A surface loop of the potato leafroll virus coat protein is involved in virion assembly, systemic movement, and aphid transmission

Two acidic domains of the Potato leafroll virus (PLRV) coat protein, separated by 55 amino acids and predicted to be adjacent surface features on the virion, were the focus of a mutational analysis. Eleven site-directed mutants were generated from a cloned infectious cDNA of PLRV and delivered to plants by Agrobacterium-mediated mechanical inoculation. Alanine substitutions of any of the three amino acids of the sequence EWH (amino acids 170 to 172) or of D177 disrupted the ability of the coat protein to assemble stable particles and the ability of the viral RNA to move systemically in four host plant species. Alanine substitution of E109, D173, or E176 reduced the accumulation of virus in agrobacterium-infiltrated tissues, the efficiency of systemic infection, and the efficiency of aphid transmission relative to wild-type virus, but the mutations did not affect virion stability. A structural model of the PLRV capsid predicted that the amino acids critical for virion assembly were located within a depression at the center of a coat protein trimer. The other amino acids that affected plant infection and/or aphid transmission were predicted to be located around the perimeter of the depression. PLRV virions play key roles in phloem-limited virus movement in plant hosts as well as in transport and persistence in the aphid vectors. These results identified amino acid residues in a surface-oriented loop of the coat protein that are critical for virus assembly and stability, systemic infection of plants, and movement of virus through aphid vectors.     

A search for evidence of virus/transgene interactions in potatoes transformed with the potato leafroll virus replicase and coat protein genes

A search was conducted to detect evidence for interactions between potato leafroll virus (PLRV)-derived transgenes expressed in Russet Burbank potato and viruses to which the transgenic plants were exposed and by which they were infected. More than 25000 plants in 442 lines transformed with 16 different coat protein gene (CP) constructs and nearly 40000 plants in 512 lines transformed with seven different replicase gene (Rep) constructs of PLRV were exposed to field infection over a 6-year period. These plants were individually inspected for type and severity of virus symptoms. Heterologous viruses found infecting the plants were identified and examined for alterations in transmission characteristics, serological affinity, host range, and symptoms. Selected isolates of PLRV from field-infected plants were examined for unusual symptoms produced in diagnostic hosts and for alteration in sedimentation properties in density gradient tubes. Viruses that were propagated in selected transgenic lines in a greenhouse were examined for similar alterations. Transmission characteristics and serological properties were not altered when they replicated in potatoes containing CP constructs in the field or greenhouse. Potato plants expressing CP or Rep constructs of PLRV were not infected in the field or in the greenhouse with viruses that do not normally infect potato. New viruses or viruses with altered sedimentation characteristics, symptoms, or host range were not detected in field-exposed or greenhouse-inoculated potato plants expressing CP or Rep gene constructs of PLRV.     

Amino Acids in the Capsid Protein of Tomato Yellow Leaf Curl Virus That Are Crucial for Systemic Infection, Particle Formation, and Insect Transmission

A functional capsid protein (CP) is essential for host plant infection and insect transmission in monopartite geminiviruses. We studied two defective genomic DNAs of tomato yellow leaf curl virus (TYLCV), Sic and SicRcv. Sic, cloned from a field-infected tomato, was not infectious, whereas SicRcv, which spontaneously originated from Sic, was infectious but not whitefly transmissible. A single amino acid change in the CP was found to be responsible for restoring infectivity. When the amino acid sequences of the CPs of Sic and SicRcv were compared with that of a closely related wild-type virus (TYLCV-Sar), differences were found in the following positions: 129 (P in Sic and SicRcv, Q in Sar), 134 (Q in Sic and Sar, H in SicRcv) and 152 (E in Sic and SicRcv, D in Sar). We constructed TYLCV-Sar variants containing the eight possible amino acid combinations in those three positions and tested them for infectivity and transmissibility. QQD, QQE, QHD, and QHE had a wild-type phenotype, whereas PHD and PHE were infectious but nontransmissible. PQD and PQE mutants were not infectious; however, they replicated and accumulated CP, but not virions, in Nicotiana benthamiana leaf discs. The Q129P replacement is a nonconservative change, which may drastically alter the secondary structure of the CP and affect its ability to form the capsid. The additional Q134H change, however, appeared to compensate for the structural modification. Sequence comparisons among whitefly-transmitted geminiviruses in terms of the CP region studied showed that combinations other than QQD are present in several cases, but never with a P129.     

In vitro assembly of Sindbis virus core-like particles from cross-linked dimers of truncated and mutant capsid proteins

A nucleic acid-bound capsid protein dimer was previously identified using a Sindbis virus in vitro nucleocapsid assembly system and cross-linking reagents. Cross-link mapping, in combination with a model of the nucleocapsid core, suggested that this dimer contained one monomer from each of two adjacent capsomeres. This intercapsomere dimer is believed to be the initial intermediate in the nucleocapsid core assembly mechanism. This paper presents the purification of cross-linked dimers of a truncated capsid protein and the partial purification of cross-linked dimers of a full-length assembly-defective mutant. The assembly of core-like particles from these cross-linked capsid protein dimers is demonstrated. Core-like particles generated from cross-linked full-length mutant CP(19-264)L52D were examined by electron microscopy and appeared to have a morphology similar to that of wild-type in vitro-assembled core-like particles, although a slight size difference was often visible. Truncated cross-linked CP(81-264) dimers generated core-like particles as well. These core-like particles could subsequently be disassembled when reversible cross-linking reagents were used to form the dimers. The ability of the covalent intercapsomere cross-link to rescue capsid proteins with assembly defects or truncations in the amino-terminal region of the capsid protein supports the previous model of assembly and suggests a possible role for the amino-terminal region of the protein.     

Structural analysis of human immunodeficiency virus type 1 Gag protein interactions, using cysteine-specific reagents.

We have examined structural interactions of Gag proteins in human immunodeficiency virus type 1 (HIV-1) particles by utilizing cysteine mutagenesis and cysteine-specific modifying reagents. In immature protease-minus but otherwise wild-type (wt) particles, precursor Pr55Gag proteins did not form intermolecular cystines naturally but could be cross-linked at cysteines, and cross-linking appeared to occur across nucleocapsid (NC) domains. Capsid (CA) proteins in wt mature viruses possess cysteines near their carboxy termini at gag codons 330 and 350, but these residues are not involved in natural covalent intermolecular bonds, nor can they be intermolecularly cross-linked by using the membrane-permeable cross-linker bis-maleimido hexane. The cysteine at gag codon 350 (C-350) is highly reactive to thiol-specific modifying reagents, while the one at codon 330 (C-330) appears considerably less reactive, even in the presence of ionic detergent. These results suggest that the HIV-1 CA C terminus forms an unusually stable conformation. Mutagenesis of C-350 to a serine residue in the mutant C350S (C-350 changed to serine) virtually eliminated particle assembly, attesting to the importance of this region. We also examined a C330S mutant, as well as mutants in which cysteines were created midway through the capsid domain or in the C-terminal section of the major homology region. All such mutants appeared wt on the basis of biochemical assays but showed greatly reduced infectivities, indicative of a postassembly, postprocessing replicative block. Interestingly, capsid proteins of mature major homology region mutant particles could be cysteine cross-linked, implying either that these mutations permit cross-linking of the native C-terminal CA cysteines or that major homology regions on neighbor capsid proteins are in close proximity in mature virions.     

Site-directed mutagenesis and generation of chimeric viruses by homologous recombination in yeast to facilitate analysis of plant-virus interactions

A yeast homologous recombination system was used to generate mutants and chimeras in the genome of Potato leafroll virus (PLRV). A yeast-bacteria shuttle vector was developed that allows mutants and chimeras generated in yeast to be transformed into Escherichia coli for confirmation of the mutations and transformed into Agrobacterium tumefaciens to facilitate agroinfection of plants by the mutant PLRV genomes. The advantages of the system include the high frequency of recovered mutants generated by yeast homologous recombination, the ability to generate over 20 mutants and chimeras using only two restriction endonuclease sites, the ability to introduce multiple additional sequences using three and four DNA fragments, and the mobilization of the same plasmid from yeast to E. coli, A. tumefaciens, and plants. The wild-type PLRV genome showed no loss of virulence after sequential propagation in yeast, E. coli, and A. tumefaciens. Moreover, many PLRV clones with mutations generated in the capsid protein and readthrough domain of the capsid protein replicated and moved throughout plants. This approach will facilitate the analysis of plant-virus interactions of in vivo-generated mutants for many plant viruses, especially those not transmissible mechanically to plants.     

Chimeric Virus as a Source of the Potato Leafroll Virus Antigen

Large quantities of potato leafroll virus (PLRV) antigen are difficult to obtain because this virus accumulates in plants at a low titer. To overcome this problem, we constructed a binary vector containing chimeric cDNA, in which the coat protein (CP) gene of the crucifer infecting tobacco mosaic virus (crTMV) was substituted for the coat protein gene of PLRV. The PLRV movement protein (MP) gene, which overlaps completely with the CP gene, was doubly mutated to eliminate priming of the PLRV MP translation from ATG codons with no changes to the amino acid sequence of the CP. The untranslated long intergenic region located upstream of the CP gene was removed from the construct. Transcribed powerful tobamovirus polymerase of the produced vector synthesized PLRV CP gene that was, in turn, translated into the protein. CP PLRV packed RNAs from the helical crTMV in spherical virions. Morphology, size and antigenic specificities of the wild-type and chimeric virus were similar. The yield of isolated chimera was about three orders higher than the yield of native PLRV. The genetic manipulations facilitated the generation of antibodies against the chimeric virus, which recognize the wild-type PLRV.     

Structural insights into viral determinants of nematode mediated Grapevine fanleaf virus transmission

Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode''s feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector.     

Identification of subunit-subunit interactions in bacteriophage P22 procapsids by chemical cross-linking and mass spectrometry

Viral capsids are dynamic structures which self-assemble and undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Chemical cross-linking analyzed by mass spectrometry was used to identify residue specific inter- and intra-subunit interactions in the P22 procapsids. All the intersubunit cross-links occurred between residues clustered in a loop region (residues 157-207) which was previously identified by mass spectrometry based on hydrogen/deuterium exchange and biochemical experiments. DSP and BS3 which have similar distance constraints (12 angstroms and 11.4 angstroms, respectively) cross-linked the same residues between two subunits in the procapsids (K183-K183), whereas DST, a shorter cross-linker, cross-linked lysine 175 in one subunit to lysine 183 in another subunit. The replacement of threonine with a cysteine at residue 182 immediately adjacent to the K183 cross-linking site resulted in slow spontaneous disulfide bond formation in the procapsids without perturbing capsid integrity, thus suggesting flexibility within the loop region and close proximity between neighboring loop regions. To build a detailed structure model, we have predicted the secondary structure elements of the P22 coat protein, and attempted to thread the prediction onto identified helical elements of cryoEM 3D reconstruction. In this model, the loop regions where chemical cross-linkings occurred correspond to the extra density (ED) regions which protrude upward from the outside of the capsids and face one another around the symmetry axes.     

Effects of point mutations in the major capsid protein of beet western yellows virus on capsid formation,virus accumulation,and aphid transmission

Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected approximately 90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a "reverse" substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid.     

Molecular diagnostics of potato infections with PVY and PLRV by immunochromatography

An immunochromatography test system has been developed for molecular diagnostics of the potato virus Y and PLRV infection. To increase a low yield of PLRV and raise antibodies against the PLRV antigen, chimerical virus was constructed comprising the PLRV coat protein and recombinant RNA of a tobamovirus, in which capsid protein gene was replaced by the PLRV coat protein gene. Binary vector containing the DNA copy of the recombinant RNA was infectious, and yield of the chimerical virus increased up to 800 times in comparison with the wild type PLRV. On the basis of experience in the development of the diagnostics of viral and viroid infections, rational tactics are proposed for the mass laboratory and field diagnosis of viral infections on the molecular level.     

Nucleic acid-dependent cross-linking of the nucleocapsid protein of Sindbis virus

The assembly of the alphavirus nucleocapsid core is a multistep event requiring the association of the nucleocapsid protein with nucleic acid and the subsequent oligomerization of capsid proteins into an assembled core particle. Although the mechanism of assembly has been investigated extensively both in vivo and in vitro, no intermediates in the core assembly pathway have been identified. Through the use of both truncated and mutant Sindbis virus nucleocapsid proteins and a variety of cross-linking reagents, a possible nucleic acid-protein assembly intermediate has been detected. The cross-linked species, a covalent dimer, has been detected only in the presence of nucleic acid and with capsid proteins capable of binding nucleic acid. Optimum nucleic acid-dependent cross-linking was seen at a protein-to-nucleic-acid ratio identical to that required for maximum binding of the capsid protein to nucleic acid. Identical results were observed when cross-linking in vitro assembled core particles of both Sindbis and Ross River viruses. Purified cross-linked dimers of truncated proteins and of mutant proteins that failed to assemble were found to incorporate into assembled core particles when present as minor components in assembly reactions, suggesting that the cross-linking traps an authentic intermediate in nucleocapsid core assembly. Endoproteinase Lys-C mapping of the position of the cross-link indicated that lysine 250 of one capsid protein was cross-linked to lysine 250 of an adjacent capsid protein. Examination of the position of the cross-link in relation to the existing model of the nucleocapsid core suggests that the cross-linked species is a cross-capsomere contact between a pentamer and hexamer at the quasi-threefold axis or is a cross-capsomere contact between hexamers at the threefold axis of the icosahedral core particle and suggests several possible assembly models involving a nucleic acid-bound dimer of capsid protein as an early step in the assembly pathway.     

The Herpesvirus capsid surface protein, VP26, and the majority of the tegument proteins are dispensable for capsid transport toward the nucleus

Upon entering a cell, alphaherpesvirus capsids are transported toward the minus ends of microtubules and ultimately deposit virus DNA within the host nucleus. The virus proteins that mediate this centripetal transport are unknown but are expected to be either viral tegument proteins, which are a group of capsid-associated proteins, or a surface component of the capsid itself. Starting with derivatives of pseudorabies virus that encode a fluorescent protein fused to a structural component of the virus, we have made a collection of 12 mutant viruses that lack either the VP26 capsid protein or an individual tegument protein. Using live-cell fluorescence microscopy, we tracked individual virus particles in axons following infection of primary sensory neurons. Quantitative analysis of the VP26-null virus indicates that this protein plays no observable role in capsid transport. Furthermore, viruses lacking tegument proteins that are nonessential for virus propagation in cell culture were also competent for axonal transport. These results indicate that a protein essential for viral propagation mediates transport of the capsid to the nucleus.     

Mutational analysis of essential interactions involved in the assembly of hepatitis E virus capsid

The hepatitis E virus (HEV) capsid consists of a single structural protein, a portion of which is engaged in isosahedral contact to form a basal shell, and another portion in dimeric contact to form the homodimers protruding from the shell. Previous studies revealed that homodimers of the truncated HEV capsid proteins, E2 (amino acids 394-606) and p239 (amino acids 368-606), model dominant antigenic determinants of HEV. Immunization with these proteins protected rhesus monkeys against the virus, and three monoclonal antibodies against the homodimers could neutralize HEV infectivity and/or immune-capture of the virus. Furthermore, homodimers of p239 further interact to form particles of 23 nm diameter, rendering it an efficacious candidate vaccine. In light of this we postulate that the interactions involved in the formation of the homodimers and particles might be similar to those involved in assembly of the virus capsid. Presently, mutational analysis was carried out to identify these sites of interactions. The site of dimeric interactions was located to a cluster of six hydrophobic amino acids residues, Ala597, Val598, Ala599, Leu601, and Ala602; furthermore, the site involved in particle formation was located at amino acids 368-394. The possibility that these sites are also involved in assembly of the virus capsid is supported by the fact that they are located at two major and highly conserved hydrophobic regions of the HEV structural protein.     

Shrimp laminin receptor binds with capsid proteins of two additional shrimp RNA viruses YHV and IMNV

Laminin receptor (Lamr) in shrimp was previously proposed to be a potential receptor protein for Taura syndrome virus (TSV) based on yeast two-hybrid assays. Since shrimp Lamr bound to the VP1 capsid protein of TSV, we were interested to know whether capsid/envelope proteins from other shrimp viruses would also bind to Lamr. Thus, capsid/envelope encoding genes from 5 additional shrimp viruses were examined. These were Penaeus stylirostris densovirus (PstDNV), white spot syndrome virus (WSSV), infectious myonecrosis virus (IMNV), Macrobrachium rosenbergii nodavirus (MrNV), and yellow head virus (YHV). Protein interaction analysis using yeast two-hybrid assay revealed that Lamr specifically interacted with capsid/envelope proteins of RNA viruses IMNV and YHV but not MrNV and not with the capsid/envelope proteins of DNA viruses PstDNV and WSSV. In vitro pull-down assay also confirmed the interaction between Lamr and YHV gp116 envelope protein, and injection of recombinant Lamr (rLamr) protein produced in yeast cells protected shrimp against YHV in laboratory challenge tests.     

Protein-protein interactions within paramyxoviruses identified by native disulfide bonding or reversible chemical cross-linking.

Analysis of native disulfide-bonded protein oligomers in paramyxoviruses showed that some viral proteins are consistently present as covalent complexes. In isolated Sendai virus the hemagglutinating protein HN is present in homodimeric and homotetrameric forms, and the minor nucleocapsid protein P exists partly as a monomer and partly as a disulfide-linked homotrimer. Similar disulfide-linked complexes were observed in Newcastle disease virus (strain HP-16), in which HN exists as a homodimer and some of the major nucleocapsid protein NP exists as a homotrimer. Noncovalent intermolecular interactions between proteins were studied with the reversible chemical cross-linkers dimethyl-3,3'-dithiobispropionimidate and methyl 3-[(p-azidophenyl)dithio]propionimidate, which contain disulfide bridges and a 1.1-nm separation between their functional groups. The same results were achieved with both reagents. The conditions of preparation, isolation, and storage of the viruses affected the protein-protein interactions observed upon cross-linking. Homooligomers of the glycoprotein F, the matrix protein M, and the major nucleocapsid protein NP were produced in both Sendai and Newcastle disease viruses after mild cross-linking of all viral preparations examined, but NP-M heterodimer formation in both viruses was most prevalent in early harvest preparations that were cross-linked soon after isolation. The ability of NP and M to form a heterodimer upon cross-linking indicates that the matrix protein layer lies in close proximity (within 1.1 nm) to the nucleocapsid in the newly formed virion. Some noncovalent intermolecular protein interactions in Sendai and Newcastle disease viruses, i.e., those leading to the formation of F, NP, and M homooliogmers upon cross-linking, are more stable to virus storage than others, i.e., those leading to the formation of an NP-M heterodimer upon cross-linking. The storage-induced loss of the ability of NP and M to form a heterodimer is not accompanied by any apparent loss of infectivity. This indicates that some spacial relationships which form during virus assembly can alter after particle formation and are not essential for the ensuing stages of the infectious process.     

Structural interactions between retroviral Gag proteins examined by cysteine cross-linking.

We have examined structural interactions between Gag proteins within Moloney murine leukemia virus (M-MuLV) particles by making use of the cysteine-specific cross-linking agents iodine and bis-maleimido hexane. Virion-associated wild-type M-MuLV Pr65Gag proteins in immature particles were intermolecularly cross-linked at cysteines to form Pr65Gag oligomers, from dimers to pentamers or hexamers. Following a systematic approach of cysteine-to-serine mutagenesis, we have shown that cross-linking of Pr65Gag occurred at cysteines of the nucleocapsid (NC) Cys-His motif, suggesting that the Cys-His motifs within virus particles are packed in close proximity. The M-MuLV Pr65Gag protein did not cross-link to the human immunodeficiency virus Pr55Gag protein when the two molecules were coexpressed, indicating either that they did not coassemble or that heterologous Gag proteins were not in close enough proximity to be cross-linked. Using an assembly-competent, protease-minus, cysteine-minus Pr65Gag protein as a template, novel cysteine residues were generated in the M-MuLV capsid domain major homology region (MHR). Cross-linking of proteins containing MHR cysteines showed above-background levels of Gag-Gag dimers but also identified a novel cellular factor, present in virions, that cross-linked to MHR residues. Although the NC cysteine mutation was compatible with M-MuLV particle assembly, deletions of the NC domain were not tolerated. These results suggest that the Cys-His motif is held in close proximity within immature M-MuLV particles by interactions between CA domains and/or non-Cys-His motif domains of the NC.     

Analysis of differential selective forces acting on the coat protein (P3) of the plant virus family Luteoviridae

The coat protein (CP) of the family Luteoviridae is directly associated with the success of infection. It participates in various steps of the virus life cycle, such as virion assembly, stability, systemic infection, and transmission. Despite its importance, extensive studies on the molecular evolution of this protein are lacking. In the present study, we investigate the action of differential selective forces on the CP coding region using maximum likelihood methods. We found that the protein is subjected to heterogeneous selective pressures and some sites may be evolving near neutrality. Based on the proposed 3-D model of the CP S-domain, we showed that nearly neutral sites are predominantly located in the region of the protein that faces the interior of the capsid, in close contact with the viral RNA, while highly conserved sites are mainly part of beta-strands, in the protein's major framework.     

Heterologous encapsidation in transmission of plant viral particles by aphid vectors

Recently, significant progress has been made in recognizing virus-aphid specificities and identifying the proteins involved in virus transmission by aphids. An essential role of the viral capsid protein in this process has been proved. Heterologous encapsidation between viruses in mixed infections may allow transmission by aphids of normally non-aphid-transmissible viruses or change virus-vector interactions. This review describes the most characteristic examples of the phenomenon. Recent findings regarding transmission by aphids of viroid encapsidated in the viral capsid protein, and of virus encapsidated in transgenic coat protein, are presented.