The vectors of plant viruses include insects, nematodes, mites, and fungi, and the majority of viruses have arthropod vectors (26
). In cases where plant virus transmission has been studied in detail, the capsid protein has been shown to be the primary determinant for transmission by vectors (13
). The interactions between virus capsid proteins and their vectors are either direct or mediated by an accessory or helper protein (22
). Transmission involves the binding and retention of virions to sites within the vector, although little is known about the ligands to which viruses bind. Genetic studies have revealed that specific viral capsid protein domains and amino acids are determinative for transmissibility and vector specificity. Examples can be found for aphid-transmitted viruses in the genera Potyvirus
), and Cucumovirus
); for the nematode-transmitted viruses in the genus Tobravirus
); and for fungus-transmitted viruses in the genera Tombusvirus
) and Benyvirus
). An emerging theme for a number of these viruses is that a minor capsid protein readthrough protein plays an important role in transmission; this protein is generated by a mechanism involving readthrough of the major capsid protein stop codon (reviewed in reference 4
Cucumber mosaic virus
(CMV) is an aphid-transmitted virus, the transmission of which has been studied in some detail (33
). It is a plus-sense RNA virus with three single-stranded genomic RNAs (32
). RNAs 1 and 2 each encode a replication-related protein (15
). CMV RNA 2 also encodes an 11- to 13-kDa protein (2b) that influences virulence and functions as a suppressor of plant host-mediated gene silencing (8
). RNA 3 encodes two proteins, the 3a protein involved in the cell-to-cell movement of the virus and the 24.1-kDa capsid protein. The capsid protein is multifunctional; in addition to having a role in encapsidation, it affects virus movement in plants (19
), transmission, symptom expression, and host range (43
CMV virions have T=3 icosahedral symmetry and consist of 180 copies of the single, virus-encoded capsid protein. The 3.2-Å-resolution X-ray crystallographic structure of CMV has recently been described (46
). This structure revealed a number of remarkable differences between the A subunits (those at the fivefold axis of symmetry) and the B and C subunits (those at the threefold axis of symmetry). The most striking difference was a cluster of six amphipathic helices present in the B and C subunits and oriented down into the virion core at the threefold axis; these helices were not present in the A subunits at the fivefold axis (46
). The lateral βF-βG loop structure faces the threefold and fivefold axes of symmetry, but its structure differs in the respective hexameric and pentameric capsomeres. A similar difference in structure was observed for the βE-βF loop, which makes contact among the A subunits about the fivefold axis and forms the contact surface between the B and C subunits at the threefold axis. In contrast to the βE-βF and βF-βG loops, all of the three prominent surface loop structures, βB-βC, βD-βE, and βH-βI, have nearly identical structures in the A, B, and C subunits.
The transmission of CMV in nature is dependent upon aphid vectors, and virions are transmitted in a nonpersistent or stylet-borne manner (10
). The CMV capsid protein has been shown to be the primary determinant for vector transmission (5
); no additional helper component is known to be required for the transmission of virions (27
). Molecular and genetic analyses of strains that are defective in aphid transmission have revealed that different regions of the viral capsid protein variably affect transmission and that the efficiency of transmission of CMV varies with the species of aphid vector. Amino acids that affect virus transmission by the aphid Aphis gossypii
have been mapped to two capsid protein positions, 129, located in the βE-βF loop, and 162, located in the βF-βG loop (36
). Three additional capsid protein amino acid positions affect transmission by the aphid Myzus persicae
). Surprisingly, most of these amino acids that affect transmission are buried in the folded polypeptide or between subunits in assembled virions. In examining what capsid protein domains are clearly exposed on the virus, it was noted that the amino acid sequence of one of three prominent surface loops was conserved among cucumoviruses. This report describes the amino acid conservation of the βH-βI loop and how mutations in this CMV surface domain affect vector transmission.
The conservation of an amino acid sequence on the surface of a virus suggests an essential role beyond that of encapsidation. Results presented in this study are consistent with our hypothesis that the conserved CMV βH-βI loop structure plays a role in aphid vector transmission. While this loop forms a conspicuous, negatively charged electrostatic field on the surfaces of virions, the two other prominent surface domains, the βB-βC and βD-βE loops, make minimal charge contributions (46
). In studies of spontaneous mutants defective in aphid transmission, amino acids in two other loop structures (βE-βF and βF-βG) were shown to impact transmission; these loops do not appear to alter the surface charge on virions but do affect virion stability (29
). It is useful to compare and contrast what is observed in CMV with what is seen in the genetically related CCMV. Although the capsid structure of CCMV is quite similar to that of CMV, CCMV is transmitted by beetles and not by aphids. As the capsid protein is a primary determinant for vector transmission, comparative analyses may provide insights into the underlying structural basis of vector specificity. In CCMV, the βH-βI loop structure is the same length but has fewer acidic residues (2 versus 5). Analogous to the arrangement in CMV, the two other prominent surface loop structures in CCMV do not contribute significantly to the surface charge density (47
). Our working hypothesis is that a unique structure and charge density present on the surface of CMV facilitate the reversible binding of virions in the aphid vector.
The presentation of a putative vector binding domain on the surface of a virion must be fully integrated and compatible with other capsid protein functions such as RNA binding, encapsidation, cell-to-cell and systemic movement within the host, and any essential interactions with plant factors (4
). Although six of the βH-βI loop mutations did not give rise to detectable phenotypic differences beyond those of their transmission defects, two of the mutants induced altered symptoms on tobacco and a third mutation interfered with replication and/or movement. Of particular interest is the D192K mutation, which induces necrosis. The induction of a similar type of necrosis has been reported for CMV capsid protein mutants with amino acid substitutions at position 129 (49
). The necrotic phenotype is dependent on the specific amino acid replacement, as some substitutions at position 129 result in symptoms of chlorosis or a wild-type mottle-mosaic (44
). It is not known whether virions, the free capsid protein, or assembly intermediates are the entities responsible for the induction of necrosis or chlorosis. Interestingly, position 129 mutations also affect the aphid transmission phenotype, reducing or eliminating vector transmission (35
). With regard to the structure of CMV, amino acid position 129 and the βE-βF loop on which it is located face a depression surrounding the pentameric and hexameric capsomeres. The βE-βF loop is at or proximal to the surfaces of virions, and this region would be accessible to factors in the plant host or the aphid vector. In contrast, although amino acid position 192 is clearly on the surfaces of virions and accessible to factors in the host or vector, its effects on symptoms or transmission may be indirect. Position 192 is implicated in the binding of a metal cation (46
), and mutations that affect metal binding may influence the structure or presentation of the βH-βI loop or affect subunit-subunit interactions. Capsid protein mutations that interfere with aphid transmission have also been shown to reduce the stability of virions (29
). The stabilities of virions of the βH-βI loop mutants are comparable to that of the wild-type virus (J. C. Ng, C. Josefsson, and K. L. Perry, unpublished results). Therefore, we believe that the defect in aphid transmission is most likely due to an alteration of virion binding (or release) from sites in the aphid vector.
A second mutant with an altered symptom phenotype was the L194A mutant. The induced yellow chlorosis was similar to that described for CMV strains M (28
) and Y (51
), but it differed in the subsequent appearance of necrotic flecks within the chlorotic, mottled areas. In a subgroup II strain of CMV, a capsid protein change of K193N or K193S also radically altered symptoms (50
); these changes are of interest because they are in the βH-βI loop, although the amino acid at position 193 is not conserved among CMVs. All three CMV RNAs encode symptom determinants (8
), but the mechanisms by which viral proteins induce symptom expression are not understood. It is known that expression of the CMV capsid protein alone in a viral vector does not induce symptoms characteristic of the CMV infection (50
A D197K mutation appeared to be lethal, as multiple attempts to infect plants with transcripts of the D197K mutant failed. It is not known whether this mutation affects replication, movement, or both. The only recoverable mutant with the D197K change was a virus (the D197K∗ mutant) with a compensatory second-site mutation located 4 amino acid residues away in the same loop structure. Compensatory second-site mutations were also described by Suzuki et al. (49
) in their study of an engineered mutant (the Phe-129 mutant); this mutant induced necrotic local lesions but did not move systemically. Remarkably, in that study both the introduced and second-site mutations were in the same loop structure (βE-βF); this situation is analogous to the results described above for the D197K∗ mutant. The results from both of these studies suggest that the perturbation of a structural motif can be compensated for by a second, proximal amino acid change that restores a structure or charge density.
Consistent with a predicted alteration of a surface loop in the capsid protein and a reduction in the net negative charge on virions, the migration of mutant virions in agarose gels was retarded. It should be noted that the three D-to-A mutants (the D191A, D192A, and D197A mutants) did not migrate to the same position in the gel, suggesting that there may be conformational differences in the capsid protein of each mutant. A similar inference can be made to account for the faster migration of the L194A mutant relative to that of the wild-type virus. Three of the mutants with introduced lysine residues (the D191K, D192K, and E195K mutants) did not migrate in the gel; for each of these mutants, material stained by both ethidium bromide and Coomassie blue was observed in the well. This may have been due to aggregation; alternatively, the mutations may have conferred a net neutral charge upon virions. Wild-type-like virions of these lysine mutants were purified and observed by negative staining, indicating that a perturbation in virion formation was not responsible for the defect in transmission. The one feature that all of the mutants had in common was a predicted alteration in their surface charge, and this property appears to underlie the defect in vector transmission.
One issue that remains to be addressed is the extent to which amino acid residues in other surface loop structures can be modified and the extent to which they affect viability, symptomatology, and aphid vector transmission. Although it would not be surprising to find that the mutation of any surface residue gives rise to a phenotypic change, altering conserved amino acids (such as those found in the βH-βI loop) would be expected to have the most profound effects. From the present study, it is clear that disrupting amino acid residues in a conserved surface loop structure reduces or eliminates vector transmissibility without grossly affecting virion formation. The availability of these transmission-defective mutants will facilitate studies aimed at identifying ligands in the aphid vector and understanding molecular mechanisms underlying the vector transmission of a plant virus.