance. Sequence analysis of the M RNA segment of TSWV-A, -D, and the type isolate BR-01 revealed multiple differences in the coding and noncoding regions, which prevented identification of the resistance-breaking nucleotide sequences.     

Engineered resistance to tomato spotted wilt virus in transgenic peanut expressing the viral nucleocapsid gene

The nucleocapsid gene of tomato spotted wilt virus Hawaiian L isolate in a sense orientation, and the GUS and NPTII marker genes, were introduced into peanut (Arachis hypogaea cv. New Mexico Valencia A) using Agrobacterium-mediated transformation. Modifications to a previously defined transformation protocol reduced the time required for production of transformed peanut plants. Transgenes were stably integrated into the peanut genome and transmitted to progeny. RNA expression and production of nucleocapsid protein in transgenic peanut were observed. Progeny of transgenic peanut plants expressing the nucleocapsid gene showed a 10- to 15-day delay in symptom development after mechanical inoculations with the donor isolate of tomato spotted wilt virus. All transgenic plants were protected from systemic tomato spotted wilt virus infection. Inoculated non-transformed control plants and plants transformed with a gene cassette not containing the nucleocapsid gene became systemically infected and displayed typical tomato spotted wilt virus symptoms. These results demonstrate that protection against tomato spotted wilt virus can be achieved in transgenic peanut plants by expression of the sense RNA of the tomato spotted wilt virus nucleocapsid gene     

Amphipathic alpha-helix AH2 is a major determinant for the oligomerization of hepatitis C virus nonstructural protein 4B

Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. It induces a specific membrane rearrangement, designated membranous web, that serves as a scaffold for the HCV replication complex. However, the mechanisms underlying membranous web formation are poorly understood. Based on fluorescence resonance energy transfer (FRET) and confirmatory coimmunoprecipitation analyses, we provide evidence for an oligomerization of NS4B in the membrane environment of intact cells. Several conserved determinants were found to be involved in NS4B oligomerization, through homotypic and heterotypic interactions. N-terminal amphipathic α-helix AH2, comprising amino acids 42 to 66, was identified as a major determinant for NS4B oligomerization. Mutations that affected the oligomerization of NS4B disrupted membranous web formation and HCV RNA replication, implying that oligomerization of NS4B is required for the creation of a functional replication complex. These findings enhance our understanding of the functional architecture of the HCV replication complex and may provide new angles for therapeutic intervention. At the same time, they expand the list of positive-strand RNA virus replicase components acting as oligomers.     

Dengue virus nonstructural protein 1 is expressed in a glycosyl-phosphatidylinositol-linked form that is capable of signal transduction.

Dengue virus nonstructural protein 1 (NS1) is expressed on the surface of infected cells and is a target of human antibody responses to dengue virus infection. We show here that dengue virus uses the cellular glycosyl-phosphatidylinositol (GPI) linkage pathway to express a GPI-anchored form of NS1 and that GPI anchoring imparts a capacity for signal transduction in response to binding of NS1-specific antibody. This study is the first to identify GPI linkage of a virus-encoded protein. The GPI anchor addition signal for NS1 was identified, by transfection of HeLa cells with dengue cDNA constructs, as a downstream hydrophobic domain in NS2A. GPI linkage of NS1 in both transfected and infected cells was demonstrated by cleavage of NS1 from the surface by PI-specific phospholipase C and by metabolic incorporation of the GPI-specific components ethanolamine and inositol. In common with other GPI-anchored proteins, addition of specific antibody resulted in signal transduction, as evidenced by tyrosine phosphorylation of cellular proteins. Antibody-induced signal transduction by GPI-linked NS1 suggests a mechanism of cellular activation that may contribute to the pathogenesis of human dengue disease. Signal transduction by a GPI-anchored viral antigen interacting with a specific antibody that it induces is a new concept in the pathogenesis of viral disease.     

Resistance to tomato spotted wilt virus infection in transgenic tobacco expressing the viral nucleocapsid gene.

A recombinant plasmid containing the entire tomato spotted with virus (TSWV) nucleocapsid gene, with the exception of nucleotide encoding three N-terminal amino acids, was isolated by screening a complementary DNA library, prepared against random primed viral RNA, using a specific monoclonal antibody. The insert contained in plasmid pTSW1 was repaired and amplified by polymerase chain reaction, and the complete nucleocapsid protein gene was introduced into Nicotiana tabacum 'Samsun' by leaf disk transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral nucleocapsid protein were resistant to subsequent infection following mechanical inoculation with TSWV as indicated by a lack of systemic symptoms and little or no systemic accumulation of virus as determined by double antibody sandwich enzyme-liked immunosorbent assay. These results further extend the applicability of coat protein-mediated resistance, as previously demonstrated for a number of simple plant viruses composed of a positive-sense RNA genome encapsidated with a single species of coat protein, to a membrane-encapsidated, multi-component, negative-sense RNA virus.     

Changes in ultrastructure and protein metabolism of tobacco plants infected by tomato spotted wilt virus

As the result of electron microscope investigation of ultra-thin sections of the tissues infected by tomato spotted wilt virus it was shown that ultrastructural changes in the cells depend on the virus virulence. The isolate with low virulence induces mostly virus-specific changes (virus particles and virus inclusion bodies); the isolate with high virulence besides the virus-specific changes causes essential non-specific violation of cell organelle structure that could be the consequence of pathological action of the virus. It was determined that severe virus infection results in the decrease of general content of the proteins in the leaves. At the same time it induces formation of at least three pathogenesis-associated proteins (PR-proteins) and two antiviral factors of the types AVF (6) and IVR (7) active towards tobacco mosaic virus.     

Evidence that the Rous sarcoma virus transforming gene product is associated with glycerol kinase activity

This communication provides biochemical, immunological, and genetic evidence that pp60src, the Rous sarcoma virus transforming gene product, is associated with glycerol kinase activity. Our investigations demonstrated that the compound phosphorylated by pp60src or by glycerol kinase (EC 2.7.1.30) from Candida mycoderma share the same electrophoretic and chromatographic mobilities. The glycerol kinase and protein kinase activities of pp60src were inhibited similarly by preincubation with immune IgG. Both activities were reduced 6-9-fold in pp60src preparations derived by immunoaffinity chromatography from cells which were infected with NY68, a temperature-sensitive transformation mutant of Rous sarcoma virus. The thermolability at 41 degrees C of the glycerol kinase activity of pp60src from the mutant virus-infected cells was greater (t/2 = 1.3 min) than the same activity in pp60src preparations from wild type virus-infected cells (t/2 = 4.8 min).     

Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism

A recombinant infectious bronchitis virus (IBV), BeauR-M41(S), was generated using our reverse genetics system (R. Casais, V. Thiel, S. G. Siddell, D. Cavanagh, and P. Britton, J. Virol. 75:12359-12369, 2001), in which the ectodomain region of the spike gene from IBV M41-CK replaced the corresponding region of the IBV Beaudette genome. BeauR-M41(S) acquired the same cell tropism phenotype as IBV M41-CK in four different cell types, demonstrating that the IBV spike glycoprotein is a determinant of cell tropism.