Dissociated Coat Protein Research Articles

Engineering Thermal Stability in RNA Phage Capsids via Disulphide Bonds

The RNA bacteriophages, a group that includes phages Qbeta and MS2, have a number of potential bionanotechnological applications, including cell specific drug delivery and as substrates for the formation of novel materials. Despite extensive sequence identity between their coat protein subunits, and an almost identical three-dimensional fold, Qbeta and MS2 capsids have dramatically different thermal stabilities. The increased stability of Qbeta has been correlated with the inter-subunit disulphide bonds present in that capsid and not present in MS2. We have tested this hypothesis directly using mass spectrometry. Analysis of the dissociated coat protein subunits suggests that inter-molecular disulphides are formed at the capsid five-fold but may not be at the three-fold axes. This conclusion has been tested by engineering disulphide cross-links into either the five-fold or three-fold positions of the recombinant MS2 capsid. Five-fold cross-linking results in a mutant with stability properties similar to those of Qbeta. Three-fold cross-linking results in a mutant unable to assemble T = 3 shells, implying that five-fold structures are on pathway to capsid assembly in these phages. The results demonstrate how it is possible to redesign the physical properties of phage shells and may be of general relevance to future applications of viruses and virus-like particles.

IDENTIFICATION AND PARTIAL CHARACTERIZATION OF AN ISOLATE OF APRICOT LATENT VIRUS FROM PALESTINE

A filamentous virus was isolated by mechanical sap transmission from GF 305 seedlings showing asteroid ringspots to Nicotiana occidentalis. The GF 305 seedlings were graft-inoculated with buds from an apricot tree cv Mistikawi, from Palestine. The electrophoretic dsRNA pattern from symptomatic GF 305 and N. occidentalis showed a major band of about 9.5 kbp. Bundles of elongated virus-like particles were present in the cytoplasm of infected N. occidentalis. Dissociated coat protein from partially purified virus preparations consisted of a single subunit with an estimated Mr of ca. 50 kDa. RT-PCR using primers specific for Apricot latent virus (ApLV) RNA amplified a DNA product of about 200 bp from infected GF 305 and N. occidentalis plants. Sequence analysis of this fragment showed 90-93% identity in the encoded amino acid sequence with corresponding sequences from different ApLV isolates. This is the first record of ApLV from the southern Mediterranean. An ApLV-specific digoxygenin-labelled riboprobe was produced and used for molecular hybridisation tests. A survey of apricot orchards in Southern Italy did not show the presence of ApLV.

Dispensability of 3′ tRNA-like sequence for packaging cowpea chlorotic mottle virus genomic RNAs

The 3′ ends of three genomic RNAs (gRNAs) of cowpea chlorotic mottle virus (CCMV) terminate in a highly conserved tRNA-like structure (3′TLS). To examine the intrinsic role played the 3′TLS in packaging, the competence of each gRNA lacking the 3′ TLS (ΔTLS-gRNA) to interact with dissociated coat protein (CP) subunits and form virions was assayed in vitro. In contrast to the well established requirement for the participation of either viral 3′TLS or host-tRNAs in the assembly of RNA-containing virions in brome mosaic virus (BMV; Choi, Y, G., Dreher, T. W., Rao, A. L. N. 2002. tRNA elements mediate the assembly of an icosahedral RNA virus. Proc. Natl. Acad. Sci. 99, 655–660), CCMV CP does not require the presence of viral TLS in cis or in trans. Similar in vitro assembly assays showed that CCMV CP subunits also packaged BMV RNAs lacking 3′ TLS as well as two other non-bromoviral RNAs although with lesser efficiency. To characterize sequences of CCMV RNA3 (C3) required for packaging, a series deletions was engineered into C3 and their effect on virus assembly was examined. It was observed that, unlike BMV RNA3 whose packaging requires a bipartite signal (Choi, Y. G., Rao, A. L. N. 2003. Packaging of brome mosaic virus RNA3 is mediated through a bipartite signal. J. Virol. 77, 9750–9757), packaging of C3 is independent of either movement protein (MP) ORF or CP ORF or 3′ non-coding regions. Based on the differential prerequisites identified in this study for the assembly of BMV and CCMV, we hypothesize that the adaptive condition for movement in monocotyledonous host has made packaging a necessary co-requirement for BMV.

Detection of Poinsettia mosaic virus Infecting Poinsettias (Euphorbia pulcherrima) in Venezuela.

Poinsettia mosaic virus (PnMV), a putative member of the tymoviruses, was detected in several cultivars of vegetatively propagated poinsettias grown in commercial nurseries in Estado Miranda, Venezuela. Symptoms associated with the affected plants consisted of severe mottling and distortion of leaves and bracteoles. The suspect virus was mechanically transmitted to Nicotiana benthamiana. Leaf extracts and thin sections of affected leaf tissue were analyzed by transmission electron microscopy. Spherical virus particles (30 nm diameter) were observed in samples from symptomatic poinsettia plants. Ultrastructural analyses of virus-infected cells revealed aggregates of virus particles in the cytoplasm and central vacuole. The virus was purified twice from infected N. benthamiana, resulting in yields as high as 12 mg/100 g. Dissociated coat protein contained a single 24-kDa protein species. The virus was not serologically related to Carnation mottle, Bean rugose mosaic, Cowpea mosaic, Cucumber mosaic, Pea enation mosaic, Prunus necrotic ringspot, Apple mosaic, Tobacco streak, Maize rayado fino, Tomato ringspot, Bean southern mosaic, Sowbane mosaic, Andean potato latent, Belladona mottle, Scrophularia or Turnip yellow mosaic viruses, but did react positively in enzyme-linked immunosorbent assay and western blot analysis with antiserum (ATCC PVAS-476) to PnMV. Based on these results, the virus is considered to be PnMV. To our knowledge, this is the first report of PnMV infecting poinsettias in Venezuela.

The antigenicity of tobacco mosaic virus.

The antigenic properties of the tobacco mosaic virus (TMV) have been studied extensively for more than 50 years. Distinct antigenic determinants called neotopes and cryptotopes have been identified at the surface of intact virions and dissociated coat protein subunits, respectively, indicating that the quaternary structure of the virus influences the antigenic properties. A correlation has been found to exist between the location of seven to ten residue-long continuous epitopes in the TMV coat protein and the degree of segmental mobility along the polypeptide chain. Immunoelectron microscopy, using antibodies specific for the bottom surface of the protein subunit, showed that these antibodies reacted with both ends of the stacked-disk aggregates of viral protein. This finding indicates that the stacked disks are bipolar and cannot be converted directly into helical viral rods as has been previously assumed. TMV epitopes have been mapped at the surface of coat protein subunits using biosensor technology. The ability of certain monoclonal antibodies to block the cotranslational disassembly of virions during the infection process was found to be linked to the precise location of their complementary epitopes and not to their binding affinity. Such blocking antibodies, which act by sterically preventing the interaction between virions and ribosomes may, when expressed in plants, be useful for controlling virus infection.

A single amino acid substitution at N-terminal region of coat protein of turnip mosaic virus alters antigenicity and aphid transmissibility.

The antigenic activity of the N-terminal region of coat protein of turnip mosaic virus (TuMV) aphid transmissible strain 1 and non-transmissible strain 31 was examined by using a panel of monoclonal antibodies (MAbs) raised against the two virus strains as well as antisera raised against several synthetic peptides from the N-terminal region of the protein. The reactivity of these antibodies was tested in ELISA and in a biosensor system (BIAcore Pharmacia) using virus particles, dissociated coat protein and synthetic peptides as antigens. Substitution of a single amino acid at position 8 in the coat protein of TuMV strain 1 abolished any cross-reactivity between MAbs to strain 1 and the substituted peptide (strain 31) in ELISA although some cross-reactivity was apparent in BIAcore inhibition experiments. In reciprocal tests with MAbs to strain 31 no cross-reactivity with the heterologous peptide was detected in either type of assay. The amino acid residue present at position 8 appears to play a critical role in the binding capacity of MAbs specific for the N-terminal region of TuMV. Antiserum to a synthetic peptide corresponding to residues 1-14 of the protein of TuMV strain 1 was found to react strongly with dissociated coat protein and intact virus particles and was able to inhibit the aphid transmission of the virus. Antiserum to the corresponding peptide of strain 31 did not have this capacity.

Inhibition of in vitro Cotranslational Disassembly of Tobacco Mosaic Virus by Monoclonal Antibodies to the Viral Coat Protein

It has been shown by others that translation of tobacco mosaic virus (TMV) RNA may begin before uncoating of particles is complete. We provide evidence that this cotranslational disassembly can be inhibited by incubating TMV treated at pH 8 with certain monoclonal antibodies (MAbs) specific for TMV coat protein. The most efficient inhibition was achieved by incubation with some anti-metatope MAbs known to bind to the TMV extremity that becomes disassembled first and contains the 5' end of the RNA, as well as with some anti-cryptotope MAbs that bind only to dissociated coat protein subunits.

Possible members of the potyvirus group transmitted by mites or whiteflies share epitopes with aphid-transmitted definitive members of the group

There are at least ten viruses identified in the literature that resemble definitive potyviruses in having flexuous filamentous particles and inducing the formation of "pinwheel" cytoplasmic inclusions in infected cells but that are transmitted by eriophyid mites, whiteflies or soil fungi and not by aphids, the vectors of the definitive potyviruses. The taxonomic status of these viruses is uncertain at present. Using a broadly cross-reactive antiserum raised against the dissociated coat protein core (residues 68-285) of a definitive potyvirus (Johnsongrass mosaic virus), we have shown that wheat streak mosaic virus which is transmitted by mite and sweet potato mild mottle virus which is transmitted by whitefly have coat proteins that share epitopes with definitive potyviruses. This finding further supports their classification as definitive members of the potyvirus group. The cross-reactive antiserum used here had been shown previously to react with coat proteins of fifteen different definitive potyviruses. The antiserum did not react with coat proteins of potexviruses and tobamoviruses.

Production of cDNA Clones from the MAV Isolate of Barley Yellow Dwarf Virus

Summary A library of cDNA clones was produced from the approximately 6 kb RNA of the MAV isolate of barley yellow dwarf virus (BYDV) in bacteriophage λgtl 1, by a method that involved random priming and cloning ds cDNAs of between 1·0 and 2·5 kbp. Screening with antiserum to the dissociated coat protein of the MAV isolate showed that approximately 2·5% of the recombinants were capable of expressing this protein. After subcloning some inserts into the plasmid pUC18, restriction endonuclease mapping showed that they collectively represented at least 85% (a total of 5·1 kbp) of the BYDV genome. We did not attempt to determine which, if any, of the immunologically positive clones expressed the entire coat protein, but of the nine examined, all shared a region of approximately 1000 bp, located between 750 bp and 1750 bp from the 3′ terminus of the restriction map. Sequence homology among different isolates of BYDV was examined by using selected MAV cDNA clones as probes in viral nucleic acid hybridization studies. Hybridization specificity varied according to the origin of the clones within the BYDV genome. Those from the putative coat protein-coding region hybridized well only to the homologous MAV isolate; those from elsewhere hybridized also with another isolate from the same subgroup (P-PAV). No clones hybridized significantly to a third isolate (RPV), which is in another subgroup of BYDV. The sensitivity of detection was related to probe size; the larger clones detected as little as 70 µg of purified virus (1·4 ng/ml in a 50 µl sample), and with these the sensitivity of virus detection in plant extracts by dot-blot hybridization was greater than that of ELISA.

Some Physicochemical Properties of Maize Rayado Fino Virus

SUMMARY Maize rayado fino virus (RFV) was purified from infected maize (Zea mays) leaves. The virus had two components: empty shells and complete virus particles with sedimentation coefficients of 54S and 120S respectively, and buoyant densities of 1.28 and 1.46 g/ml in CsCl and 1.24 and 1.37 g/ml in Cs2SO4. Virus particles were stable from pH 7 to pH 4 but not at pH 3. Isoelectric focusing of purified empty capsids revealed a single band in the pH range of 6 to 6.2. RFV contained 33 to 36% of single-stranded (ss)RNA of mol. wt. approx. 2 × 106. Dissociated coat protein migrated in acrylamide gel electrophoresis as a main band with an average mol. wt. of 2.1 × 104. RFV shares several biological and physicochemical properties with oat blue dwarf virus. These viruses appear to represent a new group of small RNA viruses, transmitted propagatively by their cicadellid vectors.

Purification and characterization of potato leafroll virus

Potato leafroll virus (PLRV) was purified from infected potato ( Solanum tuberosum L.) with yields of 0.4–0.6 mg/kg of foliage. The virus sedimented as a single component of 127 S. An antiserum prepared against purified virus had a maximum titer of 1:1024 in agar gel double diffusion tests. PLRV had a buoyant density of 1.39 g/ml and an estimated nucleic acid content of 28%. The nucleic acid had a molecular weight of 2.0 × 10 6 and was degraded by RNase but not by DNase, indicating that the PLRV nucleic acid is RNA. The sedimentation coefficient of the RNA molecule was 34.5 S and after treatment with formaldehyde, 20.7 S. Dissociated coat protein migrated as a single band in polyacrylamide gel electrophoresis and the average subunit molecular weight was 26,300. PLRV should be considered a member of the luteovirus group.

A pH-dependent conformational change in the coat protein subunits from potato virus X

Both the circular dichroism and fluorescence spectra of the dissociated coat protein subunits from potato virus X changed substantially over the pH range 8 to 4, irreversible changes resulted below pH 4, with tyrosyl and tryptophanyl residues affected most. The titration curves show a p K a of about 5.6 and do not require cooperative interactions between the coat protein subunits, thus they are in marked contrast to titrations of tobacco mosaic virus A-protein. The spectra of the intact virus were little changed between pH 8 and 4 and suggested that the coat protein was locked into a conformation similar to that of the subunits in solution at pH 7. It is proposed that the pH induced conformational change is responsible for determining the acidic branch of the pH profile for reconstitution of potato virus X from its dissociated coat protein subunits and RNA.