Characterization of polymorphism displayed by the coat protein mutants of tomato bushy stunt virus

Expression of full-length and N-terminal deletion mutants of the coat protein (CP) of tomato bushy stunt virus (TBSV) using the recombinant baculovirus system resulted in spontaneously assembled virus-like particles (VLPs). Deletion of the majority of the R-domain sequence of the CP, residues 1-52 (CP-NDelta52) and 1-62 (CP-NDelta62), produced capsids similar to wild-type VLPs. Interestingly, the CP-NDelta62 mutant that retains the last 3 residues of R-domain is capable of forming both the T = 1 and T = 3 particles. However, between the two types of VLPs, formation of the T = 1 capsids appears to be preferred. Another mutant, CP-NDelta72, in which R-domain (residues 1-65) was completely removed but contains most of the beta-annulus and extended arm (betaA) regions exclusively formed T = 1 particles. These results suggest that as few as 3 residues (63-65) of the R-domain, which includes 2 basic amino acids together with the arm (betaA) and beta-annulus regions, may be sufficient for the formation of T = 3 particles. However, anywhere between 4 to 13 residues of the R-domain may be required for proper positioning of betaA and beta-annulus structural elements of the C-type subunits to facilitate an error free assembly of T = 3 capsids.     

Complementation of coat protein-defective TMV mutants in transgenic tobacco plants expressing TMV coat protein

Transgenic tobacco plants (Nicotiana tabacum cv. Xanthi) which express tobacco mosaic virus (TMV) U1 strain coat protein (CP) can complement both the assembly and the long-distance spread of CP-defective (DT1) or coat proteinless (DT1G) mutants of TMV. Both mutants arose spontaneously from PM2 and exist only as unencapsidated RNA in the inoculated leaves of control tobacco plants, where they are unable to form virus particles or to spread systemically. TMV CP expressed in transgenic tobacco plants [CP+ line 3404; P. Powell Abel, R. S. Nelson, B. De, N. Hoffman, S. G. Rogers, R. T. Fraley, and R. N. Beachy, 1986, Science 232, 738-743] was able to package some of either mutant viral RNA into TMV-like particles in vivo and resulted in the long-range spread of infection. In vivo encapsidated DT1 RNA was recovered and reinoculated onto control or new CP+ transgenic tobacco plants. Localized infection of control plants confirmed that no RNA recombination or reversion of the mutant RNA to wild-type had occurred during passage in the first CP+ plant. In contrast, encapsidated DT1 RNA was unable to produce even local infection in CP+ transgenic plants confirming that CP-mediated protection operates during the early stages of virus infection, including particle uncoating. By positive complementation, these results also confirm that TMV CP is required for the long-distance spread of infection.     

Coat protein-mediated resistance to TMV infection of Nicotiana tabacum involves multiple modes of interference by coat protein

Expression of tobacco mosaic virus (TMV) coat protein (CP) restricts virus disassembly and alters the accumulation of the movement protein (MP). To characterize the role of structure of transgenic CP in regulating virus disassembly and production of MP, we generated CPs with mutations at residues Glu50 and Asp77, located in the interface between juxtaposed CP subunits. In transgenic Nicotiana tabacum and BY-2 cells, three categories of coat protein-mediated resistance (CP-MR) levels were identified: wild-type CP-MR; elevated CP-MR; and no CP-MR. Mutant CPs that interfered with the accumulation of virus replication complexes conferred very high levels of protection to TMV, except by CP(E50D) which provided no protection in the systemic host (Xanthi-nn) but high CP-MR in the local lesion host (Xanthi-NN). In transgenic BY-2 cells CP(E50D) strongly reduced accumulation of MP:GFP. In general, there was a strong correlation between the capacity for CP to assemble to pseudovirions and CP-MR, while there was not strong correlation with packaging viral RNA and CP-MR. The data demonstrate that interference with one or more steps in virus infection and replication by wild type and mutant CP determine the degree of CP-MR.     

The importance of alfalfa mosaic virus coat protein dimers in the initiation of replication

Deletion and substitution mutations affecting the oligomerization of alfalfa mosaic virus (AMV) coat protein (CP) were studied in protoplasts to determine their effect on genome activation, an early step in AMV replication. The CP mutants that formed dimers, CPDeltaC9 and CPC-A(R)F, were highly active in initiating replication with 63-84% of wild-type (wt) CP activity. However, all mutants that did not form dimers, CPDeltaC18, CPDeltaC19, CPC-WFP, and CPC-W, were much less active with 19-33% of wt CP activity. The accumulation and solubility of mutant CPs expressed from a virus-based vector in Nicotiana benthamiana were similar to that of wt CP. Analysis of CP-RNA interactions indicated that CP dimers and CP monomers interacted very differently with AMV RNA 3' ends. These results suggest that CP dimers are more efficient for replication than CP monomers because of differences in RNA binding rather than differences in expression and accumulation of the mutant CPs in infected cells.     

Membrane activity of the southern cowpea mosaic virus coat protein: the role of basic amino acids, helix-forming potential, and lipid composition

Southern cowpea mosaic virus (SCPMV) is a spherical RNA virus with T = 3 icosahedral symmetry. The particle is composed of 180 subunits of the coat protein (CP) and one copy of the positive-sense viral RNA. The CP has two domains, the random (R) domain formed by the N-terminal 64 aa and the shell (S) domain (aa 65--260). The R domain is highly charged, with 11 of the N-terminal 30 residues being basic. It is localized to the interior of the native particle where it may interact with the viral RNA, but under certain pH and salt conditions the topology of the particle changes to externalize the R domain. Since the CPs of several spherical RNA viruses have been shown to interact with host membranes during infection, we have begun investigating the membrane interactions of the SCPMV CP using the artificial liposome membranes. Both the native CP and the R domain overexpressed in Escherichia coli were observed to interact with liposomes. The interaction between the R domain and liposomes required either anionic phospholipids or non-bilayer-forming lipids and involved electrostatic interactions since it was shown to be both pH and ionic strength dependent. The analysis of four different deletion and six different site-directed substitution mutations partially mapped the region responsible for this interaction to residues 1--30. Analysis of this region of the R domain by circular dichroism indicated that it assumes an alpha-helical structure when exposed to liposomes composed of anionic lipids. Mutations, which extend the helical nature of this region, promoted an increased interaction. The possible role of the CP/lipid interaction in the SCPMV infection is discussed.     

Molecular studies on bromovirus capsid protein. VII. Selective packaging on BMV RNA4 by specific N-terminal arginine residuals

An arginine-rich RNA-binding motif (ARM) found at the N-proximal region of Brome mosaic virus (BMV) coat protein (CP) adopts alpha-helical conformation and shares homology with CPs of plant and insect RNA viruses, HIV-Rev and Tat proteins, bacterial antiterminators, and ribosomal splicing factors. The ARM of BMV CP, consisting of amino acids 9 through 21 with six arginine residues, is essential for RNA binding and subsequent packaging. In this study analysis of the alpha-helical contents of wild-type and mutant peptides by circular dichroism spectra identified protein determinants required for such conformation. Electrophoretic mobility-shift assays between viral RNA and BMV CP peptides with either proline or alanine substitutions revealed that the interaction is nonspecific. Expression in vivo of mature full-length BMV CP subunits, having the same substitutions for each arginine within the ARM, derived from biologically active clones was found to be competent to assemble into infectious virions and cause visible symptom phenotypes in whole plants. However, analysis of virion progeny RNA profiles of CP variants and subsequent in vitro reassembly assays between mutant CP and four BMV RNAs unveiled the ability of arginine residues at positions 10, 13, or 14 of the ARM to confer selective packaging of BMV RNA4. Thus, BMV CP contains determinants that specifically interact with RNA4 to ensure selective packaging.     

Cucumovirus- and bromovirus-encoded movement functions potentiate cell-to-cell movement of tobamo- and potexviruses

Cucumber mosaic virus (CMV, a cucumovirus) and Brome mosaic virus (BMV, a bromovirus) require the coat protein (CP) in addition to the 3a movement protein (MP) for cell-to-cell movement, while Cowpea chlorotic mottle virus (CCMV, a bromovirus) does not. Using bombardment-mediated transcomplementation assays, we investigated whether the movement functions encoded by these viruses potentiate cell-to-cell movement of movement-defective Tomato mosaic virus (ToMV, a tobamovirus) and Potato virus X (PVX, a potexvirus) mutants in Nicotiana benthamiana. Coexpression of CMV 3a and CP, but neither protein alone, complemented the defective movement of ToMV and PVX. A C-terminal deletion in CMV 3a (3a Delta C33) abolished the requirement of CP in transporting the ToMV genome. The action of 3a Delta C33 was inhibited by coexpression of wild-type 3a. These findings were confirmed in tobacco with ToMV-CMV chimeric viruses. Either BMV 3a or CCMV 3a alone efficiently complemented the movement-defective phenotype of the ToMV mutant. Therefore, every 3a protein examined intrinsically possesses the activity required to act as MP. In transcomplementation of the PVX mutant, the activities of BMV 3a, CCMV 3a, and CMV 3a Delta C33 were very low. The activities of the bromovirus 3a proteins were enhanced by coexpression of the cognate CP but the activity of CMV 3a Delta C33 was not. Based on these results, possible roles of cucumo- and bromovirus CPs in cell-to-cell movement are discussed.     

In vivo particle polymorphism results from deletion of a N-terminal peptide molecular switch in brome mosaic virus capsid protein

The interaction between brome mosaic virus (BMV) coat protein (CP) and viral RNA is a carefully orchestrated process resulting in the formation of homogeneous population of infectious virions with T=3 symmetry. Expression in vivo of either wild type or mutant BMV CP through homologous replication never results in the assembly of aberrant particles. In this study, we report that deletion of amino acid residues 41-47 from the N-proximal region of BMV CP resulted in the assembly of polymorphic virions in vivo. Purified virions from symptomatic leaves remain non-infectious and Northern blot analysis of virion RNA displayed packaging defects. Biochemical characterization of variant CP by circular dichroism and MALDI-TOF, respectively, revealed that the engineered deletion affected the protein structure and capsid dynamics. Most significantly, CP subunits dissociated from polymorphic virions are incompetent for in vitro reassembly. Based on these observations, we propose a chaperon-mediated mechanism for the assembly of variant CP in vivo and also hypothesize that (41)KAIKAIA(47) N-proximal peptide functions as a molecular switch in regulating T=3 virion symmetry.     

Complementation of the movement-deficient mutations in potato virus X: potyvirus coat protein mediates cell-to-cell trafficking of C-terminal truncation but not deletion mutant of potexvirus coat protein

The cell-to-cell movement of the GUS-tagged potato virus X (PVX) coat protein (CP) movement-deficient mutant was restored by potyviral CPs of potato virus A (PVA) and potato virus Y (PVY) in Nicotiana benthamiana leaves in transient cobombardment experiments. Viral cell-to-cell movement of PVX CP mutant was complemented in Nicotiana tabacum cv. SR1 transgenic plants expressing PVY CP: PVX RNA and polymerase were detected in the PVX CP mutant-inoculated leaves of transgenic plants. These findings demonstrated the ability of the PVX CP-deficient mutant to move from cell to cell but not long distances in the transgenic plants and suggest that CPs of potex- and potyviruses display complementary activities in the movement process. Potyviral CP alone is not able to carry out these activities, since the mutated PVX CP is indispensable for restored movement. No trans-encapsidation between potyviral CP and PVX RNA was observed. Therefore, potyviral CP facilitates the PVX CP mutant movement by the mechanism that cannot be explained by coat protein substitution. Our data also suggest that CP functioning in cell-to-cell movement is not restricted to a simple passive role in forming virions.     

The role of the C-terminal region of olive latent virus 1 coat protein in host systemic infection

Summary. A full-length cDNA clone of olive latent virus 1 (OLV-1), a member of the genus Necrovirus, family Tombusviridae, was subjected to site-directed mutagenesis, and coat protein gene mutants were constructed. A mutant clone, denoted Δ3297, was obtained by deleting the nucleotide in position 3297, thus inducing a frameshift and replacing the last 49 amino acids of the viral coat protein (CP) by a shorter sequence of 39 amino acids. This mutant was viable, stable, able to synthesize a smaller CP, and able to give rise to the formation of apparently intact virus particles. Cell-to-cell movement of Δ3297 in Nicotiana benthamiana leaves was not affected, but, contrary to wild type OLV-1, it failed to spread systemically. These results indicate that virion formation is necessary but not sufficient for long-distance movement for OLV-1 and highlights the role of the CP carboxy-terminal domain in systemic infection.     

In vitro analysis of an RNA binding site within the N-terminal 30 amino acids of the southern cowpea mosaic virus coat protein

Southern cowpea mosaic virus (SCPMV) is a positive-sense RNA virus with T = 3 icosahedral symmetry. The coat protein (CP) has two domains, the random (R) domain and the shell (S) domain. The R domain is formed by the N-terminal 64 amino acids (aa) and is localized to the interior of the particle where it is expected to interact with the viral RNA. The R domain (aa 1--57) was expressed in Escherichia coli as a recombinant protein (rWTR) containing a nonviral C-terminal extension with two histidine tags. The RNA binding site of the R domain was identified by Northwestern blotting and electrophoretic mobility shift assay (EMSA) using recombinant wild-type and mutant R domain proteins. Deletions within the R domain revealed that the RNA binding site is localized to its N-terminal 30 aa. RNA binding by this element was found to be nonspecific with regard to RNA sequence and was sensitive to high salt concentrations, suggesting that electrostatic interactions are important for RNA binding by the R domain. The RNA binding site includes 11 basic residues, eight of which are located in the arginine-rich region between aa 22 and 30. It was demonstrated using alanine substitution mutants that the basic residues of the arginine-rich region but not those present at positions 3, 4, and 7 are necessary for RNA binding. None of the basic residues within the arginine-rich region are specifically required for RNA binding, but the overall charge of the N-terminal 30 aa is important. Proline substitution mutations within the N-terminal 30 aa, and alanine substitutions for prolines at positions 18, 20, and 21, did not affect the RNA binding activity of the R domain. However, it was demonstrated by circular dichroism (CD) that the conformation of the N-terminal 30 aa of the R domain changes from a random coil to an alpha-helix in the presence of 50% trifluoroethanol (TFE). The possible role for this structural change in RNA binding by the R domain is discussed.     

Symptom attenuation by a satellite RNA in vivo is dependent on reduced levels of virus coat protein.

Many plant RNA viruses provide replication and encapsidation functions for one or more satellite RNAs (sat-RNAs) that can modulate the symptoms of the associated helper virus. Sat-RNA C, a virulent sat-RNA associated with turnip crinkle virus (TCV), normally intensifies symptoms but can attenuate symptoms if the TCV coat protein (CP) is replaced with that of cardamine chlorotic fleck carmovirus [Kong et al. (1995) Plant Cell 7, 1625-1634] or if TCV contains an alteration in the CP initiation codon (TCV-CPm) [Kong et al. (1997b) Plant Cell 9, 2051-2063]. To further elucidate the mechanism of symptom attenuation by sat-RNA C, the composition of the CP produced by TCV-CPm (CPCPm) was determined. Our results reveal that CPCPm likely has two additional amino acids at its N-terminus compared with wild-type TCV CP. TCV-CPm produces reduced levels of CP, and this reduction, not the two additional residues at the CP N-terminus, is responsible for symptom attenuation by sat-RNA C.     

The hypersensitive response to cucumber mosaic virus in Chenopodium amaranticolor requires virus movement outside the initially infected cell

Cucumber mosaic virus (CMV) expressing the green fluorescent protein (GFP), and lacking either the 3a movement protein or the coat protein (CP), failed to induce a hypersensitive response producing local lesions in inoculated leaves of Chenopodium amaranticolor. Cytological analysis showed that both viral-encoded proteins are required for cell-to-cell movement of the virus and the simultaneous appearance of cellular necrosis. In the absence of either or both proteins, infection was confined to single, non-necrotized, epidermal cells. CMV with a mutation in the 3a protein (M8 CMV) could infect tobacco systemically but did not induce necrotic lesions in C. amaranticolor. In this host, the mutated 3a protein was unable to promote viral movement out of the initially infected epidermal cell. Movement-deficient CMV expressing wild-type (WT) 3a protein as a fusion to the GFP, as well as WT CP, also failed to induce necrosis. Finally, single epidermal cells infected with a movement-deficient CMV expressing WT 3a protein, WT CP, and free GFP did not show necrosis. These data indicate that viral movement out of the initially infected epidermal cell, and not the simultaneous expression in this cell of the 3a protein and the CP, is required for the induction of cell death.     

Aggregation of TMV CP plays a role in CP functions and in coat-protein-mediated resistance

Tobacco mosaic virus (TMV) coat protein (CP) in absence of RNA self-assembles into several different structures depending on pH and ionic strength. Transgenic plants that produce self-assembling CP are resistant to TMV infection, a phenomenon referred to as coat-protein-mediated resistance (CP-MR). The mutant CP Thr42Trp (CP(T42W)) produces enhanced CP-MR compared to wild-type CP. To establish the relationship between the formation of 20S CP aggregates and CP-MR, virus-like particles (VLPs) produced by TMV variants that yield high levels of CP-MR were characterized. We demonstrate that non-helical structures are found in VLPs formed in vivo by CP(T42W) but not by wild-type CP and suggest that the mutation shifts the intracellular equilibrium of aggregates from low to higher proportions of non-helical 20S aggregates. A similar shift in equilibrium of aggregates was observed with CP(D77R), another mutant that confers high level of CP-MR. The mutant CP(D50R) confers a level of CP-MR similar to wild-type CP and aggregates in a manner similar to wild-type CP. We conclude that increased CP-MR is correlated with a shift in intracellular equilibrium of CP aggregates, including aggregates that interfere with virus replication.