Studies of virus structure by laser-Raman spectroscopy. II. MS2 phage, MS2 capsids and MS2 RNA in aqueous solutions.

Laser-Raman spectra of the bacteriophage MS2, and of its isolated coat-protein and RNA components, have been obtained as a function of temperature in both H₂O and D₂O (deuterium oxide) solutions. The prominent Raman lines in the spectra are assigned to the amino acid residues and polypeptide backbone of the viral coat protein and to the nucleotide residues and ribosyl-phosphate backbone of the viral RNA. The Raman frequencies and intensities, and their temperature dependence, indicate the following features of MS2 structure and stability. Coat-protein molecules in the native phage maintain a conformation determined largely by regions of β-sheet (~60%) and random-chain (~40%) structures. There are no disulfide bridges in the virion and all sulfhydryl groups are accessible to solvent molecules. Protein-protein interactions in the virion are stable up to 50 °C. Release of viral RNA from the virion does not affect either the conformation of the coat-protein molecules or the thermal stability of the capsid. MS2 RNA within the virion contains a highly ordered secondary structure in which most (~85%) of the bases are either paired or stacked or both paired and stacked and in which the RNA backbone assumes a geometry of the A-type. When RNA is partially or fully released from the virion its overall secondary structure at 32 °C is unchanged. However, the exposed RNA is more susceptible to changes in secondary structure promoted by increasing the temperature. Thus the viral capsid exerts a significant stabilizing effect on the secondary structure of MS2 RNA. This stabilization is ionic-strength dependent, being more pronounced in solutions containing high concentrations of KCl. Raman intensity profiles as a function of temperature reveal that disordering of the MS2 RNA backbone and rupture of hydrogen-bonding between complementary bases are gradual processes, the major portions of which occur above 40 °C. However, the unstacking of purine and pyrimidine bases is a more co-operative phenomenon occurring almost exclusively above 55 °C.     

Turnip yellow mosaic virus as a chemoaddressable bionanoparticle

Viruses and virus-like particles (VLPs) have been demonstrated to be robust scaffolds for the construction of nanomaterials. In order to develop new nanoprobes for time-resolved fluoroimmuno assays as well as to investigate the two-dimensional self-assembly of viruses and VLPs, the icosahedral turnip yellow mosaic virus (TYMV) was investigated as a potential building block in our study. TYMV is an icosahedral plant virus with an average diameter of 28 nm that can be isolated inexpensively in gram quantities from turnips or Chinese cabbage. There are 180 coat protein subunits per TYMV capsid. The conventional N-hydroxysuccinimide-mediated amidation reaction was employed for the chemical modification of the viral capsid. Tryptic digestion with sequential MALDI-TOF MS analysis identified that the amino groups of K32 of the flexible N-terminus made the major contribution for the reactivity of TYMV toward N-hydroxysuccinimide ester (NHS) reagents. The reactivity was also monitored with UV-vis absorbance and fluorescence, which revealed that approximately 60 lysines per particle could be addressed. We hypothesized that the flexible A chain contains the reactive lysine because the crystal structure of TYMV has shown that chain A is much more flexible compared to B and C, especially at the N-terminal region where the Lys-32 located. In addition, about 90 to 120 carboxyl groups, located in the most exposed sequence, could be modified with amines catalyzed with 1-(3-dimethylaminopropyl-3-ethylcarbodiimide) hydrochloride (EDC) and sulfo-NHS. TYMV was stable to a wide range of reaction conditions and maintained its integrity after the chemical conjugations. Therefore, it can potentially be employed as a reactive scaffold for the display of a variety of materials for applications in many areas of nanoscience.     

Replication and encapsidation of recombinant Turnip yellow mosaic virus RNA

Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus that infects mainly Cruciferae plants. In this study, the TYMV genome was modified by inserting an extra subgenomic RNA promoter and a multiple cloning site. This modified TYMV was introduced into Nicotiana benthamiana using a Agrobacterium-mediated T-DNA transfer system (agroinfiltration). When a gene encoding beta-glucuronidase or green fluorescent protein was expressed using this modified TYMV as a vector, replication of the recombinant viruses, especially the virus containing beta-glucuronidase gene, was severely inhibited. The suppression of replication was reduced by co-expression of viral silencing suppressor genes, such as tombusviral p19, closteroviral p21 or potyviral HC-Pro. As expected, two subgenomic RNAs were produced from the recombinant TYMV, where the larger one contained the foreign gene. An RNase protection assay revealed that the recombinant subgenomic RNA was encapsidated as efficiently as the genuine subgenomic RNA.     

Chemical accessibility of tyrosyl and lysyl residues in turnip yellow mosaic virus capsids.

The chemical accessibility of tyrosyl residues in TYMV capsids was studied by spectrophotometric titration and with the nitrating agent tetranitromethane. That of the lysyl residues was probed with trinitrobenzenesulfonate. Attempts to test their accessibility in virions were also made. Since some of these reactions were accompanied by structural changes, degradation of the particles were monitored with ultracentrifugation and light-scattering measurements. Alkaline titration of TYMV capsids induced ionization of two of the three tyrosyl residues per subunit at pH 11.3, but the third tyrosyl ionized with an apparent pK of 12.65, concomitantly with the degradation of the capsids. Reaction with tetranitromethane suggested that one tyrosyl residue per subunit can easily be nitrated and initiates degradation, after which the remaining residues also react. In intact capsids, five out of seven lysyl residues per subunit reacted readily with trinitrobenzenesulfonate. The other two lysyl residues were trinitrophenylated only after degradation of the capsids. On the other hand, all seven lysyl residues per subunit were easily trinitrophenylated in virions, during which reaction the virions disintegrated. The demonstrated chemical inaccessibility of specific numbers of tyrosyl and lysyl residues in TYMV capsids and the observed structural consequences to the capsids when the residues were made to react are consistent with previously published properties of the cysteinyl and tryptophanyl residues. The findings suggest that in the capsid the central region of the TYMV polypeptide chain is buried and might represent a site of contact between neighboring subunits.     

A Turnip yellow mosaic virus infection system in Arabidopsis suspension cell culture

Turnip yellow mosaic virus (TYMV) is a positive-strand RNA virus able to infect Arabidopsis thaliana. To establish a TYMV infection system in Arabidopsis cell culture, TYMV replicons with the capsid protein gene replaced by a reporter gene expressing the Sh ble protein conferring zeocin resistance were used to transfect Arabidopsis cells. Zeocin-resistant Arabidopsis calli were used to generate a suspension cell culture. Detection of viral proteins and RNAs after 18 months in culture demonstrated persistent replication of the replicon. The Arabidopsis cell culture yielded soluble, active replication complexes, providing a useful tool to study host factors involved in TYMV replication.     

Modification of Turnip yellow mosaic virus coat protein and its effect on virion assembly

Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus. We have modified TYMV coat protein (CP) by inserting a c-Myc epitope peptide at the N- or C-terminus of the CP, and have examined its effect on assembly. We introduced the recombinant CP constructs into Nicotiana benthamiana leaves by agroinfiltration. Examination of the leaf extracts by agarose gel electrophoresis and Western blot analysis showed that the CP modified at the N-terminus produced a band co-migrating with wild-type virions. With C-terminal modification, however, the detected bands moved faster than the wild-type virions. To further examine the effect, TYMV constructs producing the modified CPs were prepared. With N-terminal modification, viral RNAs were protected from RNase A. In contrast, the viral RNAs were not protected with C-terminal modification. Overall, the results suggest that virion assembly and RNA packaging occur properly when the N-terminus of CP is modified, but not when the C-terminus is modified. [BMB Reports 2013; 46(10): 495-500]     

Replication and packaging of Turnip yellow mosaic virus RNA containing Flock house virus RNA1 sequence

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. In this study, RNA1 sequence of Flock house virus (FHV) was inserted into the TYMV genome to test whether TYMV can accommodate and express another viral entity. In the resulting construct, designated TY-FHV, the FHV RNA1 sequence was expressed as a TYMV subgenomic RNA. Northern analysis of the Nicotiana benthamiana leaves agroinfiltrated with the TY-FHV showed that both genomic and subgenomic FHV RNAs were abundantly produced. This indicates that the FHV RNA1 sequence was correctly expressed and translated to produce a functional FHV replicase. Although these FHV RNAs were not encapsidated, the FHV RNA having a TYMV CP sequence at the 3’-end was efficiently encapsidated. When an eGFP gene was inserted into the B2 ORF of the FHV sequence, a fusion protein of B2-eGFP was produced as expected. [BMB Reports 2014; 47(6): 330-335]     

Arabidopsis DRB4 protein in antiviral defense against Turnip yellow mosaic virus infection

RNA silencing is an important antiviral mechanism in diverse eukaryotic organisms. In Arabidopsis DICER‐LIKE 4 (DCL4) is the primary antiviral Dicer, required for the production of viral small RNAs from positive‐strand RNA viruses. Here, we showed that DCL4 and its interacting partner dsRNA‐binding protein 4 (DRB4) participate in the antiviral response to Turnip yellow mosaic virus (TYMV), and that both proteins are required for TYMV‐derived small RNA production. In addition, our results indicate that DRB4 has a negative effect on viral coat protein accumulation. Upon infection DRB4 expression was induced and DRB4 protein was recruited from the nucleus to the cytoplasm, where replication and translation of viral RNA occur. DRB4 was associated with viral RNA in vivo and directly interacted in vitro with a TYMV RNA translational enhancer, raising the possibility that DRB4 might repress viral RNA translation. In plants the role of RNA silencing in viral RNA degradation is well established, but its potential function in the regulation of viral protein levels has not yet been explored. We observed that severe infection symptoms are not necessarily correlated with enhanced viral RNA levels, but might be caused by elevated accumulation of viral proteins. Our findings suggest that the control of viral protein as well as RNA levels might be important for mounting an efficient antiviral response.     

Turnip yellow mosaic virus RNA is aminoacylated in vivo in Chinese cabbage leaves

Turnip yellow mosaic virus (TYMV) contains a tRNA-like structure as an integral part of its genome. This structure is located at the extreme 3' end of the viral RNA and is the acceptor of valine after 3'-terminal adenylation. It is known that in vitro (with bacterial, yeast, or plant systems) and in vivo (upon microinjection into Xenopus laevis oocytes) a series of tRNA-specific enzymes can recognize this structure in the viral RNA. We report that TYMV RNA is valylated and consequently adenylated in vivo in its natural host, Chinese cabbage leaves. This suggests that the acylated form of the viral RNA could play an important role in the life-cycle of the virus.     

A circular dichroism study of the Turnip yellow mosaic virus-RNA

We examined the circular dichroism spectra of intact Turnip yellow mosaic virus, freezed/thawed virus, empty capsid particles, and phenol extracted RNA. The circular dichroism signal of the empty capsid was found to contribute for less than 1% to the circular dichroism of the virus. Differences in the circular dichroism spectra indicate that TYMV-RNA exhibits different conformations when it is in situ in the virus, when it has been ejected by freezing/thawing and when it has been phenol extracted. Increase of the ionic strength up to 0.1 M NaCl led to conformational change of the RNA either freezed/thawed ejected or phenol extracted but not in situ in the capsid. Addition of spermidine (3 mM) induced a conformational change only for the phenol extracted RNA. These results are discussed with respect to the origin of the various conformational states of viral RNA.     

芜菁花叶病毒分子生物学研究进展

芜菁花叶病毒(Turnip mosaic virus,TuMV)是马铃薯Y病毒属(Potyvirus)的一个重要成员, 在世界上分布广泛,主要危害十字花科植物。本文对芜菁花叶病毒的分子生物学相关研究,如基因组结构和功能、株系变异与进化、寄主植物抗性以及抗病毒转基因工程等的研究进展作一阐述。     

Turnip yellow mosaic virus RNAs with anticodon loop substitutions that result in decreased valylation fail to replicate efficiently.

Single and multiple nucleotide substitutions have been introduced into the anticodon loop of the tRNA-like structure of turnip yellow mosaic virus (TYMV) genomic RNA. We studied the effects of these mutations on in vitro valylation and on replication in Chinese cabbage protoplasts and plants. Only those mutants capable of efficient and complete valylation showed efficient replication in protoplasts and gave rise to systemic symptoms in whole plants. Mutants that accepted valine inefficiently (in some cases Vmax/Km values were less than 10(-3) relative to wild-type values) replicated to levels 200- to 500-fold below wild-type levels in protoplasts (estimated on the basis of coat protein and genomic RNA levels). These mutants could not support systemic spread in plants. In one plant inoculated with TYMC-A55 RNA, which replicates poorly in protoplasts, systemic symptoms developed after a delay. The reversion in replication was accompanied by improved valine acceptance and the appearance of a U57 second-site mutation. Our results indicate a correlation between valine acceptance activity and viral yield. Possible roles for valylation are discussed, and the results are compared with those of similar studies with brome mosaic virus which suggested that tyrosylation is not crucial for brome mosaic virus replication (T. W. Dreher, A. L. N. Rao, and T. C. Hall, J. Mol. Biol. 206:425-438, 1989).     

The Ubiquitin-Proteasome System Regulates the Accumulation of Turnip yellow mosaic virus RNA-Dependent RNA Polymerase during Viral Infection

Replication of positive-strand RNA viruses, the largest group of plant viruses, is initiated by viral RNA-dependent RNA polymerase (RdRp). Given its essential function in viral replication, understanding the regulation of RdRp is of great importance. Here, we show that Turnip yellow mosaic virus (TYMV) RdRp (termed 66K) is degraded by the proteasome at late time points during viral infection and that the accumulation level of 66K affects viral RNA replication in infected Arabidopsis thaliana cells. We mapped the cis-determinants responsible for 66K degradation within its N-terminal noncatalytic domain, but we conclude that 66K is not a natural N-end rule substrate. Instead, we show that a proposed PEST sequence within 66K functions as a transferable degradation motif. In addition, several Lys residues that constitute target sites for ubiquitylation were mapped; mutation of these Lys residues leads to stabilization of 66K. Altogether, these results demonstrate that TYMV RdRp is a target of the ubiquitin-proteasome system in plant cells and support the idea that proteasomal degradation may constitute yet another fundamental level of regulation of viral replication.     

Electron microscopy of double-stranded RNA induced by turnip yellow mosaic virus and tobacco mosaic virus

Summary Double-stranded RNA isolated by phenol extraction from turnip yellow mosaic virus-infected chinese cabbage leaves and from tobacco mosaic virus-9nfected tobacco leaves was rotary shadowed and examined in the electron microscope. The TYMV and TMV molecules are similar in appearance, having uniform width and a linear configuration similar to that previously described for double-stranded RNA and double-stranded DNA molecules. More than 99.5% of the molecules of each virus fall within the range 0.1 to 2.2 , there being a predominance of smaller molecules in both cases (TYMV mean=0.24 , TMV mean 0.42 ). The mode of the larger molecules of TYMV 1.92 and of TMV 1.8 . These values are close to the expected lengths of whole molecules, calculated from biophysical data. Apparently branched molecules were observed in preparations of both TYMV and TMV double-stranded RNA. It was found, however, that the number of such branches per unit length of RNA decreases with a decrease in density of the RNA in the fields examined.     

Electrostatic properties of cowpea chlorotic mottle virus and cucumber mosaic virus capsids

Electrostatic properties of cowpea chlorotic mottle virus (CCMV) and cucumber mosaic virus (CMV) were investigated using numerical solutions to the Poisson-Boltzmann equation. Experimentally, it has been shown that CCMV particles swell in the absence of divalent cations when the pH is raised from 5 to 7. CMV, although structurally homologous, does not undergo this transition. An analysis of the calculated electrostatic potential confirms that a strong electrostatic repulsion at the calcium-binding sites in the CCMV capsid is most likely the driving force for the capsid swelling process during the release of calcium. The binding interaction between the encapsulated genome material (RNA) inside of the capsid and the inner capsid shell is weakened during the swelling transition. This probably aids in the RNA release process, but it is unlikely that the RNA is released through capsid openings due to unfavorable electrostatic interaction between the RNA and capsid inner shell residues at these openings. Calculations of the calcium binding energies show that Ca(2+) can bind both to the native and swollen forms of the CCMV virion. Favorable binding to the swollen form suggests that Ca(2+) ions can induce the capsid contraction and stabilize the native form.