Cucumovirus- and bromovirus-encoded movement functions potentiate cell-to-cell movement of tobamo- and potexviruses

Cucumber mosaic virus (CMV, a cucumovirus) and Brome mosaic virus (BMV, a bromovirus) require the coat protein (CP) in addition to the 3a movement protein (MP) for cell-to-cell movement, while Cowpea chlorotic mottle virus (CCMV, a bromovirus) does not. Using bombardment-mediated transcomplementation assays, we investigated whether the movement functions encoded by these viruses potentiate cell-to-cell movement of movement-defective Tomato mosaic virus (ToMV, a tobamovirus) and Potato virus X (PVX, a potexvirus) mutants in Nicotiana benthamiana. Coexpression of CMV 3a and CP, but neither protein alone, complemented the defective movement of ToMV and PVX. A C-terminal deletion in CMV 3a (3a Delta C33) abolished the requirement of CP in transporting the ToMV genome. The action of 3a Delta C33 was inhibited by coexpression of wild-type 3a. These findings were confirmed in tobacco with ToMV-CMV chimeric viruses. Either BMV 3a or CCMV 3a alone efficiently complemented the movement-defective phenotype of the ToMV mutant. Therefore, every 3a protein examined intrinsically possesses the activity required to act as MP. In transcomplementation of the PVX mutant, the activities of BMV 3a, CCMV 3a, and CMV 3a Delta C33 were very low. The activities of the bromovirus 3a proteins were enhanced by coexpression of the cognate CP but the activity of CMV 3a Delta C33 was not. Based on these results, possible roles of cucumo- and bromovirus CPs in cell-to-cell movement are discussed.     

The movement protein gene is involved in the virus-specific requirement of the coat protein in cell-to-cell movement of bromoviruses

Summary.  Brome mosaic virus (BMV) requires the coat protein (CP) for cell-to-cell movement whereas Cowpea chlorotic mottle virus (CCMV), from the same genus, does not. Chimeric viruses created by exchanging the movement protein (MP) gene between the viruses can move from cell to cell. We show that interference in CP expression impaired the movement of the chimeric CCMV with the BMV MP gene but not of the chimeric BMV with the CCMV MP gene. We thus conclude that the MP gene plays a crucial role in determination of the virus-specific CP requirement in bromovirus cell-to-cell movement. Received September 19, 2002; accepted November 5, 2002     

Coat protein-independent cell-to-cell movement of bromoviruses expressing brome mosaic virus movement protein with an adaptation-related amino acid change in the central region

Summary. The movement protein (MP) of Brome mosaic virus (BMV) depends on the coat protein (CP) to mediate the cell-to-cell movement of BMV and CCMV(B3a), a recombinant Cowpea chlorotic mottle virus (CCMV) expressing BMV MP. Previous studies identified gain-of-function mutations in the central region of BMV MP that enable CCMV(B3a) to adapt to a resistant host. This study demonstrates that all adaptation-related MPs can partially or almost fully mediate the cell-to-cell movement of CCMV(B3a) and BMV without CP. Based on these results, we discuss adaptation mechanisms of CCMV(B3a) and the role of the central region of MP in the determination of virus movement mode.     

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.     

Molecular characterization of a strain of cucumber mosaic virus based on coat protein and movement protein genes

Cucumber mosaic virus (CMV) A strain (CMV-A) isolated from Amaranthus tricolor was partially characterized at molecular level. Complete coat protein (CP) and movement protein (MP) ORFs were cloned and sequenced. The 657 bp region of CP gene and the 840 bp region of MP gene encode 218 and 276 amino acids, respectively. CP, at nucleotide level, showed 90-98% sequence identity with the CMV subgroup I and less than 80% with the CMV subgroup II, it showed at amino acid level 92-96% identity with the subgroup I and 74-87% with the subgroup II. The nucleotide and amino acid sequence identities of MP ranged in 91-94% and 92-96%, respectively with the subgroup I but in 81-83% with the subgroup II. Phylogenetic trees generated from nucleotide and amino acid sequences of both CP and MP genes identified the virus strain as a member of the subgroup IB. CMV-A CP also displayed a remarkably higher homology with Indian strains of CMV than with other CMV strains and formed a separate cluster within the subgroup IB.     

A plastid-targeted heat shock cognate 70 kDa protein interacts with the Abutilon mosaic virus movement protein

The movement protein (MP) of bipartite geminiviruses facilitates cell-to-cell as well as long-distance transport within plants and influences viral pathogenicity. Yeast two-hybrid assays identified a chaperone, the nuclear-encoded and plastid-targeted heat shock cognate 70 kDa protein (cpHSC70-1) of Arabidopsis thaliana, as a potential binding partner for the Abutilon mosaic virus (AbMV) MP. In planta, bimolecular fluorescence complementation (BiFC) analysis showed cpHSC70-1/MP complexes and MP homooligomers at the cell periphery and co-localized with chloroplasts. BiFC revealed cpHSC70-1 oligomers associated with chloroplasts, but also distributed at the cellular margin and in filaments arising from plastids reminiscent of stromules. Silencing the cpHSC70 gene of Nicotiana benthamiana using an AbMV DNA A-derived gene silencing vector induced minute white leaf areas, which indicate an effect on chloroplast stability. Although AbMV DNA accumulated within chlorotic spots, a spatial restriction of these occurred, suggesting a functional relevance of the MP-chaperone interaction for viral transport and symptom induction.     

The hypersensitive response to cucumber mosaic virus in Chenopodium amaranticolor requires virus movement outside the initially infected cell

Cucumber mosaic virus (CMV) expressing the green fluorescent protein (GFP), and lacking either the 3a movement protein or the coat protein (CP), failed to induce a hypersensitive response producing local lesions in inoculated leaves of Chenopodium amaranticolor. Cytological analysis showed that both viral-encoded proteins are required for cell-to-cell movement of the virus and the simultaneous appearance of cellular necrosis. In the absence of either or both proteins, infection was confined to single, non-necrotized, epidermal cells. CMV with a mutation in the 3a protein (M8 CMV) could infect tobacco systemically but did not induce necrotic lesions in C. amaranticolor. In this host, the mutated 3a protein was unable to promote viral movement out of the initially infected epidermal cell. Movement-deficient CMV expressing wild-type (WT) 3a protein as a fusion to the GFP, as well as WT CP, also failed to induce necrosis. Finally, single epidermal cells infected with a movement-deficient CMV expressing WT 3a protein, WT CP, and free GFP did not show necrosis. These data indicate that viral movement out of the initially infected epidermal cell, and not the simultaneous expression in this cell of the 3a protein and the CP, is required for the induction of cell death.     

Mimicking carboxyterminal phosphorylation differentially effects subcellular distribution and cell-to-cell movement of Tobacco mosaic virus movement protein

Phosphorylation of Tobacco mosaic virus movement protein (TMV-MP) at three carboxyterminal Ser/Thr sites negatively regulates TMV-MP gating function and viral spread in Nicotiana tabacum but not in Nicotiana benthamiana, indicating a host dependant inactivation strategy. Here, we examine the effect of mimicking carboxyterminal phosphorylation on cell-to-cell transport of TMV-MP protein itself in host plants Nicotiana clevelandii, N. benthamiana, Nicotiana glutinosa and N. tabacum. Since TMV-MP transport function was inactivated only in N. tabacum, this host was chosen to explore the contribution of individual carboxyterminal phosphorylation sites. Selective mimicking of phosphorylation at one site enhances TMV-MP cell-to-cell transport, whereas a negative effect requires mimicking of phosphorylation at two or three sites. Potentially, during viral infection in N. tabacum, MP phosphorylation may occur sequentially: first, MP phosphorylation at a single site might ensure effective viral movement; only thereafter, further phosphorylation events may lead to inactivation of TMV-MP transport function.     

Mutational analysis of the cowpea mosaic virus movement protein

Cowpea mosaic virus moves from cell-to-cell in a virion form through tubular structures that are assembled in modified plasmodesmata. Similar tubular structures are formed on the surface of protoplasts inoculated with cowpea mosaic virus. The RNA 2-encoded movement protein (MP) is responsible for the induction and formation of these structures. To define functional domains of the MP, an alanine-substitution mutagenesis was performed on eight positions in the MP, including two conserved sequence motifs, the LPL and D motifs. Results show that these two conserved motifs as well as the central region of the MP are essential for cell-to-cell movement. Several viruses carrying mutations in the N- or C-terminal parts of their MP retained infectivity on cowpea plants. Coexpression studies revealed that mutant MPs did not interfere with the activity of wild-type MP and could not mutually complement their defects.     

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     

Effects of replacing the movement protein gene of Tobacco mosaic virus by that of Tomato mosaic virus

The broad bean strain of Tobacco mosaic virus (TMV-B) infects Nicotiana tabacum White Burley systemically whereas the tomato strain of T. mosaic virus (ToMV-S1) induces necrotic local lesions and is restricted to inoculated leaves. To examine the possible role of the viral movement protein (MP) in these symptom differences, a chimaeric virus (T/OMP) was produced in which the TMV-B MP gene was replaced by the ToMV-S1 MP gene. T/OMP induced the same symptoms as TMV-B in N. tabacum White Burley. However, in N. tabacum Samsun NN and other plants containing the N resistance gene, T/OMP caused necrotic lesions that were smaller than those produced by TMV-B but similar in size to those of ToMV-S1. We conclude that ToMV MP gene can substitute functionally for the TMV-B MP gene, and that the MP gene influences the size of necrotic local lesions on N-containing hosts.     

Trans-complementation of long-distance movement of White clover mosaic virus triple gene block (TGB) mutants: phloem-associated movement of TGBp1.

The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Both cell-to-cell and long-distance movement of White clover mosaic virus in which individual, combinations, or all movement functions were mutated could be rescued by transgenic Nicotiana benthamiana expressing complementary viral products. To address the importance of TGB functions in vascular transport, we used an experimental system based on grafted plants and trans-complementation, to define co-translocated viral products and the minimal requirements for viral exit from the plant vasculature. Evidence is presented that TGBp1 is co-translocated with viral RNA and CP and that, once viral RNA is loaded into the phloem translocation stream, it can exit in sink tissues and replicate in the absence of TGBp2-3. These results are discussed in the context of the recent finding that TGBp1 can mediate the suppression of signaling involved in systemic gene silencing.     

Development of a new cucumber mosaic virus-based plant expression vector with truncated 3a movement protein

We have developed a Cucumber mosaic virus (CMV)-based expression vector for the production of heterologous proteins in plants. Cell-to-cell movement of CMV is dependent on the presence of coat protein (CP). Previous studies have shown that deletion of 33 amino acids (aa) from the carboxy-terminus of the 3a movement protein facilitates cell-to-cell movement that is independent of CP. The CMV-based expression vector that we have designed utilizes this truncated 3a protein, allowing the expression of target genes from the strong CP subgenomic promoter and without the need for providing CP in trans for cell-to-cell spread. Using this vector we achieved expression levels of ~ 450 mg/kg leaf tissue of green fluorescent protein (GFP) when the vector was delivered into Nicotiana benthamiana plants by agroinfiltration. Human growth hormone (hGH), on the other hand, accumulated to ~ 170 mg/kg of leaf tissue when the same approach was used to deliver the vector.     

Identification of a novel tobacco DnaJ-like protein that interacts with the movement protein of tobacco mosaic virus

The movement protein (MP) of tobacco mosaic virus (TMV) mediates the transport of viral RNA from infected cells to neighboring uninfected cells via plasmodesmata by interacting with putative host factors. To find such host factors, we screened tobacco proteins using the yeast two-hybrid system. NtMPIP1, a novel subset of DnaJ-like proteins, was identified from a tobacco cDNA library, and its specific interaction with TMV MP was confirmed with an in vitro filter-binding assay. In a deletion analysis, using a series of truncated TMV MPs and NtMPIP1s, at least two regions of TMV MP, amino acid residues 65–86 and 120–185, conferred the ability to interact with the C-terminal domain of NtMPIP1, which is thought to be involved in substrate binding. Virus-induced gene silencing of NtMPIP1 significantly inhibited the spread of TMV. Therefore, it is reasonable to consider that endogenous NtMPIP1 is a host factor involved in virus cell-to-cell spread by interacting with TMV MP. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB092334.     

The complex subcellular distribution of satellite panicum mosaic virus capsid protein reflects its multifunctional role during infection

Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus for replication and movement in host plants. The positive-sense single-stranded genomic RNA of SPMV encodes a 17-kDa capsid protein (CP) to form 16-nm virions. We determined that SPMV CP accumulates in both cytosolic and non-cytosolic fractions, but cytosolic accumulation of SPMV CP is exclusively associated with virions. An N-terminal arginine-rich motif (N-ARM) on SPMV CP is used to bind its cognate RNA and to form virus particles. Intriguingly, virion formation is dispensable for successful systemic SPMV RNA accumulation, yet this process still depends on an intact N-ARM. In addition, a C-terminal domain on the SPMV CP is necessary for self-interaction. Biochemical fractionation and fluorescent microscopy of green fluorescent protein-tagged SPMV CP demonstrated that the non-cytosolic SPMV CP is associated with the cell wall, the nucleus and other membranous organelles. To our knowledge, this is the first report that a satellite virus CP not only accumulates exclusively as virions in the cytosol but also is directed to the nucleolus and membranes. That SPMV CP is found both in the nucleus and the cell wall suggests its involvement in viral nuclear import and cell-to-cell transport.     

Viral cell-to-cell movement requires formation of cortical punctate structures containing Red clover necrotic mosaic virus movement protein

Movement protein (MP) of Red clover necrotic mosaic virus (RCNMV) forms punctate structures on the cortical endoplasmic reticulum (ER) of Nicotiana benthamiana cells, which are associated with viral RNA1 replication (Kaido et al., Virology 395, 232-242. 2009). We investigated the significance of ER-targeting by MP during virus movement from cell to cell, by analyzing the function of a series of MPs with varying length deletions at their C-terminus, either fused or not fused with green fluorescent protein (GFP). The C-terminal 70 amino acids were crucial to ER-localization of MP-GFP and cell-to-cell movement of the recombinant virus encoding it. However, C-terminal deletion did not affect MP functions, such as increasing the size exclusion limit of plasmodesmata, single-stranded RNA binding in vitro, and MP interacting in vivo. We discuss the possible role of this MP region in virus movement from cell to cell.