Convenient Auto-Processing Vector Based on Bamboo Mosaic Virus for Presentation of Antigens Through Enzymatic Coupling

We have developed a new binary epitope-presenting CVP platform based on bamboo mosaic virus (BaMV) by using the sortase A (SrtA)-mediated ligation technology. The reconstructed BaMV genome harbors two modifications: 1) a coat protein (CP) with N-terminal extension of the tobacco etch virus (TEV) protease recognition site plus 4 extra glycine (G) residues as the SrtA acceptor; and 2) a TEV protease coding region replacing that of the triple-gene-block proteins. Inoculation of such construct, pKB5G, on Nicotiana benthamiana resulted in the efficient production of filamentous CVPs ready for SrtA-mediated ligation with desired proteins. The second part of the binary platform includes an expression vector for the bacterial production of donor proteins. We demonstrated the applicability of the platform by using the recombinant envelope protein domain III (rEDIII) of Japanese encephalitis virus (JEV) as the antigen. Up to 40% of the BaMV CP subunits in each CVP were loaded with rEDIII proteins in 1 min. The rEDIII-presenting BaMV CVPs (BJLPET5G) could be purified using affinity chromatography. Immunization assays confirmed that BJLPET5G could induce the production of neutralizing antibodies against JEV infections. The binary platform could be adapted as a useful alternative for the development and mass production of vaccine candidates.

Download Full-text

Subcellular Localization of the Barley Stripe Mosaic Virus Triple Gene Block Proteins

Journal of Virology ◽

10.1128/jvi.01862-09 ◽

2009 ◽

Vol 83 (21) ◽

pp. 11413-11413 ◽

Cited By ~ 1

Author(s):

Hyoun-Sub Lim ◽

Jennifer N. Bragg ◽

Uma Ganesan ◽

Steven Ruzin ◽

Denise Schichnes ◽

...

Keyword(s):

Subcellular Localization ◽

Mosaic Virus ◽

Triple Gene Block ◽

Barley Stripe Mosaic Virus ◽

Gene Block

Download Full-text

A Thioredoxin Domain-Containing Protein Interacts with Pepino mosaic virus Triple Gene Block Protein 1

International Journal of Molecular Sciences ◽

10.3390/ijms19123747 ◽

2018 ◽

Vol 19 (12) ◽

pp. 3747

Author(s):

Matthaios Mathioudakis ◽

Souheyla Khechmar ◽

Carolyn Owen ◽

Vicente Medina ◽

Karima Ben Mansour ◽

...

Keyword(s):

Mosaic Virus ◽

Triple Gene Block ◽

Polyclonal Antiserum ◽

Post Inoculation ◽

Mrna Levels ◽

In Planta ◽

Pepino Mosaic Virus ◽

Gene Block ◽

Triple Gene Block Protein

Pepino mosaic virus (PepMV) is a mechanically-transmitted tomato pathogen of importance worldwide. Interactions between the PepMV coat protein and triple gene block protein (TGBp1) with the host heat shock cognate protein 70 and catalase 1 (CAT1), respectively, have been previously reported by our lab. In this study, a novel tomato interactor (SlTXND9) was shown to bind the PepMV TGBp1 in yeast-two-hybrid screening, in vitro pull-down and bimolecular fluorescent complementation (BiFC) assays. SlTXND9 possesses part of the conserved thioredoxin (TRX) active site sequence (W__PC vs. WCXPC), and TXND9 orthologues cluster within the TRX phylogenetic superfamily closest to phosducin-like protein-3. In PepMV-infected and healthy Nicotiana benthamiana plants, NbTXND9 mRNA levels were comparable, and expression levels remained stable in both local and systemic leaves for 10 days post inoculation (dpi), as was also the case for catalase 1 (CAT1). To localize the TXND9 in plant cells, a polyclonal antiserum was produced. Purified α-SlTXND9 immunoglobulin (IgG) consistently detected a set of three protein bands in the range of 27–35 kDa, in the 1000 and 30,000 g pellets, and the soluble fraction of extracts of healthy and PepMV-infected N. benthamiana leaves, but not in the cell wall. These bands likely consist of the homologous protein NbTXND9 and its post-translationally modified derivatives. On electron microscopy, immuno-gold labelling of ultrathin sections of PepMV-infected N. benthamiana leaves using α-SlTXND9 IgG revealed particle accumulation close to plasmodesmata, suggesting a role in virus movement. Taken together, this study highlights a novel tomato-PepMV protein interaction and provides data on its localization in planta. Currently, studies focusing on the biological function of this interaction during PepMV infection are in progress.

Download Full-text

Triple Gene Block Protein Interactions Involved in Movement of Barley Stripe Mosaic Virus

Journal of Virology ◽

10.1128/jvi.02586-07 ◽

2008 ◽

Vol 82 (10) ◽

pp. 4991-5006 ◽

Cited By ~ 42

Author(s):

Hyoun-Sub Lim ◽

Jennifer N. Bragg ◽

Uma Ganesan ◽

Diane M. Lawrence ◽

Jialin Yu ◽

...

Keyword(s):

Mosaic Virus ◽

Triple Gene Block ◽

Cell Movement ◽

Wild Type Virus ◽

Barley Stripe Mosaic Virus ◽

Wild Type ◽

In Planta ◽

Gene Block ◽

Physical Interactions

ABSTRACT Barley stripe mosaic virus (BSMV) encodes three movement proteins in an overlapping triple gene block (TGB), but little is known about the physical interactions of these proteins. We have characterized a ribonucleoprotein (RNP) complex consisting of the TGB1 protein and plus-sense BSMV RNAs from infected barley plants and have identified TGB1 complexes in planta and in vitro. Homologous TGB1 binding was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs but not by mutations in two C-terminal helicase motifs. The TGB2 and TGB3 proteins were not detected in the RNP, but affinity chromatography and yeast two-hybrid experiments demonstrated that TGB1 binds to TGB3 and that TGB2 and TGB3 form heterologous interactions. These interactions required the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing these amino acid substitution were unable to move from cell to cell. Infectivity experiments indicated that TGB1 separated on a different genomic RNA from TGB2 and TGB3 could function in limited cell-to-cell movement but that the rates of movement depended on the levels of expression of the proteins and the contexts in which they are expressed. Moreover, elevated expression of the wild-type TGB3 protein interfered with cell-to-cell movement but movement was not affected by the similar expression of a TGB3 mutant that fails to interact with TGB2. These experiments suggest that BSMV movement requires physical interactions of TGB2 and TGB3 and that substantial deviation from the TGB protein ratios expressed by the wild-type virus compromises movement.

Download Full-text

Immunogenicity and Efficacy of Zika Virus Envelope Domain III in DNA, Protein, and ChAdOx1 Adenoviral-Vectored Vaccines

Vaccines ◽

10.3390/vaccines8020307 ◽

2020 ◽

Vol 8 (2) ◽

pp. 307 ◽

Cited By ~ 3

Author(s):

César López-Camacho ◽

Giuditta De Lorenzo ◽

Jose Luis Slon-Campos ◽

Stuart Dowall ◽

Peter Abbink ◽

...

Keyword(s):

Immune Responses ◽

Dengue Virus ◽

Zika Virus ◽

Neutralizing Antibodies ◽

Vaccine Candidate ◽

Protein Domain ◽

Virus Envelope ◽

Zikv Infection ◽

Domain Iii ◽

Open Question

The flavivirus envelope protein domain III (EDIII) was an effective immunogen against dengue virus (DENV) and other related flaviviruses. Whether this can be applied to the Zika virus (ZIKV) vaccinology remains an open question. Here, we tested the efficacy of ZIKV-EDIII against ZIKV infection, using several vaccine platforms that present the antigen in various ways. We provide data demonstrating that mice vaccinated with a ZIKV-EDIII as DNA or protein-based vaccines failed to raise fully neutralizing antibodies and did not control viremia, following a ZIKV challenge, despite eliciting robust antibody responses. Furthermore, we showed that ZIKV-EDIII encoded in replication-deficient Chimpanzee adenovirus (ChAdOx1-EDIII) elicited anti-ZIKV envelope antibodies in vaccinated mice but also provided limited protection against ZIKV in two physiologically different mouse challenge models. Taken together, our data indicate that contrary to what was shown for other flaviviruses like the dengue virus, which has close similarities with ZIKV-EDIII, this antigen might not be a suitable vaccine candidate for the correct induction of protective immune responses against ZIKV.

Download Full-text

Topological properties of the triple gene block protein 2 of Bamboo mosaic virus

Virology ◽

10.1016/j.virol.2008.06.019 ◽

2008 ◽

Vol 379 (1) ◽

pp. 1-9 ◽

Cited By ~ 17

Author(s):

Hsiu-Ting Hsu ◽

Yuan-Lin Chou ◽

Yang-Hao Tseng ◽

Yu-Hsing Lin ◽

Tzung-Min Lin ◽

...

Keyword(s):

Mosaic Virus ◽

Triple Gene Block ◽

Topological Properties ◽

Gene Block ◽

Triple Gene Block Protein

Download Full-text

Interactions of the TGB1 Protein during Cell-to-Cell Movement of Barley Stripe Mosaic Virus

Journal of Virology ◽

10.1128/jvi.75.18.8712-8723.2001 ◽

2001 ◽

Vol 75 (18) ◽

pp. 8712-8723 ◽

Cited By ~ 49

Author(s):

Diane M. Lawrence ◽

A. O. Jackson

Keyword(s):

Subcellular Localization ◽

Mosaic Virus ◽

Fluorescent Protein ◽

Triple Gene Block ◽

Cell Movement ◽

Barley Stripe Mosaic Virus ◽

Helicase Domain ◽

Chenopodium Amaranticolor ◽

Gene Block ◽

Green Fluorescent

ABSTRACT We have recently used a green fluorescent protein (GFP) fusion to the γb protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The γb protein is involved in expression of the triple gene block (TGB) proteins encoded by RNAβ but is not essential for cell-to-cell movement. The GFP fusion appears not to compromise replication or movement substantially, and mutagenesis experiments demonstrated that the three most abundant TGB-encoded proteins, βb (TGB1), βc (TGB3), and βd (TGB2), are each required for cell-to-cell movement (D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65–75, 2001). We have now extended these analyses by engineering a fusion of GFP to TGB1 to examine the expression and interactions of this protein during infection. BSMV derivatives containing the TGB1 fusion were able to move from cell to cell and establish local lesions in Chenopodium amaranticolor and systemic infections of Nicotiana benthamiana and barley. In these hosts, the GFP-TGB1 fusion protein exhibited a temporal pattern of expression along the advancing edge of the infection front. Microscopic examination of the subcellular localization of the GFP-TGB1 protein indicated an association with the endoplasmic reticulum and with plasmodesmata. The subcellular localization of the TGB1 protein was altered in infections in which site-specific mutations were introduced into the six conserved regions of the helicase domain and in mutants unable to express the TGB2 and/or TGB3 proteins. These results are compatible with a model suggesting that movement requires associations of the TGB1 protein with cytoplasmic membranes that are facilitated by the TGB2 and TGB3 proteins.

Download Full-text

First Report of Potato mop-top virus on Potato from the United States

Plant Disease ◽

10.1094/pdis.2003.87.7.872a ◽

2003 ◽

Vol 87 (7) ◽

pp. 872-872 ◽

Cited By ~ 27

Author(s):

D. H. Lambert ◽

L. Levy ◽

V. A. Mavrodieva ◽

S. B. Johnson ◽

M. J. Babcock ◽

...

Keyword(s):

Triple Gene Block ◽

The United States ◽

Russet Burbank ◽

Enzyme Linked Immunosorbent Assay ◽

Coding Region ◽

Rt Pcr ◽

Protein Coding ◽

Gene Block ◽

Elisa Testing ◽

Potato Mop Top Virus

Potato mop-top virus (PMTV) is a tripartite pomovirus vectored by the powdery scab plasmodiophoromycete Spongospora subterranea pv. subterranea (1). PMTV occurs on potato (Solanum tuberosum) in Europe, the Andes, Asia, and Canada. Internal necrotic arc and fleck tuber symptoms (“spraing”) may reduce commercial acceptance of some cultivars (3). PMTV symptoms were discovered in ‘Shepody’ tubers at the Aroostook Research Farm, Presque Isle, ME in May 2002 and subsequently in ‘Russet Burbank’ tubers in commercial storage from the 2001 Maine crop. Symptomatic tubers exhibited single or multiple concentric necrotic arcs that were partial or complete, but exhibited no distinct external symptoms. The presence of PMTV in eight ‘Shepody’ tubers was indicated by positive enzyme-linked immunosorbent assay (ELISA; Adgen, Ltd., Auchincruive, Ayr, Scotland) and confirmed by reverse transcription polymerase chain reaction (RT-PCR). ‘Russet Burbank’ potatoes were visually diagnosed, and the corresponding halves of 128 symptomatic tubers were forwarded to the University of Maine and APHIS (Beltsville, MD). Of these, ELISA readings in Maine were strongly positive (>3 × background) for 88, ambiguous (1.5-3 × background) for 13, and negative for 27. Subsamples from these three categories were positive by PCR in 17 of 17, 9 of 9, and 12 of 14 cases, respectively. A similar rating, positive or ambiguous, in ELISA testing was identical for all but one case at Beltsville. Confirmation of PMTV required PCR testing, resulting in a characteristic PCR product of 401 bp that was generated from the coat protein coding region on RNA 2 (2) using the primer pair PMTV 1 5′-GCAGCCGTCGAGAATAGATA-3′ (RNA nucleotides 316–335) and PMTV 4 5′-GCGAGTTGATGTGCC ACATT-3′ (complementary to RNA 2 nucleotides 716–697). An immunocapture RT-PCR using this primer set and the coating antibody from the Adgen ELISA kit was also successful in detecting PMTV. In separate reactions, a second product of 646 bp was generated from the triple gene block on RNA 3 (4) using the primer pair PMTV 5 5′-GGTGAACACGAGGACAAGGT-3′ (RNA 3 nucleotides 1417–1436) and PMTV 7 5′-AACAGTCCGGTCTTGTGAAC-3′ (complementary to RNA 3 nucleotides 2063–2044). The sequence of these products was 98 to 100% identical to PMTV published sequences. The discovery of this virus will result in adjustments to U.S. and Canadian seed potato certification standards and symptom characterization for common North American cultivars. References: (1) R. A. C. Jones and B. D. Harrison. Ann. Appl. Biol 63:1, 1969. (2) S. Kashiwazak et al. Virology 206:701, 1995. (3) M. Sandgren et al. Am. J. Potato Res. 79:205, 2002. (4) K. P. Scott et al. J. Gen. Virol.75:3561, 1994.

Download Full-text

Genetic Characterization of Pepino Mosaic Virus Isolates from Morocco

The Open Virology Journal ◽

10.2174/1874357901913010018 ◽

2019 ◽

Vol 13 (1) ◽

pp. 18-28

Author(s):

Amal Souiri ◽

Mustapha Zemzami ◽

Hayat Laatiris ◽

Saaid Amzazi ◽

Moulay M. Ennaji

Keyword(s):

Sequence Analysis ◽

Mosaic Virus ◽

Triple Gene Block ◽

Cross Protection ◽

Nucleotide Polymorphisms ◽

High Sequence Identity ◽

Tomato Production ◽

Pepino Mosaic Virus ◽

Gene Block ◽

Shared Identity

Introduction: Throughout the past few years, Pepino Mosaic Virus (PepMV) has rapidly evolved from an emerging virus to endemic pathogen that causes significant losses in tomato crops worldwide. Reliable detection and molecular characterization are very important tools to support disease control. Cross-protection can also be an effective strategy, but the efficacy depends strongly on the genotype. The genetic composition of the PepMV population in Morocco has not yet been determined. Aims: The current study aims to genetically characterize twelve PepMV isolates (PepMV-MA), all from different Moroccan tomato production areas, by analyzing nucleotide sequences of a part of the RNA-dependent RNA polymerase (RdRp), Triple Gene Block (TGB) and Coat Protein (CP) genes. Results: The sequence analysis of the twelve PepMV-MA isolates shows minor nucleotide differences between them, which implies a homogenous population. The phylogenetic analysis, based on the comparison with the major genotypes, showed that Moroccan PepMV populations share a very high sequence identity, 98%, with the Chilean strain (CH2), while the shared identity with the European strains (EU) is only 85%. Interestingly, Moroccan isolates reveal specific single nucleotide polymorphisms, some of which lead to amino acids changes. These mutations have never been described before, suggesting distinct variants that may enhance aggressiveness and symptomatology. Conclusion: Our careful sequence analysis and genotype determination, which placing homogenous Moroccan PepMV strains into CH2 genotype, would be a prerequisite for deploying effective cross-protection strategies for controlling the pathogen in the field.

Download Full-text