Structure and Noncanonical Activities of Coat Proteins of Helical Plant Viruses

Transcription

1 ISSN , Biochemistry (Moscow), 2016, Vol. 81, No. 1, pp Pleiades Publishing, Ltd., Original Russian Text V. V. Makarov, N. O. Kalinina, 2016, published in Biokhimiya, 2016, Vol. 81, No. 1, pp REVIEW Structure and Noncanonical Activities of Coat Proteins of Helical Plant Viruses V. V. Makarov and N. O. Kalinina* Lomonosov Moscow State University, Belozersky Institute of Physico-Chemical Biology, Moscow, Russia; fax: +7 (495) ; Received June 30, 2015 Revision received August 17, 2015 Abstract The main function of virus coat protein is formation of the capsid that protects the virus genome against degradation. However, besides the structural function, coat proteins have many additional important activities in the infection cycle of the virus and in the defense response of host plants to viral infection. This review focuses on noncanonical functions of coat proteins of helical RNA-containing plant viruses with positive genome polarity. Analysis of data on the structural organization of coat proteins of helical viruses has demonstrated that the presence of intrinsically disordered regions within the protein structure plays an important role in implementation of nonstructural functions and largely determines the multifunctionality of coat proteins. DOI: /S Key words: helical plant viruses, coat protein, structure, intrinsically disordered regions, noncanonical functions Plant viruses with the helical symmetry capsids are nonenveloped RNA-containing viruses with the virion being an empty tube nm in diameter and nm in length depending on the genomic RNA size [1]. About 500 species of helical phytoviruses are known, and many of them are dangerous pathogens of important crops [2, 3]. Thus, potyviruses potato virus Y and plum poxvirus annually cause economic damage to harvests of potato and stone fruits up to billions of dollars [4]. Studies on pathogenesis of these viruses in plants is of help for developing new approaches for obtaining virusresistant agricultural plants, which is especially important for crop in developing countries [5]. Helical viruses are used recently in biotechnology as a base for creating vectors to express epitopes of various human and animal Abbreviations: BSMV, barley stripe mosaic virus; CP, coat or capsid protein; HR, hypersensitive response; MP, movement protein; MT, microtubules; PD, plasmodesmata; PR-genes, pathogenesis-related genes; PVA, potato virus A; PVX, potato virus X; RNP complex, ribonucleoprotein complex; RTM resistance, resistance associated with restricting long-distance movement of tobacco etch virus; SL1, stem-loop 1; TGB, triple gene block; TGB1, protein encoded by the first gene of TGB; TMV, tobacco mosaic virus; ToMV, tomato mosaic virus; 5 UTR, 5 -untranslated region; VRC, virus replicative complex. * To whom correspondence should be addressed. pathogens in order to prepare vaccines or for effectively producing in plants heterologous proteins for different purposes [6]. Helical viruses are also used as promising platforms for creating various nanomaterials for modern medicine, microelectronics, and other fields of human activities [7-11]. Helical plant viruses are small RNA-containing viruses with genomes carrying a limited number of genes (from three to twelve in members of different taxonomic groups). Despite the limited coding capacity of the genome, these viruses can realize all functions necessary for overcoming defense mechanisms of the host plant and development of productive infection. The limited number of virus proteins suggests their multifunctionality, and this phenomenon has been under active study during recent decades. The RNA genome of all helical plant viruses encodes a special structural protein the capsid protein or coat protein (CP), which forms virions and is necessary for protection of the viral RNA against degradation. The majority of virions of plant helical viruses, unlike viruses with an icosahedral virion, consist of one type of protein capsomers (CP subunits) that encapsidate the singlestranded RNA molecule into a multimeric complex with helical symmetry [1]. Sometimes, e.g. in the case of tobraviruses, hordeiviruses, and pomoviruses, the viral genome is segmented and consists of several (two or 1

2 2 MAKAROV, KALININA three) RNA molecules. In such cases, several virus particles are formed according to the number of genomic RNAs, which as a rule have different length. All plant viruses with helical symmetry can be subdivided by virion morphology into rod-shaped and flexible filamentous ones [12]. The rod-shaped viruses include the Tobamovirus, Tobravirus, Furovirus, Pomovirus, and Hordeivirus genera, whereas members of the Alphaflexiviridae, Potyviridae, and Closteroviridae families are typical representatives of flexible filamentous viruses. In total, the rod-shaped viruses belong to eight genera, whilst flexible helical viruses belong to about 20 genera [2, 3]. Subdivision of helical viruses into rod-shaped and flexible ones is based first of all on differences in their CPs structure. Comparison of the amino acid sequences of helical plant viruses CPs also allows to divide them into two groups corresponding to viruses with rod-shaped or flexible virions [13]. The CP structure of the rod-shaped viruses is based on a specific protein folding a small barrel of four α-helices with up-down topology [14, 15], whereas capsomers of flexible filamentous viruses seem to consist of seven short α-helices with complicated topology [16]. At the same time, amino acid sequences of helical viruses CPs can be quite different even within the same group. The intrinsically disordered regions within the N- and C-terminal sequences and in the loop sequences between the α-helices are the most variable. The presence in a protein of elongated disordered structural elements usually indicates multifunctionality of the protein. In fact, CPs of helical plant viruses are responsible for many various functions in addition to formation of virions. They are involved in regulation of replication, transcription, and translation of viral RNA, the transport of the virus within a plant, and in determination of the host plant range and of infectivity, pathogenicity, and expression of symptoms. Coat proteins also participate in the transmission of a virus from plant to plant by natural vectors and in formation of both the antiviral response and viral counter defense [17]. In this review, we analyze the available data on the structural organization of CPs of helical viruses and their noncanonical (nonstructural) functions in order to assess the role of the secondary/tertiary structure elements and of internally disordered regions within CP important for these functions. STRUCTURAL ORGANIZATION OF COAT PROTEINS OF PLANT HELICAL VIRUSES Based on the similarity of amino acid sequences, all CPs of helical viruses can be subdivided into two large groups: one group including CPs of envelopes with rigid rod-shaped virions, and the other group including CPs of flexible filamentous viruses. The coat proteins of rodshaped viruses (tobamo-, tobra-, pomo-, hordei-, and furoviruses) are significantly more conservative within their group than the CPs of flexible viruses [13]. Based on the similarity of their sequences, flexible helical plant viruses can be subdivided into two subgroups, one including potex-, carla-, and allexiviruses, and the other subgroup including potyviruses [13]. Notwithstanding the absence of pronounced homology, CPs of both filamentous and rod-shaped viruses seem to have once had a mutual ancestor, but now their structural organization is significantly different [13]. Nevertheless, the CPs still retain some structural features, such as formation of salt bridges through conservative residues of charged amino acids or conservative hydrophobic amino acids, which form the stable nucleus of the protein molecule (Fig. 1). The retention of these common structural features in groups separated long ago during evolution seems to indicate the importance of these structural elements allowing CPs to perform their major function to form the virion and protect the viral RNA against degradation. For many years, tobacco mosaic virus (TMV) was one of the best-studied phytopathogens [18]. Discovered more than 100 years ago, TMV has long been the only helical plant virus with resolved structure of the capsid (Fig. 2a) [19]. X-Ray crystallographic analysis of 20S discs consisting of the TMV coat protein and parallel studies on TMV virions using X-ray diffraction on the threads revealed the TMV structure with resolution of 4 Å [20, 21]. Further progress in structural studies was achieved nearly three decades later, when higher resolutions were obtained using the same approach of X-ray diffraction on threads (2.9 Å) and using cryoelectron microscopy (4.6 Å) [22, 23]. However, even such resolution was insufficient for interpretation of some nuances in the structure and self-assembly mechanism of TMV. Subunits of the TMV CP consist of one structural domain containing four α-helices packed as a so-called fourhelix bundle [20-22]. Two of the main α-helices are located radially in the plane perpendicular to the virion axis and are called left radial helix ( a.a.) and right radial helix (73-86 a.a.), whereas the two other main helices are located at an angle to this plane and are called left-slewed helix (19-33 a.a.) and right-slewed helix (37-52 a.a.). Moreover, TMV CP also contains two small α-helices located at the border of the major domain and a short β-sheet consisting of several residues. The N- and C-terminal regions of CP are exposed on the outer surface of the TMV capsid. The inner surface of the TMV virions with tightly packed neighboring subunits has a very solid structure fixed by a series of hydrogen bonds and is stabilized by β-turn in the amino acid chain [24]. Structural elements responsible for binding with RNA are also characterized for the TMV CP. This process occurs with involvement of residues Arg41, Arg90, Arg92, and Asp116. Electrostatic interactions between negatively charged groups are also important for intersubunit interactions. Carboxyl groups of Glu50 and Asp77 of the

3 TMV ORSV CGMMV TRV PEBV BSMV CWMV TMV ORSV CGMMV TRV PEBV BSMV CWMV TMV ORSV CGMMV TRV PEBV BSMV CWMV FEATURES OF COAT PROTEINS OF HELICAL PHYTOVIRUSES 3 LS RS RS RR LR TMV ORSV CGMMV TRV PEBV BSMV CWMV Fig. 1. Multiple alignment of amino acid sequences of coat proteins of plant helical viruses. The sequences were aligned using the BLASTP algorithm. The figure presents amino acid sequences of coat proteins of the following viruses: TMV, tobacco mosaic virus (Tobamovirus genus); ORSV, odontoglossum ringspot virus (Tobamovirus genus); CGMMV, cucumber green mottle mosaic virus (Tobamovirus genus); TRV, tobacco rattle virus (Tobravirus genus); PEBV, pea early browning virus (Tobravirus genus); BSMV, barley stripe mosaic virus (Hordeivirus genus); CWMV, Chinese wheat mosaic virus (Furovirus genus). Asterisks indicate identical amino acid residues in all sequences, one point indicates homologous substitutions, and two points show semiconservative substitutions of amino acid residues. Frames indicate regions corresponding to conservative core α-helices within molecules of the coat proteins; α-helices are denoted as LR (left radial helix), RR (right radial helix), LS (left-slewed helix), and RS (right-slewed helix). protein neighboring subunits separated in the axial direction are immediately close to each other [25, 26]. It seems also that there are interactions between Glu106 of one subunit and Glu95, Glu97, and Asp109 of the adjacent subunit in the transverse direction on the low radius [21, 26-28]. It is very likely that interactions between Glu106/Glu95/Glu97/Asp109 and between the phosphate backbone of RNA and Asp116 are realized through binding calcium ions [22, 24, 29]. These electrostatic interactions and binding sites play important roles in the assembly and disassembly of virions. High-resolution structures are also available for other tobamoviruses, such as the tobacco mild green mosaic virus, cucumber green mottle mosaic virus, ribgrass mosaic virus, and odontoglossum ringspot virus [29-32]. However, the structures of these viruses are not much different from the TMV structure. Assembly of the TMV virion is specific and is initiated on the origin assembly sequence of the viral RNA, which has the structure of a stem-loop and is located in

4 4 MAKAROV, KALININA a b LS RR RS LR Fig. 2. Spatial structure of coat proteins of rod-shaped and flexible filamentous helical plant viruses: a) tobacco mosaic virus (Tobamovirus genus), α-helices are indicated as LR (left radial helix), RR (right radial helix), LS (left-slewed helix), and RS (right-slewed helix); b) papaya mosaic virus (Potexvirus) genus, α-helices are designated with numbers 1-7. the 3 -terminal region of the RNA [33, 34]. The TMV virion has high stability in the external medium. In the plant cell, the virion is destabilized. This is caused by weaker interactions of the CP with the 5 -terminal leader sequence of RNA because of absence in this region of a guanidine residue in the third position of the triplet responsible for firm interaction with CP molecules in the major part of the virion. Microfluctuations initiate removal of several CP molecules from the 5 -end of the encapsidated RNA. The subsequent disassembly of the virus particle occurs cotranslationally [34-36]. Attempts to resolve structure using cryoelectron microscopy did not reveal in hordeiviruses significant differences from the TMV structure, but a detailed analysis of the barley stripe mosaic virus (BSMV) virions using approaches of structural analysis indicated that the BSMV virions are significantly more labile than the TMV virions and occupy an intermediate position between the flexible and rod-shaped viruses [37]. The situation is much worse with the high-resolution structures of flexible filamentous viruses. Only one structure of the papaya mosaic virus CP obtained using the X- ray crystallography approach and having resolution of 2.7 Å is available (Fig. 2b) [38]. The packing of this virus was shown to differ significantly from that of rod-shaped viruses and to consist of seven α-helices with rather complicated topology. However, it should be noted that this structure was obtained not for CP of the native virus within the virion, but for the recombinant protein lacking six amino acid residues from the N-terminus and crystallized as a dimer [38]. Attempts to determine high-resolution structure of the papaya mosaic virus by cryoelectron microscopy were unsuccessful. Moreover, the structure obtained by X-ray crystallography was in poor correlation with the data of cryoelectron microscopy. The structure of the virion of flexible helical potex- and potyviruses using diffraction on filaments is also lacking high resolution [39, 40]. Another model of the tertiary structure of CP of flexible helical plant viruses was created on the base of data obtained by tritium bombardment of potato virus X (PVX) virions and studies on physicochemical features of the CP within the virus particle and in a free state [41-43]. The PVX CP was supposed to have complicated topology of two domains, one consisting of four α-helices and the other including of three α-helices and four β-sheets. Similar topology was also supposed for the CP of the potyvirus potato virus A (PVA) [43, 44]. However, subsequent studies revealed that PVA CP is organized otherwise and seems to be significantly disordered [45, 46].

5 FEATURES OF COAT PROTEINS OF HELICAL PHYTOVIRUSES 5 INTRINSICAL DISORDER IN STRUCTURE OF COAT PROTEINS OF HELICAL PLANT VIRUSES Another important specific feature of CPs of plant helical viruses that is common for both rod-shaped and flexible viruses is their intrinsically disordered regions polypeptide regions unable to produce in aqueous solution a unique spatial structure characteristic for globular proteins but capable of performing their inherent functions [47-50]. This feature of proteins or of their individual regions is a natural feature of a specific polypeptide chain determined by its amino acid sequence. The intrinsically disordered regions in the native structure of a protein can be manifested by the impossibility of obtaining protein crystals, the absence of pronounced structure of circular dichroism spectra in the near UV region, large hydrodynamic size, abnormal electrophoretic mobility, and impossibility of determining by X-ray crystallography of atomic coordinates of some amino acids of the polypeptide chain [47]. Disordered proteins can be widely represented in eukaryotic cells because unfolded proteins, due to their multifunctionality, allow the eukaryotic cell to be satisfied by the same number of proteins as the prokaryotic cell [51]. In fact, some recent works have shown that only disordered proteins can perform various functions associated with regulation and control of biological processes due to their unique ability to interact with a great number of different partners [52]. Just this specific feature seems to be extremely important also in the case of CPs of plant helical viruses because it determines their multifunctionality. Many current works characterize CPs of plant helical viruses as proteins with a significant fraction of intrinsically disordered regions [37, 42, 45, 46]. Indirect methods of structural analysis, such as circular dichroism, tryptophan fluorescence, Raman spectroscopy, differential scanning calorimetry, etc. have shown that CPs of filamentous plant viruses, such as potex- and potyviruses, have a significant fraction of intrinsically disordered regions that result in a significantly greater lability of their virions than thought earlier [42, 45, 46]. To study stability of the tertiary structure of CPs of plant helical viruses within the virion, differential scanning calorimetry was used to show the temperature dependence of changes in thermal capacity of the protein molecule. Melting curves of various helical plant viruses were analyzed, and the tobamovirus TMV was found to have the most stable virions, whereas the hordeivirus BSMV virions had lability comparable to that of virions of the potexviruses (PVX), and the thermal stability of the potyvirus PVA virions was the lowest (Fig. 3) [46, 53]. These results correlate with data of both bioinformatic analysis and of structural analysis of CPs in a free state. According to these data, the content of intrinsically disordered regions in virion CP increases in the following series: tobamoviruses < hordeiviruses < potexviruses < potyviruses. These data on the CP structure are consistent С р ех, kcal mol deg PVA BSMV PVX TMV Temperature, C Fig. 3. Melting curves of virions of plant helical viruses of different taxonomic groups determined using differential scanning calorimetry: tobacco mosaic virus (TMV, Tobamovirus genus), barley stripe mosaic virus (BSMV, Hordeivirus genus), potato virus X (PVX, Potexvirus genus), potato virus A (PVA, Potyvirus genus) [45, 53]. The curves show the dependence of heat capacity of the protein on temperature changes and characterize the thermal stability of tertiary structure of the virus coat protein within the virion. with data on their functions, because most frequently just the N- and C-terminal regions are responsible for noncanonical functions of plant helical virus CPs other than the canonical structural function of virion formation and interaction with nucleic acid. FUNCTIONS OF COAT PROTEINS DURING TRANSPORT OF FLEXIBLE FILAMENTOUS VIRUSES IN PLANTS An important stage in the development of viral infection is the spread of the viral genome from the initially infected plant cell into uninfected cells through the plasmodesmata (PD), cytoplasmic channels of the cell walls connecting neighboring cells of the mesophyll and epidermis (cell-to-cell or short-distance movement) and then through the plant vascular system phloem (phloem or long-distance movement) [54-57]. The longdistance transport encompasses the movement of a transport form of viral genome through a system of specialized cells into the phloem sieve elements, transfer with the flow of assimilates, and exit from sieve elements backwards through the same cell system into mesophyll and epidermal cells of uninfected leaves [56]. It is known that for the majority of plant viruses, CP is necessary for both cell-to-cell and long-distance movement due primarily to the fact that virions or virus-like particles are the transport forms of these viruses. Members of the potex- and potyvirus genera with flexible filamentous virions are typical examples of such

6 6 MAKAROV, KALININA viruses. The interaction of the CP with viral RNA is mainly determined by its ability to produce a competent transport form of the viral genome (natural virion or virus-like particle). According to data obtained in vitro, the formation of virus-like particle is a result of a specific interaction of the potexvirus PVX CP with elements of the secondary structure of PVX RNA denoted as a stemloop 1 (SL1) in the 5 -untranslated region (5 UTR) [58]. Functional analysis of 5 UTR of the genomic PVX RNA revealed its important role in replication, encapsidation, translation, and cell-to-cell movement of the virus [59]. The ability of cymbidium mosaic potexvirus to systemically infect various plants was shown to be mainly due to efficiency of the interaction of the CP with the viral RNA [60]. Two virus isolates, M1 and M2, could infect their natural host Phalaenopsis orchids but only the M1 isolate could systemically infect Nicotiana benthamiana plants, whereas isolate M2 movement was restricted to the initially infected cells. The CPs of M1 and M2 are different only by four amino acid residues, and only two substitutions, Gly82Ala and Leu89Pro in the CP of the M2 isolate, are necessary and apparently sufficient for systemic infection of N. benthamiana. The M1 isolate CP binds RNA in vitro with higher affinity than the M2 CP, and the two substitutions increase the efficiency of viral RNA binding by the mutant M2 CP [60]. Additionally it was found that requirements to other proteins of the viral transport system became more or less restricted depending on efficiency of the interaction between the CP and the viral RNA. Nevertheless, it was shown long ago that there are mutations that do not influence the CP encapsidation ability but inhibit viral transport, both cell-to-cell and long-distance. Thus, a mutant CP of white clover mosaic virus lacking five C-terminal amino acid residues was able to form virions, but it did not support virus cell-to-cell movement [61]. Similar data were obtained for PVX CP shortened from the C-terminus: 18 amino acid residues were important for the viral movement but not for assembly of the viral particle [62]. In addition to CP, the transport of potexviruses is known to require three movement proteins (MPs) that are encoded by a special gene module the triple gene block (TGB) [57]. One of the C-terminus functions was detected in vitro: it was shown that the PVX TGB1 movement protein specifically bound with the 5 -end of the partially or completely encapsidated virion interacting with 10 C-terminal amino acid residues and producing the so-called polar virus particle. This interaction was in vitro accompanied by remodeling and translational activation of the virion or a virus-like particle [63-65]. Moreover, the C-terminal regions of CPs of potexviruses bamboo mosaic virus (BaMV) and foxtail mosaic virus (FoMV) interacted with the helicase domain of the viral replicase [66]. The mutations Ala209Gly and Asn210Ser in BMV CP and the corresponding mutation in Ala230 (conservative in potexviruses) of FoMV CP were accompanied by a decrease in the CP affinity for the helicase domain and inhibited cell-to-cell movement of viruses, but did not influence the interaction of the CPs with RNA and the assembly of virions [66]. Thus, the C- terminal regions of the CPs of potexviruses contain several overlapping functional domains. It is very likely that many functions that are inherent to the C-terminal region of potexviruses CP are still unknown. Thus, under in vitro conditions a free PVX CP and CP in the content of virion displayed ATPase activity, which was abolished by deletion of 18 C-terminal amino acid residues [67]. Interestingly, the transport functions of the C-terminal determinants can be complemented by CPs of other filamentous viruses (poty- and closteroviruses) and by CPs of icosahedral sobemovirus and some nonfunctional mutants of the tobamovirus TMV MP [62, 68]. However, these proteins are unable to complement the transport function on deletion of the full-size gene encoding the PVX CP [68]. Nevertheless, the transport system of PVX, namely the genes encoding the CP and TGB proteins, can be functionally replaced by the only MP of a tobamoor umbravirus [69, 70]. Probably one of functions performed by the major part of the CP necessary for movement is the formation of a competent transport form (virion or virus-like particle). According to a model based on recent experimental data, replication and cell-to-cell movement of the viral genome of potexviruses are linked and realized in specialized structures at the cell wall PD entrance: viral movement protein TGB1 recruited by proteins TGB2/TGB3 is responsible for the incorporation of PVX CP into the PD channel as a virus particle (transport complex) [71]. The authors suggest that movement proteins of other filamentous viruses (potyviruses and closteroviruses) interacting asymmetrically with CP on the virion 5 -end and forming polar virions [72, 73] can provide a spatially combined process of replication and transport (coreplicational insertion of viral RNA into PD). In addition to the C-terminal region, the N-terminus of potexviruses CP also participates in transport. In addition to the conservative core region, the CPs of potexviruses have a variable N-terminal region of various length [74]. This seems to be an explanation of some contradictions in the data obtained for different representatives of potexviruses. The N-terminal region of the PVX CP (deletion of 30 a.a.) is dispensable for cell-to-cell movement [75, 76], and the PVX virions treated with trypsin and lacking 20 a.a. from the N-terminus or having the N-terminus shortened by a.a. (depending on the strain) as a result of the natural CP proteolysis are infective, although this infectivity is slightly reduced [77]. At the same time, it has been shown that the N-terminal region of the Plantago asiatica mosaic potexvirus (14 a.a. and the Leu3 residue), which is shorter than the N-terminal PVX CP region, is specifically involved in transport [78]. The N-terminal region of potexviruses is supposed

7 FEATURES OF COAT PROTEINS OF HELICAL PHYTOVIRUSES 7 to be subdivided into two functional domains: the extra N-region (Nex) that is not required for cell-to-cell movement and virion assembly but possibly can be responsible for host plant specificity, and the adjacent core N-region (Nc) that is required for cell-to-cell movement but not for assembly of a viral particle [78]. Interesting data were obtained by microscopic studies on the progression of Nicotiana benthamiana infection with PVX mutant variants with deletions in the N-terminal CP region or with chimeric virus particles with inserted in this region peptides selectively affecting cell-to-cell and phloem movement. The authors found that the cell-to-cell movement was maintained by chimeric CPs incapable of virion assembly, whereas long-distance movement could be promoted only by those CP variants that provided the assembly of virions [79]. These results re-raised the question whether the transport form of potexviruses for cell-to-cell movement is a modified polar virion or an RNP complex that includes CP, TGB1 movement protein, virus replicase, and virus RNA. A number of cellular proteins have been found that interact with CPs of potexviruses, but regions within the protein molecules responsible for these interactions have not been mapped for all of them. Experiments in vitro and in vivo showed that PVX CP interacts with the NbPCIP1 protein from N. benthamiana, and studies on overexpression or expression inhibition of this protein-coding gene revealed that NbPCIP1 stimulated replication and systemic transport of the virus [80]. For this interaction, the N-terminal region of PVX CP containing both disordered elements and elements of core α-helices is important [81]. The cellular NbDnaJ protein, a co-chaperone of HSP70, also influenced PVX replication and viral movement interacting with PVX CP and with the structural element SL1 on the 5 UTP of minus-strand PVX RNA (SL1( )RNA), but just the opposite compared with the action of NbPCIP1 protein its overexpression in plants inhibited both processes [82]. The NbDnaJ protein is supposed to be a negative regulator of PVX replication and transport. Moreover, the NbMPB2Cb and NbMBF1 proteins from the Nicotiana benthamiana that bind in vitro to both SL1(+/ )RNA of PVX, and the NbCPIP2a protein interacting only with SL1(+)RNA were revealed. It was shown that the PVX CP could bind also to both SL1(+/ )RNAs [83]. The pepino mosaic potexvirus CP directly interacts both in vitro and in vivo with the Hsc70 protein related to HSP70 from the tomato Solanum lycopersicum [84, 85]. Transmission electron microscopy revealed that the Hsc70 protein is co-located with virions in the phloem of tomato leaves infected with the virus and is purified together with virions from infected plants [84, 85]. It is supposed that the ATPase activity of Hsc70 can be used during virion translocation through PDs. Data obtained on protoplasts also indicate that Hsc70 is necessary for the replication of the viral RNA [85]. It has been shown in vitro that the PVX CP specifically interacts with microtubules (MTs) and causes rapid polymerization of tubulin with generation of aberrant structures. It is supposed that in vivo this process can result in subsequent degradation of these proteins under the influence of cellular proteolytic systems or induce local disorganization of MTs due to inhibition of viral genome transport in the cell [86]. In contrast, the PVX virions induced in vitro polymerization of tubulin with generation of morphologically normal bundles of microtubules, which can serve as in vivo centers of organization and stabilization of MTs by promoting intracellular transport of viral genome [86]. Coat proteins play the major role in cell-to-cell and long-distance movement of potyviruses. Mutants of the tobacco etch virus not competent in virion assembly did not maintain cell-to-cell movement [87]. Apparently, as in the case of potexviruses, potyviruses have as a transport form modified virions, so-called tailed or polar particles [88], or RNP complexes associated with CP. It should be noted that movement of potyviruses involves, in addition to CPs, several virus-specific proteins, whereas the main protein protein interactions are provided by VPg protein covalently bound with the 5 -end of the viral RNA [55, 89]. Previous studies on functions of tobacco etch potyvirus CP revealed that deletions of its N- and C- terminal regions affected the long-distance movement and partially the cell-to-cell movement efficiency, but not the assembly of virions [87, 90]. These data confirmed that the encapsidation of the viral RNA and transport of potyviruses are controlled by different regions/domains of CPs. Similarly, small deletions at the C-terminus of both major and minor closterovirus CPs inhibit virus transport, and this suggests that definite determinants responsible for the viral transport are localized in the C- terminal region of the closterovirus CP [91]. Later, it was shown that the N-terminal CP region of viruses from family Potyviridae was an independent determinant of long-distance (systemic) movement specific for host plant and virus strain. This finding was demonstrated on mutants of plum pox virus (PPV) CP whose variable N- terminal fragment was responsible for the host range in the case of multiple infections of herbaceous and woody hosts [92]. Similar results were obtained in studies on functions of the N-terminal CP region of another potyvirus, the wheat streak mosaic virus (WSMV). Amino acid sequences of CPs of the WSMV Sidney 81 and Type strains were 98.7% homologous; nevertheless, the infection symptoms observed on the maize line SDp2 were different: on infecting SDp2 with the Type strain, there was no systemic infection. Analysis of mutant CPs revealed that five of 11 differing amino acid residues in the N-terminal region (74 a.a.) of the proteins are directly involved in manifestation of the infection symptoms. On the substitutions Ala20Asp/Ser21Pro, Gln30Leu, and Ala50Val/ Gly52Asp, an experimental strain with the N-terminal fragment of the WSMV Type CP re-acquired the ability for long-distance movement. Note that the most pronounced

8 8 MAKAROV, KALININA symptoms of the infection developed with the single substitution Gln30Leu in the CP [93]. It is known that, similar to potexviruses, the N-terminal region of the potyvirus CP is significantly variable in both length and sequence among individual species and even among strains within the same species. Such variability of the N-terminal CP fragment exposed on the virion surface seems to promote virus interaction with specific factors of the host plant involved in the cell-to-cell and long-distance movement. Recently the role of the C-terminal region of WSMV CP in virus movement and host specificity was studied by arbitrarily mutating six of seven aspartic acid (Asp) (negatively charged amino acid) residues in the C-terminal 69 a.a. and examining the ability of these mutants to infect wheat and SDp2 maize. It has been shown that these amino acids are dispensable for virion assembly, but mutation of Asp at positions 289, 290, and 326 debilitated longdistance transport in maize but not in wheat. These amino acids likely facilitate expansion of WSMV host range through host-specific long-distance movement by allowing virus ingress into the vascular system of maize [94]. The resistance of Arabidopsis thaliana plants to potyviruses infection (tobacco etch virus, lettuce mosaic virus, and PPV) associated with RTM genes is especially interesting [95, 96]. Note that RTM resistance does not influence replication and cell-to-cell movement of viruses and does not trigger mechanisms of hypersensitive response or of acquired systemic resistance, but it inhibits the long-distance transport of potyviruses. This form of resistance is provided at least by five genes, which seem to be an atypical class of R genes of resistance [97]. The RTM resistance is supposed to be a novel form of plant defense response against viral infection, which is manifested in phloem-associated tissues. The N-terminal CP region was shown to be a key factor/determinant allowing virus to overcome RTM resistance [97]. However, the amino acid residue or the region responsible for removal of the RTM resistance has still not been accurately located [97]; moreover, neither RTM protein directly interacts with CPs [98]. Perhaps additional cellular or viral proteins mediate this interaction with CPs or virions, and CPs of potyviruses can be considered as avirulent factors. Recognition of this factor is accompanied by an inhibition of long-distance movement because of sequestering virions or virus RNP structures by multimeric complexes formed by RTM proteins in the vascular tissue [98]. Moreover, the RTM complexes may compete with or block the host factors that (i) are necessary for the virus long-distance movement and (ii) interact with the N-terminal CP region. Posttranslational modifications found in the N-terminal region of potyviruses CPs (phosphorylation or glycosylation) and the composition of the amino acid sequence (including the total charge) may play important roles in the supposed interactions of CPs with partners (protein or RNA) and in the virus ability to spread in the host plant [99, 100]. FUNCTIONS OF COAT PROTEINS DURING MOVEMENT OF VIRUSES WITH RIGID ROD-SHAPED VIRIONS IN PLANTS Although movement of tobamoviruses in plants has been studied in some detail, data on the role of CP in cellto-cell movement are insufficient and often contradictory. For a long time it was thought that CP was not required for the cell-to-cell movement of tobamoviruses, and that at this stage the transport form is presented by RNP complex formed by genomic RNA and viral MP [101, 102]. However, detailed analysis of formation of virus replicative complexes (VRC) and studies on locations of viral replicase, MP, and CP in TMV RNA infected wild-type protoplasts and protoplasts from TMV-resistant transgenic plant lines expressing TMV CP or its mutant variants revealed that the wild-type CP increased the size of VRC and MP production. However, the mutant TMV CP with Thr42Trp substitution did not generate functional VRC that restricted the viral RNA replication and decreased the amount of MP [103]. Based on these data, it was supposed that TMV CP could directly or indirectly regulate the formation of viral VRCs, the production of subgenomic RNAs encoding MP, and possibly CP or the translation of these RNAs that, in turn, influenced viral genome transport [103, 104]. It is significant that the mutant CP with the Thr42Trp substitution is unable to encapsidate viral RNA [105], but it generates multiple nonhelical 20S aggregates that seem to negatively influence viral replication and as a result, virus transport [106]. The time of TMV spread from the primarily infected cells into secondary ones (18-20 h after inoculation) and from the secondary cells into tertiary ones (2-4 h) were significantly different, and, therefore, it was supposed that VRC/virus replicase should be involved in the cell-to-cell movement of viral genome [107]. Thus, TMV CP affects positively the VRC assembly, viral replication, and production of MP (and, consequently, formation of transport complexes) and increases cell-to-cell movement, but the mechanism regulating MP production under the influence of CP is not known in detail. The composition and structure of TMV transport form for cell-to-cell movement were studied only in vitro. Up to now, the supposed MP RNA complex of TMV has not been found in vivo. Nevertheless, numerous data suggest that the absence of a gene encoding tobamovirus CP (i) does not inhibit the cell-to-cell movement of a mutant TMV and (ii) is not required for TMV RNA replication, and that in the absence of CP the mutant TMV is able to form VRC-like complexes. Moreover, the TMV MP is able to functionally replace the transport systems of unrelated viruses [69, 101, 102]. Thus, the role of the CP in cell-to-cell movement remains unclear. However, CP is absolutely necessary for the systemic transport because virions are the most probable transport form of tobamoviruses competent for long-distance move-

9 FEATURES OF COAT PROTEINS OF HELICAL PHYTOVIRUSES 9 ment. It has been long known that mutations in the TMV RNA region initiating the assembly of a virus particle inhibit not only the viral RNA encapsidation, but also the systemic transport of TMV [108]. A mutant TMV with abolished expression of the CP-encoding gene could reach only cells of the vascular parenchyma of minor leaf veins and was not detected in the companion cells, which were the last barrier before loading into the phloem sieve elements [109]. These data suggest that TMV CP is required for the passage through the PDs between these cells. CP is thought to be required also for exit from the phloem: in tobacco plants with compromised synthesis of cell wall enzyme, pectin methylesterase, the TMV exit is also disturbed [110]. The role of virus replicase in systemic transport of tobamoviruses as a suppressor of the post-transcriptional gene silencing (PTGS) and a factor promoting virus entry into phloem has been demonstrated [111]. Data on plant proteins capable of direct binding with tobamovirus CPs are scarce. One such protein IP-L interacting with the tomato mosaic virus (ToMV) CP was detected by screening a N. tabacum cdna library using the yeast two-hybrid system. Inhibition of the IP-L gene expression decreased efficiency of systemic infection with ToMV [112]. It is significant that the IP-L protein is 89% homologous to NbPCIP1 from N. benthamiana, which interacts with the N-terminus of the PVX CP [80]. The IP-L protein was shown to bind with two core α-helices of the ToMV CP (21-31 and a.a.) [113]. Interestingly, that the detected function of this protein is associated not with viral genome movement, but with the mechanism of development of chlorosis the interaction of ToMV CP with thylakoid protein L is accompanied by localization of the complex on thylakoid membranes of chloroplasts, which influences the chloroplast stability and leads to chlorosis of the leaves [113]. Another cellular factor from A. thaliana is DSTM1 protein (the protein responsible for delay of tobamovirus 1 systemic transport); it seems to provide the correct morphology of the TMV strain U1 virions in the vascular system of the plant. Mutations in the large arm of the A. thaliana second chromosome breaking DSTM1 gene expression result in detection of deficient curved virions from the phloem and petioles. However, in the mesophyll of apical leaves of mutant plants, correctly shaped viral particles (rigid rods) are found; therefore, it is supposed that DSTM1 is a phloem factor necessary for maintaining the stability of the virions in the phloem [114]. It should be noted that these data were obtained on A. thaliana plants with a significantly decreased infection. Therefore, the role of this cellular protein in the natural host plant of this virus remains unclear. Unlike tobamoviruses, data on functions of other rod-shaped virus CPs not associated with assembly of virions are very scarce. For tobraviruses, it was shown that tobacco rattle tobravirus does not require CP and formation of the viral particles for both cell-to-cell movement and systemic infection of plants [115]. The transport form of this virus obviously is a viral RNP complex formed with MP. Hordeiviruses and pomoviruses also form rigid helical rod-shaped virions; the genome of these viruses is segmented and consists of three RNAs encapsidated by a single CP into individual virus particles. Similar to potexviruses, virus transport is facilitated by three MPs encoded by TGB. With some exceptions considered below, transport form of these viruses is a viral RNP complex formed by the genomic RNA and viral TGB1 protein. The potato mop-top virus (PMTV) is the best-studied representative of pomoviruses. The viral genome moves between the neighboring cells as an RNP complex formed by the viral RNA and the TGB1 movement protein. Initially, it was supposed that CP also was not required for systemic infection of plants [116, 117]. However, recent data revealed that the CP and its minor variant with elongated C-terminus, which is synthesized as a result of the translation read-through of the CP open reading frame terminating codon, were necessary for systemic transport of one of three genomic RNAs, namely the CP-encoding RNA [118]. Note that in the absence of the CP all three genomic RNAs seem to spread systemically as RNP complexes. The minor CP was shown earlier to be capable of inserting into one end of the PMTV virus particle and thus promoting the virion transmission by a vector [119]. Based on experimental data, it was supposed that two waves could exist in the systemic transport of PMTV: the first wave at the early infection stages as the RNP complexes, and the second wave at the later stages as polar virions with one end interacting with the minor CP [120]. Interactions were found between the CP and the C-terminal region of the minor CP. Thirty-three amino acid residues in the C-terminal domain of this protein but not the CP are responsible for interaction with TGB1 protein, which also seems to be a component of virions competent for long-distance movement [118]. Thus, CP of pomoviruses is necessary for encapsidation of genomic RNAs and participates in formation of polar virus particles due to binding with the minor CP and subsequent interacting with TGB1 protein. Interestingly, pomoviruses are the only examples of rod-shaped helical viruses that form polar virions similarly to filamentous helical viruses (potex-, poty-, and closteroviruses). Additional nonstructural activities of pomoviruses, hordeiviruses, and tobraviruses CPs are unknown. It is unlikely that CPs of these viruses can only take part in virion assembly, but to find their additional functions is a task for future studies. OTHER NONCANONICAL FUNCTIONS OF COAT PROTEINS OF HELICAL VIRUSES Since viruses are biotrophic parasites, their evolutionary success depends on productive infection not lead-

10 10 MAKAROV, KALININA ing to death of the host plant. During coevolution of virus and host plant, many viral proteins are involved in formation of the plant antivirus response. CPs of plant helical viruses are among them. Proteomics studies have shown that interactions of cell factors with coat proteins of some viruses (including helical ones) activate pronounced rearrangements in the intracellular metabolic pathways, which change the expression of several hundred genes. Thus, the expression level of the photosynthesis-related genes significantly decreased, whereas expression of the genes associated with energy metabolism and with protein synthesis and turnover increased. Significant changes were observed also in metabolism of sugars, cell wall proteins, reactive oxygen species, and of pathogenesis-related proteins (PR proteins) [121]. Plant resistance genes (R-genes) provide resistance to various pathogens including viruses. In many cases, viral CPs are molecules specifically recognizable by products of the R-genes. In the majority of cases, triggering of R-gene expression is associated with triggering mechanisms of programmed cell death visualized as necroses (hypersensitive response, HR). It is long known that the tobamovirus TMV CP is an elicitor (avirulence effector) of HR caused by N-genes in tobacco plants. Mutational analysis of the CP of the elicitor strain TMV P20L identified its structural regions responsible for this activity: individual amino acid residues in the CP core α-helices were located within a three-dimensional structure [34, 122]. Interestingly, deletion of the N-terminus (1-14 a.a.) and deletion of the C-terminus ( a.a.) involving residues (Tyr2, Leu150, and Trp153) that are conservative in tobamoviruses and form an important moiety of the CP hydrophobic core, abolished the hypersensitive phenotype. Thus, maintaining the integrity of the CP structure was crucial for induction of resistance [122]. The role of the CP structure in induction of a hypersensitive-like response in N. tabacum cv. Xanthi nn plants was shown using the turnip vein-clearing virus (TVCV) [123]. Only mutants in which amino acid substitutions did not disturb the CP conformation (tertiary structure) were able to induce symptoms and were effective elicitors. It was supposed that a monomer or low order CP aggregates, possibly dimers or the A-protein, might be molecules recognizable by the plant defense systems. It was also shown that CPs of tobamoviruses induced resistance due to activation of L-genes. The product of the L-gene allele (L(1a)) of green pepper can recognize CPs of two tobamoviruses tobacco mild green mosaic virus and pepper mild mottle virus (both are Japanese strains) [124]. Resistance is induced by both viruses at 24 C and by only the first at 30 C. Differences in the efficiency of elicitors are mapped in the CP domain containing the β- sheet and are associated not only with induction of L(1a)- gene mediated resistance, but also with the possibility of virion formation [124]. Note that in tobamoviruses, coat proteins are the most frequent effectors of avirulence compared to the whole bulk of viral proteins. The TMV- P(0) CP also can indirectly induce expression of the transcription factor WRKYd from Capsicum annuum, which influences development of the hypersensitive response and accumulation of the TMV CP due to increasing expression of genes responsible for resistance to pathogens (PR-genes) and of the hypersensitive response genes (HR-genes) [125]. The TMV CP can also act as a factor of negative regulation of defense response due to suppression of the plant cell defense signaling pathways mediated through PR-1 and RDR-1 proteins [126]. Moreover, the tobamovirus CP was shown to negatively regulate the defense signaling pathway mediated through salicylic acid in Arabidopsis plants by increasing stability of DELLA proteins [127]. Potexvirus PVX CP is also an avirulence effector inducing potato plant resistance to PVX infection through activation of resistance gene Rx product [128, 129]. The CP domain responsible for this activity was located by deletion and mutational analysis within the core structure of non-virion form of CP [128]. The product of the potato Rx gene a protein of the CC- NB-LRR type is in the inactive state due to intramolecular interactions. The interaction with the effector (the PVX CP) releases the cell protein and triggers the defense response [128, 130]. Post-transcriptional gene silencing, or RNA-interference, is another innate defense mechanism of plants. Viruses overcome this mechanism via encoding suppressors of silencing, which act on different stages of this process. There are only a few examples of the function of helical viral CPs as suppressors of RNA-interference (silencing). Thus, the pepino mosaic potexvirus CP was shown to be an efficient suppressor of post-transcriptional gene silencing due to prevention of silencing signal spreading both among neighboring cells and systemically [85]. Both the major and minor CPs of the citrus tristeza closterovirus display the activity of silencing suppressors [131]. Coat proteins of helical viruses act as determinants of pathogenicity. Thus, the 5 -proximal region of the gene encoding the potyvirus PVY CP determines the intensity of the infection symptoms in Physalis floridana plants [132]. Deletion of the N-terminal fragment (35 a.a.) of potexvirus BaMV CP containing a glycine-rich motif converted necrotic symptoms into chlorotic lesions on Chenopodium quinoa leaves and also reduced mosaic symptoms on N. benthamiana leaves. This region was dispensable for BaMV replication, virion formation and movement. However, the interaction of the N-terminal fragment of PVX CP with the NbPCIP1 protein from N. benthamiana increases the viral replication and thus positively influences the accumulation of the virus and manifestation of infection symptoms [81]. Comparative analysis of infection caused by numerous isolates of pepino mosaic virus obtained from different wild tomato species revealed correlation between similarities of the