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Plum pox virus (PPV) is a worldwide threat to stone fruit production. Its woody perennial hosts provide a dynamic environment for virus evolution over multiple growing seasons. To investigate the impact seasonal host development plays in PPV population structure, next generation sequencing of ribosome associated viral genomes, termed translatome, was used to assess PPV variants derived from phloem or whole leaf tissues over a range of plum leaf and bud developmental stages. Results show that translatome PPV variants occur at proportionately higher levels in bud and newly developing leaf tissues that have low infection levels while more mature tissues with high infection levels display proportionately lower numbers of viral variants. Additional variant analysis identified distinct groups based on population frequency as well as sets of phloem and whole tissue specific variants. Combined, these results indicate PPV population dynamics are impacted by the tissue type and developmental stage of their host., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In this study we use vascular specific promoters and a translating ribosome affinity purification strategy to identify phloem associated translatome responses to infection by tobacco mosaic virus (TMV) in systemic hosts Arabidopsis thaliana ecotype Shahdara and Nicotiana benthamiana. Results demonstrate that in both hosts the number of translatome gene alterations that occurred in response to infection is at least four fold higher in phloem specific translatomes than in non-phloem translatomes. This finding indicates that phloem functions as a key responsive tissue to TMV infection. In addition, host comparisons of translatome alterations reveal both similarities and differences in phloem responses to infection, representing both conserved virus induced phloem alterations involved in promoting infection and virus spread as well as host specific alterations that reflect differences in symptom responses. Combined these results suggest phloem tissues play a disproportion role in the mediation and control of host responses to virus infection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Bottom-up self-assembly methods in which individual molecular components self-organize to form functional nanoscale patterns are of long-standing interest in the field of materials sciences. Such self-assembly processes are the hallmark of biology where complex macromolecules with defined functions assemble from smaller molecular components. In particular, plant virus-derived nanoparticles (PVNs) have drawn considerable attention for their unique self-assembly architectures and functionalities that can be harnessed to produce new materials for industrial and biomedical applications. In particular, PVNs provide simple systems to model and assemble nanoscale particles of uniform size and shape that can be modified through molecularly defined chemical and genetic alterations. Furthermore, PVNs bring the added potential to "farm" such bio-nanomaterials on an industrial scale, providing a renewable and environmentally sustainable means for the production of nano-materials. This review outlines the fabrication and application of several PVNs for a range of uses that include energy storage, catalysis, and threat detection., (Copyright © 2015 Elsevier Inc. All rights reserved.)

An interaction between the Tobacco mosaic virus (TMV) 126kDa replication protein and a host-encoded Rab GDP dissociation inhibitor (GDI2) was identified and investigated for its role in infection. GDI proteins are essential components of vesicle trafficking pathways. TMV infection alters the localization of GDI2 from the cytoplasm to ER-associated complexes. Partial silencing of GDI2 results in significant increases in the number of TMV infection foci observed in inoculated tissues. However, GDI2 silencing does not affect TMV accumulation at the infection site, cell-to-cell movement, or susceptibility of the host to mechanical inoculation. Furthermore, increases in the number of successful infection foci were specific to TMV and correlated with the appearance of vesicle-like rearrangements in the vacuolar membrane. Tissue infiltrations with brefeldin A, an inhibitor of vesicle trafficking, also enhanced host susceptibility to TMV. Combined these findings suggest that the 126kDa-GDI2 interaction alters vesicle trafficking to enhance the establishment of an infection., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Mutations disrupting helicase domain motifs of the Tobacco mosaic virus 126/183-kDa proteins were investigated for their effect on replicase function and assembly. These mutations inhibited virus replication but did not affect 126-kDa induced N gene resistance or RNAi suppression. However, in vivo expressed 126-kDa motif mutants yielded two distinct cytoplasmic phenotypes that correlated with ATPase activity. Specifically, ATPase active 126-kDa proteins produced small cytoplasmic bodies that resembled the ovoid granular-like bodies found early in virus infection while 126-kDa proteins defective in ATPase activity produced large tubule containing cytoplasmic bodies similar to those observed late in infection. Additional studies indicate that the helicase ATPase activity resides predominantly within monomer and dimer helicase forms and that motifs affecting ATPase activity induce alterations in helicase assembly. Combined these findings indicate that helicase ATPase activity modulates the progression of replicase complex assembly and maturation., (Copyright 2010 Elsevier Inc. All rights reserved.)

A transient expression system using onion epidermal cells was used to investigate domains of the Tobacco mosaic virus (TMV) 126-kDa replicase protein involved in cellular localization. Initially, a nuclear localization signal (NLS), identified within the amino-terminus of the 126-kDa protein, was investigated for its functionality using fusion constructs containing the green fluorescent protein (GFP). Fusion of the amino-terminal 70 amino acids of the 126-kDa protein, containing the NLS, to a beta-glucuronidase-GFP open reading frame (ORF), directed the accumulation of fluorescence to the nucleus. In contrast, similar constructs lacking the NLS or containing a mutated NLS sequence failed to accumulate within the nucleus. Additional investigations using GFP fusion constructs containing the first 178 or 388 amino acids of the 126-kDa protein also displayed nuclear localization. However, fusion constructs encoding the first 781 amino acids or the entire 126-kDa ORF did not accumulate within the nucleus but instead associated with the endoplasmic reticulum (ER), forming spot-like inclusions. Thus, a dominant ER association domain exists between amino acids 388 and 781 of the 126-kDa protein. Interestingly, a full-length 126-kDa GFP fusion construct encoding a nonfunctional NLS mutation also localized to the ER but did not form inclusions. Furthermore, a TMV mutant containing the same nonfunctional NLS mutation failed to replicate in protoplasts. Together these findings suggest that both the NLS and the ER retention domain contribute to the functional localization of the 126-kDa protein.

The Tobacco mosaic virus (TMV) 126-kDa and read-through 183-kDa replicase-associated proteins have been shown to interact [Watanabe, T., Honda, A., Iwata, A., Ueda, S., Hibi, T., Ishihama, A. (1999). J. Virol. 73, 2633-2640]. To identify and investigate the sequence required for this interaction, five segments covering different portions of the 126/183-kDa open reading frame, including the methyl-transferase, intervening region (IR), helicase-like (HEL), and polymerase domains, were screened via the yeast two-hybrid system against a library of TMV protein segments. Only one specific interaction between the HEL domain clone and a TMV library clone, IRnHEL, encoding the C-terminal half of the IR and the N-terminal portion of the HEL domain was identified. Sequence and deletion analysis revealed that the interacting clones share a region containing the helicase NTP-binding motif and that this region was essential for the interaction. To determine the functional significance of this interaction, mutants of the HEL domain segment that conferred a temperature-sensitive (ts) defect in the yeast interaction were identified and cloned into a recombinant TMV strain. Of the five selected mutants, three (V823I/S824N/V1042M, A877V, V1087I) produced a ts replication phenotype in protoplasts while the other two (A1073V, T884I) abolished TMV replication at both the permissive and the nonpermissive temperatures. An additional mutation, K839S, designed to disrupt the shared NTP-binding motif, nearly abolished the two-hybrid interaction and prevented virus replication, suggesting that NTP-binding and/or the structure of this motif is a contributing factor in the interaction. Taken together, these results provide support for an interaction between TMV replicase-associated proteins that involves specific structural features of the HEL and IR domains., (Copyright 2001 Academic Press.)

Specific sequences of the tobacco mosaic virus (TMV) RNA-dependent RNA-polymerase (RdRp) gene were investigated for their ability to confer cross-protection. Nine overlapping segments ranging from 713 to 1070 nucleotides in length and covering the methyltransferase, helicase, and polymerase (POL) domains of the TMV RdRp open reading frame were systemically expressed in Nicotiana benthamiana using a potato X virus (PVX) vector [Chapman, S., Kavanagh, T., and Baulcombe, D. C. (1992). Plant J., 1, 549-557]. PVX-infected plants were subsequently challenge inoculated with 10 microg of wild-type TMV and monitored for TMV accumulation. Mock inoculated plants and plants preinfected with the unmodified PVX vector rapidly accumulated high levels of challenge virus. In contrast, plants preinfected with PVX vectors expressing segments of the TMV RdRp open reading frame displayed either high or low levels of protection. High protection levels were observed for PVX constructs expressing segments of the TMV POL domain, whereas low protection levels were observed for PVX constructs expressing segments covering the methyltransferase and helicase domains. Frameshift mutations that blocked protein expression from RdRp segments disrupted only the high levels of protection derived from POL segments and not the low levels derived from the other segments. However, all RdRp segments conferred similarly high levels of protection against a TMV construct with restricted local movement. Thus both RNA and protein sequences in conjunction with the speed of the infecting challenge virus can affect the protection derived from the TMV RdRp gene., (Copyright 2000 Academic Press.)

To investigate the molecular role of the tobacco mosaic tobamovirus (TMV) coat protein (CP) in conferring cross-protection, a potato X potexvirus (PVX) vector (S. Chapman, Plant J. 2, 549-557, 1992) was used to systemically express a set of TMV mutant CPs in Nicotiana benthamiana prior to challenge inoculation with TMV. PVX-expressed wild-type TMV CP delayed TMV accumulation for up to 2 weeks compared to unprotected plants or plants preinfected with the unmodified PVX vector. Similar delays in TMV accumulation were obtained using TMV CPs that were deficient in virion formation but competent to assemble into helical aggregates. In contrast, TMV CPs that were incapable of helical aggregation or unable to bind viral RNA did not delay the accumulation of TMV. Furthermore, TMV CPs with enhanced intersubunit interactions that favor helical aggregation produced significantly greater delays in the accumulation of challenge TMV than obtained from the wild-type CP. Thus the capabilities of TMV CP to interact with viral RNA and self-associate in a helical fashion appear to be essential to its ability to confer protection. Taken together, these findings support a model for CP-mediated resistance in which the protecting CP recoats the challenge virus RNA as it disassembles., (Copyright 1998 Academic Press.)

Tobacco mosaic tobamovirus (TMV) coat protein (CP) mutant E50Q lacks a repulsive intersubunit carboxylate group and can effectively inhibit the disassembly of wild-type TMV (Culver et al, 1995, Virology 206,724). To investigate the ability of this mutant CP to block disassembly, a series of second-site amino acid substitutions were added to the E50Q CP. These second-site mutations were designed to disrupt specific intersubunit stabilizing interactions involving hydrophobic or polar residues, salt bridges, and CP-RNA contacts. Results showed substitutions disrupting intersubunit interactions that face the disassembling surface of the virion dramatically reduced the ability of CP E50Q to inhibit TMV disassembly. Substitutions that disrupted the CP inner loop, RNA binding capabilities, or intersubunit interactions that faced away from the disassembling surface did not dramatically interfere with CP E50Q's ability to inhibit disassembly. Taken together, these findings suggest that intersubunit interactions made by 5' terminal E50Q subunits, not associated with RNA, provide the stabilizing forces that prevent virion disassembly. The role of these stabilizing interactions in TMV disassembly and their potential use for creating disassembly inhibiting CPs are discussed.

Cross protection is the ability of one virus to prevent or delay infection by a related challenge virus. To examine this phenomena, a potato X potexvirus (PVX) vector (Chapman et al., 1992, Plant J. 2, 549) was used to systemically express the tobacco mosaic tobamovirus (TMV) coat protein (CP) open reading frame in Nicotiana benthamiana. PVX constructs induce mild mosaic symptoms in N. benthamiana, whereas TMV infection results in rapid systemic necrosis and plant death. Healthy plants or plants preinfected with the unmodified PVX vector succumbed to necrosis within 1 week of TMV challenge inoculation. However, plants preinfected with PVX vectors expressing the TMV CP or RNA coding sequence displayed a 1- to 2-week delay in the appearance of TMV-induced necrosis along with reduced accumulations of TMV. Symptom delay and reductions in the accumulation of TMV were more pronounced in plants protected with the vector expressing the TMV CP than with the vector expressing a nontranslatable CP sequence. Protection was overcome more quickly using a 10-fold higher concentration of challenge inoculum and was ineffective against TMV RNA or a TMV construct expressing a distantly related tobamovirus CP. These findings demonstrate that both RNA and protein are involved in this cross protection phenomenon, but that CP is the main contributor to protection. Taken together, these studies indicate that virus vectors can be used to simulate both cross protection and transgene-derived coat protein-mediated protection, thus providing a new method to further investigate the mechanisms behind each.

Structural studies of tobacco mosaic tobamovirus (TMV) have identified two coat protein (CP) intersubunit carboxyl-carboxylate interactions and one CP carboxylate-RNA phosphate interaction whose electrostatic repulsion is believed to drive virion disassembly. In this study, the involvement of each interaction in the disassembly process was examined. Site-directed mutagenesis was used to replace selected negatively charged CP residues, E or D, with neutral residues, Q or N, respectively. Purified mutant CPs were assayed for their ability to inhibit wild-type TMV disassembly both in vitro and in vivo. Results indicate that the lateral carboxylate interaction made by residue E106 is much more complex than previously thought, involving three residues, E95, E97, and D109, from an adjacent subunit. Mutations at all three residues are required to inhibit disassembly significantly. Different mutant coat proteins inhibited disassembly of the wild-type virus to varying degrees. Mutant E50Q, which modified the axial intersubunit interaction, had the greatest ability to inhibit disassembly followed by mutants E95Q/E97Q/D109N and D116N, which modified the lateral and CP-RNA interactions, respectively. Within each set of interacting carboxylate groups, mutations in the face opposite the disassembling surface of the TMV virion conferred the greatest ability to inhibit disassembly. This observation is consistent with the polar nature of TMV disassembly and confirms that repulsive intersubunit interactions derived from the 5' terminal subunits provide the key controlling mechanisms for virion disassembly.

Electrostatic repulsion between carboxylate groups across subunit interfaces has for many years been recognized as important in the disassembly of simple plant viruses. In the coat protein of tobacco mosaic virus (TMV), the amino acids Glu50 and Asp77 have been proposed as examples of such carboxylate groups. Site-directed mutagenesis has been used to replace these amino acids by Gln and Asn, respectively. Increased virion stability, together with reduced infectivity and reduced capacity for long-distance transport within the host plant confirms that the negative charges on the side chains of these amino acids are involved in the disassembly of TMV. Mixing purified mutant coat proteins with wild-type virions under appropriate conditions stabilizes the virions to alkaline disassembly and reduces their infectivity. It is suggested that transgenic plants expressing such mutant coat proteins could have enhanced resistance to virus infection.

Specific nucleotide changes in the coat protein gene of tobacco mosaic virus (TMV) have been identified as responsible for the induction of the hypersensitive reaction (HR) in Nicotiana sylvestris. Each of these nucleotide changes resulted in amino acid substitutions in the coat protein. To determine if the altered viral RNA or the altered protein acted directly to elicit the HR, the coat protein translational starts were removed from full-length cDNA clones of the HR-inducing mutant TMV 25 and the systemically infecting TMV U1 strain. Infectious transcripts of these altered genomes failed to induce HR in inoculated leaves of N. sylvestris. These free-RNA mutants moved poorly out of inoculated leaves and produced a systemic mosaic symptom 9 to 12 weeks after inoculation. Infectious viral RNA, from both mutants, was recovered from inoculated and systemic mosaic leaves. Western blot analysis of both inoculated and noninoculated leaves revealed the presence of TMV-encoded 126-kDa protein and the absence of coat protein for both mutants. This study demonstrates that the coat protein of TMV 25 is an elicitor molecule responsible for the induction of HR in N. sylvestris and that the TMV coat protein is not required for the development of systemic mosaic symptoms.