Single-Molecule Force Spectroscopy Study on the Mechanism of RNA Disassembly in Tobacco Mosaic Virus

To explore the disassembly mechanism of tobacco mosaic virus (TMV), a model system for virus study, during infection, we have used single-molecule force spectroscopy to mimic and follow the process of RNA disassembly from the protein coat of TMV by the replisome (molecular motor) in vivo, under different pH and Ca²⁺ concentrations. Dynamic force spectroscopy revealed the unbinding free-energy landscapes as that at pH 4.7 the disassembly process is dominated by one free-energy barrier, whereas at pH 7.0 the process is dominated by one barrier and that there exists a second barrier. The additional free-energy barrier at longer distance has been attributed to the hindrance of disordered loops within the inner channel of TMV, and the biological function of those protein loops was discussed. The combination of pH increase and Ca²⁺ concentration drop could weaken RNA-protein interactions so much that the molecular motor replisome would be able to pull and disassemble the rest of the genetic RNA from the protein coat in vivo. All these facts provide supporting evidence at the single-molecule level, to our knowledge for the first time, for the cotranslational disassembly mechanism during TMV infection under physiological conditions.     

Tobacco mosaic virus particle structure and the initiation of disassembly

The structure of an intact tobacco mosaic virus (TMV) particle was determined at 2.9 A resolution using fibre diffraction methods. All residues of the coat protein and the three nucleotides of RNA that are bound to each protein subunit were visible in the electron density map. Examination of the structures of TMV, cucumber green mottle mosaic virus and ribgrass mosaic virus, and site-directed mutagenesis experiments in which carboxylate groups were changed to the corresponding amides, showed that initial stages of disassembly are driven by complex electrostatic interactions involving at least seven carboxylate side-chains and a phosphate group. The locations of these interactions can drift during evolution, allowing the viruses to evade plant defensive responses that depend on recognition of the viral coat protein surface.     

A kinetic Zipper model and the assembly of tobacco mosaic virus

We put forward a modified Zipper model inspired by the statics and dynamics of the spontaneous reconstitution of rodlike tobacco mosaic virus particles in solutions containing the coat protein and the single-stranded RNA of the virus. An important ingredient of our model is an allosteric switch associated with the binding of the first protein unit to the origin-of-assembly domain of the viral RNA. The subsequent addition and conformational switching of coat proteins to the growing capsid we believe is catalyzed by the presence of the helical arrangement of bound proteins to the RNA. The model explains why the formation of complete viruses is favored over incomplete ones, even though the process is quasi-one-dimensional in character. We numerically solve the relevant kinetic equations and show that time evolution is different for the assembly and disassembly of the virus, the former exhibiting a time lag even if all forward rate constants are equal. We find the late-stage assembly kinetics in the presence of excess protein to be governed by a single-exponential relaxation, which agrees with available experimental data on TMV reconstruction.     

The antigenicity of tobacco mosaic virus

The antigenic properties of the tobacco mosaic virus (TMV) have been studied extensively for more than 50 years. Distinct antigenic determinants called neotopes and cryptotopes have been identified at the surface of intact virions and dissociated coat protein subunits, respectively, indicating that the quaternary structure of the virus influences the antigenic properties. A correlation has been found to exist between the location of seven to ten residue-long continuous epitopes in the TMV coat protein and the degree of segmental mobility along the polypeptide chain. Immunoelectron microscopy, using antibodies specific for the bottom surface of the protein subunit, showed that these antibodies reacted with both ends of the stacked-disk aggregates of viral protein. This finding indicates that the stacked disks are bipolar and cannot be converted directly into helical viral rods as has been previously assumed. TMV epitopes have been mapped at the surface of coat protein subunits using biosensor technology. The ability of certain monoclonal antibodies to block the cotranslational disassembly of virions during the infection process was found to be linked to the precise location of their complementary epitopes and not to their binding affinity. Such blocking antibodies, which act by sterically preventing the interaction between virions and ribosomes may, when expressed in plants, be useful for controlling virus infection.     

Identification of a peptide of the membrane protein of tobacco mosaic virus, cross-linked with intraviral RNA under the effect of UV-radiation

As reported previously, UV-irradiation induces crosslinking between tobacco mosaic virus (TMV) coat protein molecules and intraviral RNA nucleotides. We have irradiated [3H]-uridine labeled TMV and isolated TMV coat protein subunits with the attached nucleotide label. These TMV protein subunits were hydrolyzed with trypsin. The tryptic peptides were separated by high-performance liquid chromatography and [3H]-labeled peptides were identified. The UV-irradiation of TMV was found to result in crosslinking to intraviral RNA of the T8 tryptic peptide (residues 93-112) of TMV coat protein.     

The coat protein of potato virus X is a strain-specific elicitor of Rx1-mediated virus resistance in potato

The Rx1 gene in potato confers extreme resistance to potato virus X (PVX). To investigate the mechanism and elicitation of Rx resistance, protoplasts of potato cv. Cara (Rx1 genotype) and Maris Bard (rx1 genotype) were inoculated with PVX and tobacco mosaic virus (TMV). At 24 h post-inoculation in Maris Bard protoplasts there was at least 100-fold more PVX RNA than in protoplasts of Cara. TMV RNA accumulated to the same level in both types of protoplast. However, when the TMV was inoculated together with PVX the accumulation of TMV RNA was suppressed in the Cara (Rx1 genotype) protoplasts to the same extent as PVX. The Rx1 resistance also suppressed accumulation of a recombinant TMV in which the coat protein gene was replaced with the coat protein gene of PVX. It is therefore concluded that Rx1-mediated resistance is elicited by the PVX coat protein, independently of any other proteins encoded by PVX. The domain of the coat protein with elicitor activity was localized by deletion and mutation analysis to the structural core of a non-virion form of the coat protein.     

Tobacco mosaic virus and the study of early events in virus infections

In order to establish infections, viruses must be delivered to the cells of potential hosts and must then engage in activities that enable their genomes to be expressed and replicated. With most viruses, the events that precede the onset of production of progeny virus particles are referred to as the early events and, in the case of positive-strand RNA viruses, they include the initial interaction with and entry of host cells and the release (uncoating) of the genome from the virus particles. Though the early events remain one of the more poorly understood areas of plant virology, the virus with which most of the relevant research has been performed is tobacco mosaic virus (TMV). In spite of this effort, there remains much uncertainty about the form or constituent of the virus that actually enters the initially invaded cell in a plant and about the mechanism(s) that trigger the subsequent uncoating (virion disassembly) reactions. A variety of approaches have been used in attempts to determine the fate of TMV particles that are involved in the establishment of an infection and these are briefly described in this review. In some recent work, it has been proposed that the uncoating process involves the bidirectional release of coat protein subunits from the viral RNA and that these activities may be mediated by cotranslational and coreplicational disassembly mechanisms.     

A mechanism of macroscopic (amorphous) aggregation of the tobacco mosaic virus coat protein

To gain more insight into the mechanisms of heating-induced irreversible macroscopic aggregation of the tobacco mosaic virus (TMV) coat protein (CP), the effects of pH and ionic strength on this process were studied using turbidimetry, CD spectroscopy, and fluorescence spectroscopy. At 42 degrees C, the TMV CP passed very rapidly (in less than 15s) into a slightly unfolded conformation, presumably because heating disordered a segment of the subunit where the so-called hydrophobic girdle of the molecule resides. We suppose that the amino acid residues of this girdle are responsible for the aberrant hydrophobic interactions between subunits that initiate macroscopic protein aggregation. Its rate increased by several thousands of times as the phosphate buffer molarity was varied from 20 to 70 mM, suggesting that neutralization of strong repulsive electrostatic interactions of TMV CP molecules at high ionic strengths is a prerequisite for amorphous aggregation of this protein.     

Optical rotatory dispersion by 5 viruses of the tobacco mosaic virus group and their components

Optical rotatory dispersion (ORD) spectra in 250 to 350 nm region were measured for preparations of five TMV-like viruses (TMV vulgare, HR and U2 strains of TMV dolihosenation mosaic virus and cucumber virus 4) and also for RNA and protein preparations of these viruses. The data obtained testify against the possibility that the double peak with maxima at 286 and 293 nm observed in ORD of all the five viruses is due to interaction of tryptophan residues in virus coat protein with the RNA of the virul particle. The spectra of intravirus RNA of the five viruses, calculated as the difference between ORD of the intact virus and of its coat protein, were found to differ significantly from each other and from ORD of free RNA. ORD spectra of hybrid viruses, reconstituted from RNA of one virus and coat protein of another, proved to be identical to the ORD of the virus, whose protein was used in reconstitution. We suppose that the difference in ORD of the intravirus RNA of the five viruses reflect differences of RNA-protein interactions in them.     

Swelling and Softening of the Cowpea Chlorotic Mottle Virus in Response to pH Shifts

Cowpea chlorotic mottle virus (CCMV) forms highly elastic icosahedral protein capsids that undergo a characteristic swelling transition when the pH is raised from 5 to 7. Here, we performed nano-indentation experiments using an atomic force microscope to track capsid swelling and measure the shells’ Young’s modulus at the same time. When we chelated Ca²⁺ ions and raised the pH, we observed a gradual swelling of the RNA-filled capsids accompanied by a softening of the shell. Control experiments with empty wild-type virus and a salt-stable mutant revealed that the softening was not strictly coupled to the swelling of the protein shells. Our data suggest that a pH increase and Ca²⁺ chelation lead primarily to a loosening of contacts within the protein shell, resulting in a softening of the capsid. This appears to render the shell metastable and make swelling possible when repulsive forces among the capsid proteins become large enough, which is known to be followed by capsid disassembly at even higher pH. Thus, softening and swelling are likely to play a role during inoculation.     

TIMELESS‐TIPIN and UBXN‐3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans

The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin‐RING ubiquitin ligase CUL‐2^LRR‐1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC‐48 “unfoldase”. Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome‐associated TIMELESS‐TIPIN complex is required for CUL‐2^LRR‐1 recruitment and efficient CMG helicase ubiquitylation. Aided by TIMELESS‐TIPIN, CUL‐2^LRR‐1 directs a suite of ubiquitylation enzymes to ubiquitylate the MCM‐7 subunit of CMG. Subsequently, the UBXN‐3 adaptor protein directly stimulates the disassembly of ubiquitylated CMG by CDC‐48_UFD‐1_NPL‐4. We show that UBXN‐3 is important in vivo for replisome disassembly in the absence of TIMELESS‐TIPIN. Correspondingly, co‐depletion of UBXN‐3 and TIMELESS causes profound synthetic lethality. Since the human orthologue of UBXN‐3, FAF1, is a candidate tumour suppressor, these findings suggest that manipulation of CMG disassembly might be applicable to future strategies for treating human cancer.     

An extended secondary structure model for the TMV assembly origin, and its correlation with protection studies and an assembly defective mutant

Recognition of the unique internal assembly origin on tobacco mosaic virus (TMV) RNA by the disk aggregate of the viral coat protein probably involves an extended region of the RNA (larger than that coated by a single disk) folded into a specific conformation. A secondary structure model is proposed for the RNA preferentially coated by limiting amounts of coat protein disks on the basis of partial nuclease digestion data. Part of this sequence can form three symmetrically spaced hairpins with marginally stable base paired sequences at the tips of the stems. The pattern of progressive protection of the RNA from nuclease attack during assembly suggests that these three hairpins are successively coated by the first three disks to add. The spacing of these hairpins is identical to that of three hairpins in the pseudo assembly origin (part of the coat protein gene homologous to the assembly origin). In Ni 2519, a TMV mutant whose assembly is defective at high temperature because it can no longer discriminate between the true and pseudo assembly origins, a point mutation has occurred near the tip of the third metastably base paired stem of the true assembly origin which would disrupt its structure and alter one copy of a repeated heptanucleotide. This suggests an important role for the ordered and cooperative recognition of successive loops in determining the specificity of assembly.     

Difference in the mechanism of self-assembly in vitro in tobacco mosaic virus and potato virus X

The effects of 254 nm UV-irradiation of tobacco mosaic virus (TMV) and potato virus X (PVX) RNA preparations on the RNA ability to self-assembly in vitro with the viral coat proteins were studied. It was found that while TMV RNA ability to assemble with the homologous protein is rapidly inactivated by the UV-irradiation, PVX RNA ability to be encapsidated by the PVX coat protein is quite resistant to the irradiation. More than that, the irradiation of TMV RNA with the dose strongly inhibiting its assembly with the homologous protein, did not result in any significant inhibition of this RNA ability to be coated with the PVX protein. The results testify to the profound differences in the mechanisms of RNA-protein interactions in the processes of self-assembly in vitro of tobamoviruses and potexviruses.     

Location of the cistron of the tobacco mosaic virus coat protein.

Treatment of tobacco mosaic virus (TMV) RNA with T1 RNase under mild conditions cuts the RNA molecule into a large number of fragments, only a few of which may be specifically recognized by disks of TMV protein. It has been shown elsewhere that these specifically recognized RNA fragments are a part of the coat protein cistron, the portion coding for amino acids 95 to 129 of the coat protein. It is reported that different size classes of partially uncoated virus particles were prepared by limited reconstitution between TMV RNA and protein or by partial stripping of intact virus with DMSO. Both procedures produce nucleoprotein rods in which the 5'-terminal portion of the RNA is encapsidated and the 3'-terminal region is free. The free and the encapsidated portions of the RNA were each tested for the ability to give rise to the aforesaid specifically recognized fragments of the coat protein cistron upon partial T1 RNase digestion. It was found that only the 3'-terminal third of the virus particle need to be uncoated in order to expose the portion of the RNA molecule from which these fragments are derived. We conclude, therefore, that the coat protein cistron is situated upon the 3'-terminal third of the RNA chain, i.e. within 2000 nucleotides of the 3'-end.     

Response of Membrane-bound Mg-activated ATPase of Tobacco Leaves to Tobacco Mosaic Virus

Infectious material was formed at an early stage, and migrated into the mesophyll from the epidermis of tobacco leaves (Nicotiana tabacum cv. Samsun NN) during the period of 1 to 3 hours after inoculation with tobacco mosaic virus (TMV). The activity of membrane-bound Mg²⁺-activated ATPase from the mesophyll was stimulated two to four times within 30 minutes after inoculation with 1.0 microgram per milliliter of TMV. Maximum TMV stimulation of membrane-bound Mg²⁺-activated ATPase activity in epidermis and mesophyll was observed at 0.5 and 3.0 hours after inoculation, respectively. This stimulation was also observed with ultraviolet irradiated TMV (only RNA was destroyed), whereas, the stimulation was not observed with heat-irradiated TMV (both coat and RNA were destroyed). Stimulation equal to that of TMV was observed by inoculation with cucumber green mottle mosaic virus and to a lesser extent with cucumber mosaic virus.

These results illustrate that the stimulus resulting from inoculation with TMV transfers to underlying cells faster than the migration of TMV particles. This stimulus might be closely correlated to the structure of virus, but not to the infectivity of virus.

     

Structural repeats and evolution of tobacco mosaic virus coat protein and RNA

The three-dimensional structure of the tobacco mosaic virus (TMV) coat protein disk suggests a possible pathway for the early evolution of the virus self-assembly mechanism.The coat protein contains a 2-fold repeated structural pattern in the folding of both its four alpha helices (A,B,C,D), which run alternately forward and back along the radius of the disk, and the four-stranded antiparallel pleated sheet which links these helices to the hydrophobic girdle at the outer rim of the disk. Helices A and B can be approximately superposed on C and D by a screw rotation about a molecular pseudo-dyad axis which lies nearly parallel to the plane of the protein disk. This operation relates 29 pairs of α-carbon positions with a root-mean-square deviation of 1.77 Å. A second pseudo-dyad in the pleated-sheet region relates 14 more atom pairs with a deviation of 2.32 Å and forms a distorted continuation of the relationship between the helices. The helix dyad also relates repeated pairs of functionally important amino acids which take part in intersubunit contacts.We have analysed these structural repeats and tested their significance by comparing them with repeats in other “helix quartet” proteins, cytochrome b⁵ and the hemerythrins, as well as with an irregular helix cluster in thermolysin. TMV is noticeably more repetitive than the others, including hemerythrin which is thought to have evolved by gene duplication.We propose that the primitive TMV coat protein was a dimeric structure of two smaller units paired about a 2-fold axis. Each unit was a pair of helices, linked at the inner radius of the virus rod by a short bend, where the RNA binding site formed, and connected at the outer radius by two short strands of beta sheet. A tandem gene duplication joined the two units and formed the present helix quartet. The flexible loop which now runs into the centre of the virus and connects helix C to helix D developed later. The assembly origin RNA may have evolved from part of the coat protein RNA which codes for this loop.     

A Messenger RNA for Tobacco Mosaic Virus Coat Protein in Infected Tobacco Mesophyll Protoplasts

The low molecular weight tobacco mosaic virus (TMV)-specific RNA component (LMC) was demonstrated in tobacco mesophyll protoplasts by polyacrylamide gel electrophoresis of ¹⁴C-uridine-labelled RNA from infected protoplasts. Free and membrane-bound polysomes were isolated from infected protoplasts, and RNA extracted from them was analyzed. TMV-specific RNA species including full-length viral RNA, its replicative intermediate, and LMC were found in both free and membrane-bound polysomes, but were present in free polysomes in much larger amounts. In particular, as much as 37 % of total LMC in protoplasts was found in free polysomes. Fractionation of polysomes by sedimentation in sucrose gradients showed that LMC is associated with small-sized polysomes (mono- to tetrasomes). Polysomes of this size class produced viral coat protein in a cell-free protein synthesizing system from rabbit reticulocytes. On the other hand, full-length TMV-RNA was associated predominantly with larger polysomes which produced in the cell-free system TMV-specific high molecular weight polypeptides but no coat protein. These results indicated that LMC, a subgenomic RNA of TMV, in fact functions in vivo as messenger RNA for viral coat protein, as has been postulated on the basis of in vitro studies.     

The interaction between tylophorine B and TMV RNA

Tylophorine B exhibits 60% inhibition against tobacco mosaic virus (TMV) at a concentration of 1.0 x 10(-6) g/ml. In our study, high affinity for TMV RNA and assembly origin of TMV RNA (oriRNA) was revealed, accompanied by the conformational change of RNA. Considering that TMV assembly begins with the specific recognition by the coat protein aggregate of oriRNA, and that tylophorine B has favorable interaction with oriRNA, we speculate that tylophorine B likely exerts its virus inhibition by binding to oriRNA and interfering with virus assembly initiation. This work may shed light on the possible molecular inhibition mechanism against TMV by tylophorine B, and provide clues in rational design of sequence-specific RNA binding antivirus drugs.     

The isolation of tobacco mosaic virus RNA fragments containing the origin for viral assembly

Upon mixing purified TMV RNA with limited amounts of viral coat protein in the form of the disk aggregate, a unique region of the whole RNA becomes protected from nuclease digestion. The protected RNA consists of fragments up to 500 nucleotides long in varying yields, which are found in nucleoprotein particles having a protein-nucleic acid ratio similar to the mature virus. The protected RNA, when reextracted, is able to rebind to coat protein disks rapidly, quantitatively and with high affinity, becoming once more RNAase-resistant in the process. Small aggregates of TMV protein (A protein) are inactive in formation of the nuclease-resistant complexes. On the basis of this evidence, we identify the isolated RNA fragments as portions of TMV RNA containing the origin or initiation site for in vitro reassembly, which have been protected from digestion by incorporation into assembly nucleation complexes.The yield, but not the length distribution, of the protected RNA pieces is found to double upon increasing the protein added from 1–2 disk-equivalents of protein per RNA molecule. This implies that the formation of the nucleation complexes may involve a highly cooperative initial addition of protein.