Progress 09/01/01 to 08/31/05
Outputs
The transport of viruses within plants involves cell-to-cell translocation to and through the plasmodesmata (PDs) followed by rapid systemic movement via phloem. We have shown that the transport of Beet yellows virus (BYV) requires concerted action of seven viral proteins five of which potentiate cell-to-cell movement, and two more act in the systemic transport. The former five proteins encoded by quintuple gene block (QGB) include a 6-kDa hydrophobic protein (p6), an Hsp70 homologue (Hsp70h), a 60-kDa protein (p60), a minor capsid protein (CPm), and a major capsid protein (CP). P6 is a single-span, transmembrane protein that resides in ER and functions in virus movement from cell to cell. Because p6 is not required for virus replication or assembly, it can be considered a conventional movement protein (MP). CP forms a long, helical body of the flexuous, filamentous virions, and encapsidates 95% of the viral RNA. CPm is a principal component of the short virion tail.
In addition to CPm, tail assembly requires Hsp70h and p64 that act cooperatively to facilitate incorporation of CPm and to define the proper tail length. The tails encapsidate the 5 terminal, 700 nt-long, RNA region. Atomic force microscopy of BYV revealed that tails and possess segmented structure. A tip segment of the tails contains a 20-kDa protein (p20) that is dispensable for incorporation of other virion components. A salient functional aspect of the closteroviral virions is that each of the five structural proteins is also required for virus transport. In the case of CP, it is due to the need in protecting viral RNA during its transport. However, it seems unlikely that the transport-related function of the tail is merely protective. First, CP can encapsidate the entire genome if the tail formation is prevented by mutations. Second, tail harbors Hsp70h that was found in plasmodesmata. Third, p20 is dispensable for virion assembly and cell-to-cell movement, but is specifically
required for long-distance transport of the virus through the vascular system. All this prompted interpretation of the tail as a device that evolved to facilitate cell-to-cell and systemic transport of the large closterovirus genomes. The cycles of BYV genome expression and replication, virion assembly and cell-to-cell movement, each of which takes a day, are repeated till infection reaches companion cells that have connection to phloem sieve elements. Loading to sieve tubes allows virus to be transported long distances and to unload to sink tissues of stems, leaves, and roots. Two proteins that play specific roles in systemic transport are L-Pro and p20. Because L-Pro is a replicational enhancer, it is plausible that it aids RNA accumulation in the phloem cells. Location of p20 at the tail tip suggests possible role in either stabilizing virions in the phloem, or guiding them trough PD interconnecting sieve elements with companion cells, or both. In summary, transport of BYV requires
a dedicated MP p6, five structural proteins (CP, CPm, p60, Hsp70 and p20), and one replication-related protein, L-Pro.
Impacts
The impact of fundamental work involved State of Oregon level (teaching and advising at OSU), as well as numerous presentations at the national and international levels and publications in peer-reviewed jpurnals. The impact of applied work primarily involved biotech industry (Large Scale Biology Corp., Vacaville, CA and Growers Research Group, LLC, Soledad, CA) that contracted OSU. Our work improves understanding of viral disease and facilitates development of viral gene vectors for the needs of disease control and biotechnology.
Publications
- Peng, C.-W., Peremyslov, V.V., Mushegian, A.R., Dawson, W.O., and Dolja, V.V. 2001. Functional specialization and evolution of leader proteinases in the family Closteroviridae. J. Virol. 75, 12153-12160.
- Prokhnevsky, A.I., Peremyslov, V.V., and Dolja, V.V. (2005) Actin cytoskeleton is involved in targeting of a viral Hsp70 homolog to cell periphery. J. Virol. 79, 14421-14428.
- Chiba, M., Reed, J.C., Prokhnevsky, A.I., Chapman, E.J., Mawassi, M., Koonin, E.V., Carrington, J.C., and Dolja, V.V. (2005) Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon. Virology, in press.
- Peremyslov, V.V. and Dolja, V.V. (2005) Cloning of Large Positive-Strand RNA Viruses. Current Protocols in Microbiology, in press.
- Peremyslov, V.V., Andreev, I.A., Prokhnevsky, A.I., Duncan, G.H., Taliansky, M.E. and Dolja, V.V. (2004) Complex molecular architecture of beet yellows virus particles. Proc. Natl. Acad. Sci. USA 101, 5030-5035.
- Chapman, E.J., Prokhnevsky, A.I., Gopinath, K., Dolja, V.V., and Carrington, J.C. (2004) Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev. 18, 1179-1186.
- Peremyslov, V.V., Pan, Y.-W., and Dolja, V.V. (2004) Movement Protein of a Closterovirus Is a Type III Integral Transmembrane Protein Localized to the Endoplasmic Reticulum. J. Virol. 78, 3704-3709.
- Dolja, V.V. 2003. Beet yellows virus: The importance of being different. Mol. Plant Pathol., 4:91-98.
- Peng, C.-W., Napuli, A.J., and Dolja, V.V. (2003) Leader Proteinase of the Beet Yellows Virus Functions in Long-Distance Transport. J. Virol. 77, 2843-2849.
- Napuli, A.J., Alzhanova, D.V., Doneanu,C.E., Barofsky, D.F., Koonin,E.V., and Dolja, V.V. (2003) The 64-kDa Capsid Protein Homolog of Beet Yellows Virus Is Required for Assembly of Virion Tails. J. Virol. 77, 2377-2384.
- Medina, V., Rodrigo, G., Tian, T., Juarez, M., Dolja, V.V., Achon, M.A., and Falk., B.W. (2003) Comparative cytopathology of Crinivirus infections in different plant hosts. Ann. Appl. Biol. 143, 99-110.
- Reed, J.C., Kasschau, K.D., Prokhnevsky, A.I., Gopinath, K., Pogue, G.P., Carrington, J.C. and Dolja, V.V. (2003) Suppressor of RNA silencing encoded by beet yellows virus. Virology, 306, 203 - 209.
- Prokhnevsky, A.I., Peremyslov, V.V., Napuli, A.J., and Dolja, V.V. 2002. Interaction between Long-Distance Transport Factor and Hsp70-Related Movement Protein of Beet Yellows Virus. J. Virol. 76, 11003-11011.
- Peremyslov, V.V. and Dolja, V.V. 2002. Identification of the subgenomic mRNAs that encode 6-kDa movement protein and Hsp70 homolog of Beet Yellows Virus. Virology 295, 299-306.
- Peng, C.W., V.V. Peremyslov, E.J. Snijder, and Dolja, V.V. 2002. A replication-competent chimera of plant and animal viruses. Virology 294, 75-84.
- Alzhanova, D.V., Napuli, A.J., Creamer, R., and Dolja, V.V. 2001. Cell-to-cell movement and assembly of a plant closterovirus: Roles for the capsid proteins and Hsp70 homolog. EMBO J. 20, 6997-7007.
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Progress 01/01/04 to 12/31/04
Outputs
Closteroviruses possess exceptionally long filamentous particles that mediate protection and transport of the genomic RNA within infected plants. These virions are composed of a long body and short tail whose principal components are the major and minor capsid proteins (CP and CPm), respectively. We employed biochemical, genetic, and ultrastructural analyses to dissect the molecular composition and architecture of particles of Beet yellows virus (BYV), a closterovirus. We demonstrated that the virion tails encapsidate the 5-terminal, 650 nts-long, part of the viral RNA. In addition to CPm, the viral Hsp70-homolog,a 64-kDa, and 20-kDa proteins are also incorporated into the virion tail. Atomic force microscopy of virions revealed that the tail possesses a striking, segmented morphology. The unexpectedly complex structure of closterovirus virions has important mechanistic and functional implications. In addition to four structural proteins, cell-to-cell movement of BYV
requires four a 6-kDa protein (p6) We demonstrated that either virus infection results in association of p6 with the rough endoplasmic reticulum. The p6 possesses a transmembrane, N-terminal domain and a hydrophilic, C-terminal domain that is localized on the cytoplasmic face of the endoplasmic reticulum. In the infected cells, p6 forms a disulfide bridge via a cysteine residue located near the protein N-terminus. Mutagenic analyses indicated that each of the p6 domains, as well as protein dimerization, is essential for p6 function in virus movement. RNA silencing suppressors from different plant viruses are structurally diverse. Besides inhibiting the antiviral silencing response to condition susceptibility, many suppressors are pathogenicity factors that cause disease or developmental abnormalities. Here, unrelated suppressors from multiple viruses were shown to inhibit microRNA (miRNA) activities and trigger an overlapping series of severe developmental defects in transgenic
Arabidopsis thaliana. This suggests that interference with miRNA-directed processes may be a general feature contributing to pathogenicity of many viruses. A normally labile intermediate in the miRNA biogenesis/RISC assembly pathway, miRNA*, accumulated specifically in the presence of suppressors (P1/HC-Pro, p21 or p19) that inhibited miRNA-guided cleavage of target mRNAs. Both p21 and p19, but not P1/HCPro, interacted with miRNA/miRNA* complexes and hairpin RNA-derived siRNAs in vivo. In addition, p21 bound to synthetic miRNA/miRNA* and siRNA duplexes in vitro. We propose that different suppressors act by distinct mechanisms to inhibit the incorporation of small RNAs into active RISC complexes. We have also found that BYV p21 is required for virus pathogenicity and cell-to-cell movement. Taken together, our results support an intergrative concept of virus transport that requires structural, specialized movement, and counterdefensive viral proteins for plant colonization and disease
development.
Impacts
As in previous years of this project, the impact of fundamental work involved State of Oregon level (teaching at OSU) as well as National (annual retreat at Noble Foundation) and International (Congress of Virology in Morelia, Mexico) levels. The impact of applied work primarily involved biotech industry (Large Scale Biology Corp., Vacaville, CA; one of the major players in genomics and plant biotechnology) that contracted OSU. Our work improves understanding of viral disease and facilitates development of viral gene vectors for the needs of disease control and biotechnology.
Publications
- Peremyslov, V.V., Andreev, I.A., Prokhnevsky, A.I., Duncan, G.H., Taliansky, M.E. and Dolja, V.V. 2004. Complex molecular architecture of beet yellows virus particles. Proc. Natl. Acad. Sci. USA 101, 5030-5035.
- Chapman, E.J., Prokhnevsky, A.I., Gopinath, K., Dolja, V.V., and Carrington, J.C. 2004. Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev. 18, 1179-1186.
- Peremyslov, V.V., Pan, Y.-W., and Dolja, V.V. 2004. Movement Protein of a Closterovirus Is a Type III Integral Transmembrane Protein Localized to the Endoplasmic Reticulum. J. Virol. 78, 3704-3709.
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Progress 01/01/03 to 12/31/03
Outputs
Major progress has been made during last year toward characterization of the transport machinery of Beet yellows closterovirus (BYV). The filamentous virion of BYV consists of a long body formed by major capsid protein (CP) and a short tail, which is comprised of minor capsid protein (CPm) and the virus-encoded Hsp70 homolog. Using nano-liquid chromatography/tandem mass spectrometry and biochemical analyses, we showed that BYV 64-kDa protein (p64) is a fourth integral component of the BYV virions. The N-terminal domain of p64 is exposed at the virion surface and is accessible to antibodies and mild trypsin digestion. In contrast, the C-terminal domain is embedded in the virion and is inaccessible to antibodies or trypsin. The C-terminal domain of p64 is shown to be homologous to CP and CPm. Mutation of the signature motifs of capsid proteins of filamentous RNA viruses in p64 results in the formation of tailless virions, which are unable to move from cell to cell.
These results reveal the dual function of p64 in tail assembly and BYV motility and support the concept of the virion tail as a specialized device for BYV cell-to-cell movement. In addition, we addressed the role of BYV leader proteinase (L-Pro) in virus transport. L-Pro possesses a non-conserved N-terminal domain and a conserved, papain-like, C-terminal domain. Previous work revealed that the N-terminal domain functions in RNA amplification, whereas the C-terminal domain is required for autoproteolysis. Alanine-scanning mutagenesis was applied to complete functional analysis of the L-Pro throughout the virus life cycle. This analysis indicated that the C-terminal domain of the L-Pro, in addition to being required for proteolysis, also functions in RNA amplification, and that these two functions are genetically separable. Examination of the L-Pro role in BYV cell-to-cell movement revealed that none of the 20 examined replication-competent mutants was movement-defective. In contrast,
six of the L-Pro mutations affected the long-distance transport of BYV to various degrees, whereas three mutations completely abolished the transport. Because these mutations were located throughout the protein molecule, both domains of the L-Pro function in virus transport. We conclude that in addition to previously identified functions of the L-Pro, it also serves as the BYV long-distance transport factor.
Impacts
As in previous years of this project, the impact of fundamental work involved State of Oregon level (teaching at OSU) as well as National (annual meeting of ASV) and International(International Congress of Plant Pathology, New Zealand, invited seminar in Guelph, Canada) levels. The impact of applied work primarily involved biotech industry (Large Scale Biology Corp., Vacaville, CA; one of the major players in genomics and plant biotechnology) that contracted OSU.
Publications
- Peng, C.-W., Napuli, A.J., and Dolja, V.V. (2003) Leader Proteinase of the Beet Yellows Virus Functions in Long-Distance Transport. J. Virol. 77, 2843-2849.
- Napuli, A.J., Alzhanova, D.V., Doneanu,C.E., Barofsky, D.F., Koonin,E.V., and Dolja, V.V. (2003) The 64-kDa Capsid Protein Homolog of Beet Yellows Virus Is Required for Assembly of Virion Tails. J. Virol. 77, 2377-2384.
- Medina, V., Rodrigo, G., Tian, T., Juarez, M., Dolja, V.V., Achon, M.A., and Falk., B.W. (2003) Comparative cytopathology of Crinivirus infections in different plant hosts. Ann. Appl. Biol. 143, 99-110.
- Reed, J.C., Kasschau, K.D., Prokhnevsky, A.I., Gopinath, K., Pogue, G.P., Carrington, J.C. and Dolja, V.V. (2003) Suppressor of RNA silencing encoded by beet yellows virus. Virology, 306, 203 - 209.
- Dolja, V.V. 2003. Beet yellows virus: The importance of being different. Mol. Plant Pathol., 4:91-98.
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Progress 01/01/02 to 12/31/02
Outputs
Our research of the beet yellows virus (BYV) was focused on three major directions: understanding systemic transport of BYV, identifying BYV protein responsible for suppression of RNA silencing, which is a plant defense response, and characterizing expression mechanism for two BYV proteins, Hsp70h and p6. Systemic spread of viruses involves local movement from cell to cell and long-distance transport through the vascular system. The cell-to-cell movement of BYV is mediated by a movement protein that is an Hsp70 homolog (Hsp70h). This protein is required for the assembly of movement-competent virions that incorporate Hsp70h. Using three independent approaches, we showed that the Hsp70h interacts with BYV protein p20. We further demonstrated that p20 is associated with the virions via binding to Hsp70h. Genetic analyses indicate that p20 is required for the long-distance transport of virus trough the phloem. These results reveal a novel activity for the Hsp70h that
provides a molecular link between the local and systemic spread of a plant virus by docking a long distance transport factor to virions. Using an Agrobacterium-mediated transient assay, we screened the BYV genome for proteins with RNA silencing suppressor activity. Among eight proteins tested, only a 21-kDa protein (p21) was able to suppress RNA silencing. In addition, p21 elevated the transient expression level of the mRNAs. Similar activities were detected using homologs of p21 encoded by other members of the genus Closterovirus. Computer analysis indicated that p21-like proteins constitute a novel protein family that is unrelated to other recognized suppressors of RNA silencing. Examination of the subcellular distribution in BYV-infected plants revealed that p21 is partitioned between soluble cytoplasmic form and proteinaceous inclusion bodies at the cell periphery. A tandem arrangement of the genes encoding BYV p6 and Hsp70h is conserved among the members of the Closterovirus
genus. It was not known, however, if these movement proteins are expressed from one or two subgenomic (sg) RNAs. We showed that BYV produces separate sgRNAs encoding p6 and Hsp70h. This result is further supported by generation of the recombinant BYV in which the truncated variants of these sgRNAs were resolved by Northern analysis. The 5'-termini of the p6 and Hsp70h sgRNAs are localized to BYV nucleotides G-9,402 and A-9,467, respectively. Each of the sgRNAs was generated in vitro and found to direct the expected product upon translation in wheat germ extract. Inactivation of the first start codons in these sgRNAs abolished translation of the each product. The polyclonal antibodies raised to synthetic C-terminal peptides of p6 and Hsp70h specifically recognized corresponding translation products, as well as p6 and Hsp70h produced in BYV-infected plants. Taken together with the previous work, our data demonstrate that expression of BYV genome involves formation of as many as seven
sgRNAs.
Impacts
Our work resulted in a major progress toward characterization of virus-plant interactions during infection. We identified viral proteins required for suppression of plant defense and passage of virus through the vascular system. These data improve our understanding of viral disease and help to utilize virus and its proteins for the needs of plant biotechnology.
Publications
- Peremyslov, V.V. and Dolja, V.V. (2002) Identification of the subgenomic mRNAs that encode 6-kDa movement protein and Hsp70 homolog of beet yellows virus. Virology 295, 299-306.
- Peng, C.W., Peremyslov, V.V., Snijder, E.J. and Dolja, V.V. (2002) A replication-competent chimera of plant and animal viruses. Virology 294, 75-84.
- Prokhnevsky, A.I., Peremyslov, V.V., Napuli, A.J. and Dolja, V.V. (2002) Interaction between long-distance transport factor and Hsp70-related movement protein of beet yellows virus. J. Virol. 76, 11003-11011.
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Progress 01/01/01 to 12/31/01
Outputs
We analyzed the virion assembly and cell-to-cell movement of the beet yellows closterovirus (BYV) and revealed a strong correlation between these two processes. The filamentous virions of BYV possess a long body formed by the major capsid protein (CP) and a short tail formed by the minor capsid protein (CPm). Genetic and biochemical analyses showed that functions of these virion components are distinct. A virion body is primarily required for genome protection, whereas a tail represents specialized device for cell-to-cell movement. Furthermore, the tail assembly is mediated by the viral Hsp70 homolog (Hsp70h) that becomes an integral part of the virion. Inactivation of the ATPase domain of Hsp70h results in assembly of tailless virions that are incapable of translocation. A dual role of the viral molecular chaperone Hsp70h in virion assembly and transport combined with the previous finding of this protein in intercellular channels allowed us to propose a model of
closteroviral movement from cell to cell. We also compared functions of the several leader proteinases using a gene-swapping approach. The leader proteinase (L-Pro) of the BYV was replaced with L1 or L2 proteinases of the Citrus tristeza virus (CTV, another Closterovirus), P-Pro proteinase of the Lettuce infectious yellows virus (LIYV, a Crinivirus), and HC-Pro proteinase of the Tobacco etch virus (a Potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only the L1 and P-Pro, but not the L2 and HC-Pro were able to rescue the amplification of the chimeric BYV variants. The combined expression of the L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids in which only the papain-like domain of the L-Pro was replaced with those derived from the L1, L2, P-Pro, and
HC-Pro, also revealed functional specialization of these domains. In subcellular localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of the citrus tristeza virus originated by a gene duplication event.
Impacts
Our work allowed to develop a novel concept of the closterovirus cell-to-cell movement that is potentiated by a specialized, virion-associated movement device. This device is assembled by the minor capsid protein and a movement protein Hsp70h.In addition, we conducted comparative analysis of the leader proteinases from several closteroviruses and a potyvirus and revealed a high degree of functional specialization between these viral proteins. We have also identified novel functions of the leader proteinases in virus invasiveness and cell-to-cell movement.
Publications
- Alzhanova, D.V., Napuli, A.J., Creamer, R., and Dolja, V.V. 2001. Cell-to-cell movement and assembly of a plant closterovirus: Roles for the capsid proteins and Hsp70 homolog. EMBO J. 20, 6997-7007.
- Peng, C.-W., Peremyslov, V.V., Mushegian, A.R., Dawson, W.O., and Dolja, V.V. 2001. Functional specialization and evolution of leader proteinases in the family Closteroviridae. J. Virol. 75, 12153-12160.
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