Structure of Sesbania mosaic virus at 3 A resolution.

Sesbania mosaic virus (SMV) is an isometric, ss-RNA plant virus found infecting Sesbania grandiflora plants in fields near Tirupathi, South India. The virus particles, which sediment at 116 S at pH 5.5, swell upon treatment with EDTA at pH 7.5 resulting in the reduction of the sedimentation coefficient to 108 S. SMV coat protein amino acid sequence was determined and found to have approximately 60% amino acid sequence identity with that of southern bean mosaic virus (SBMV). The amino terminal 60 residue segment, which contains a number of positively charged residues, is less well conserved between SMV and SBMV when compared to the rest of the sequence. The 3D structure of SMV was determined at 3.0 A resolution by molecular replacement techniques using SBMV structure as the initial phasing model. The icosahedral asymmetric unit was found to contain four calcium ions occurring in inter subunit interfaces and three protein subunits, designated A, B and C. The conformation of the C subunit appears to be different from those of A and B in several segments of the polypeptide. These observations coupled with structural studies on SMV partially depleted of calcium suggest a plausible mechanism for the initiation of the disassembly of the virus capsid.

Assessment of the autonomy of replicative and structural functions encoded by the luteo-phase of pea enation mosaic virus.

The genome of pea enation mosaic virus (PEMV) is composed of two taxonomically unrelated RNAs, interacting to create what has traditionally been considered a bipartite virus. The cohesiveness of this interaction was assessed by examining the autonomy of each RNA in viral replication, coat protein expression and systemic invasion. Using a pea protoplast system, in vitro transcripts of RNA1 were found to be capable of initiating RNA2-independent replication, including the formation of the distinctive nuclear membrane-based replication complex associated with wild-type PEMV infection. Western blotting and electron microscopic analysis demonstrated that the synthesis of the RNA1-encoded coat protein, as well as virion assembly, was also independent of RNA2-directed functions. Mechanical inoculations with transcripts of RNA1 failed to establish a systemic RNA1 infection, whereas inoculations with RNA2 were able to establish a largely asymptomatic systemic infection. Combined inoculum containing RNA1 and RNA2 transcripts were able to recreate wild-type PEMV symptomatology, demonstrating the dependence of RNA1 on RNA2 for mechanical passage. With the notable exception of the adaptation of PEMV to establish a true systemic invasion, these data further strengthen the analogy between PEMV and the helper-dependent complexes associated with members of the luteovirus group.

Failure of long-distance movement of southern bean mosaic virus in a resistant host is correlated with lack of normal virion formation.

Sunn-hemp mosaic tobamovirus (SHMV) facilitated the spread of the cowpea strain of southern bean sobemovirus (SBMV-C) only in inoculated leaves of common bean (Phaseolus vulgaris L. cv. Bountiful), a resistant host for SBMV-C. Tissue prints of bean primary leaves doubly inoculated with SHMV and SBMV-C, developed by Western blotting, showed the presence of the SBMV-C capsid antigen in the mesophyll and epidermis, but no antigen was detected in the conducting bundles. Typical SBMV-C virions were not seen in electron micrographs of immunogold-labelled mesophyll cells; instead, specifically labelled, amorphous protein clumps were found in the vacuole. Particles of smaller diameter than that of typical SBMV-C virions were specifically trapped by SBMV antibodies following immunosorbent electron microscopy of extracts from doubly infected leaves. SBMV-C coat protein from infected Vigna unguiculata L. (cowpea) and bean plants showed no difference in its mobility following electrophoresis in denaturing SDS-polyacrylamide gels. Lack of efficient assembly of SBMV-C virions does not impede cell-to-cell movement of the virus in doubly infected leaves of bean, yet it is probably an important factor in determining the inability of SBMV-C to move into and/or through the vascular system of this host.

Signal for potyvirus-dependent aphid transmission of potato aucuba mosaic virus and the effect of its transfer to potato virus X.

A British isolate of potato aucuba mosaic potexvirus (PAMV) was transmitted by aphids (Myzus persicae) which had fed previously on a source of potato Y potyvirus (PVY). Nucleotide sequence analysis of the PAMV coat protein gene indicated that amino acid residues 14 to 16 from the N terminus of the coat protein have the sequence DAG, which is also found in the coat proteins of potyviruses and is required for their aphid transmissibility. A recombinant virus isolate (TXPA7) was produced in which a segment of the coat protein gene of PAMV encoding the 40 N-terminal amino acids was inserted in the genome of potato X potexvirus (PVX) in place of the segment encoding the 28 N-terminal amino acids of PVX coat protein. This isolate, and a second similar recombinant (TXPA5) in which the DAG motif was changed to YTS, were mechanically transmissible to intact plants, in which they caused slightly milder symptoms than PVX. Particles of TXPA7 reacted in immunosorbent electron microscopy with PVX- and PAMV-specific antibodies and so were antigenically distinguishable from PAMV and PVX particles, which reacted only with their homologous antibody, and from TXPA5 particles, which reacted only with the PVX antibody. Recombinant TXPA7 was transmitted by aphids that had already fed on a source of PVY whereas TXPA5 and PVX were not. TXPA7 was not transmitted by aphids that had not fed on a PVY source. It is concluded that (i) the potyvirus-dependent aphid transmissibility of PAMV results from possession of a domain which includes the DAG motif and is located near the N terminus of the virus coat protein, and (ii) potyvirus-dependent aphid transmissibility can be conferred on PVX, a non-aphid-borne potexvirus, by substituting this domain for the N-terminal part of its coat protein.

[Optimization of gold immunolabeling techniques on ultrathin slides obtained from samples fixed with glutaraldehyde and osmium tetroxide and enclosed in epoxy resin]. / Optimisation de la technique de l'immunomarquage à l'or sur sections ultrafines réalisées à partir d'échantillons fixés à la glutaraldéhyde et au tétroxyde d'osmium et inclus dans une époxy-résine.

An immunogold labeling technique was carried out on plants infected with CMV, fixed with glutaraldehyde and osmium tetroxide and embedded in araldite CY 212. The effect of the type of support-film used, the resin and manipulations of the grids during immunogold steps, were studied and are discussed. The antigenic activity of virus was restored by treating the sections with sodium metaperiodate. The very high non-specific reactions observed with the support-film or with the resin were eliminated by adding powdered skimmed milk or non-purified albumin into the buffers. Purified bovine serum albumin (grade V) or chicken albumin (grade III to V) were inefficient in reducing this non-specific background.

Serotype A and B strains of bean common mosaic virus are two distinct potyviruses.

The serological relationships among strains of bean common mosaic virus (BCMV) (genus Potyvirus, family Potyviridae) were investigated by testing 13 isolates of the 10 known BCMV pathotypes with two monoclonal antibodies and six antisera to BCMV strains. In addition, other properties of serologically distinct BCMV strains were compared. Two groups of BCMV strains were obtained by ELISA and Western blot serology: serotype A contained the BCMV strains NL3, NL5, and NL8 and serotype B contained the BCMV strains NL1, NL2, NL4, NL6, US4, NL7, NY15, and Fla. SDS polyacrylamide gel electrophoresis and Western blotting of freshly purified preparations, and of extracts from leaves infected with eleven BCMV strains showed that the apparent molecular mass of the capsid protein of the serotype A isolates NL3, NL5, and NL8 are lower (about M(r) 33,000) than those of the serotype B isolates (M(r) 34,500 to 35,000). The normal lengths of the particles of the serotype A isolates were shorter (810-818 nm) than those of most isolates (except NL6 and NY15) of serotype B (847-886 nm). All isolates studied induced cytoplasmic pinwheel and scroll inclusions. Cells infected with serotype A isolates contained a specific type of proliferated endoplasmic reticulum which was never found in cells infected with serotype B isolates. The capsid protein gene of a representative member of each serotype was cloned and sequenced. Molecular mass calculations based upon nucleotide sequence-derived amino acid sequences yielded M(r) of 29,662 and 32,489 for the capsid proteins of the serotype A isolate NL8 and the serotype B isolate NL4, respectively. Comparison of the coat-protein sequences showed considerable differences at the N-termini whereas the core regions and the C-termini appeared to be highly conserved. Marked differences were also observed within the 3' non-coding regions of cloned cDNAs of NL 4 and NL 8. The striking differences between the two serotypes of BCMV strongly suggest that they be classified as two distinct potyviruses which naturally infect Phaseolus beans.

Characterization of bean mild mosaic virus: particle morphology, composition and RNA cell-free translation.

Virions of bean mild mosaic virus (BMMV) are built of 180 subunits of a single protein species of MW 40 x 10(3) [coat protein CP], packed into a T = 3 surface lattice. The capsomers on the five-fold symmetry axes protrude 2-3 nm from the particle surface. The virions encapsidate genome-size [approximately 4,200 nucleotides (nt)] as well as some heterogeneous RNAs of subgenomic size approximately 1,000-2,000 nt. In cell-free systems from Krebs-2 ascites cell extracts and rabbit reticulocyte lysates, genome-size RNA directed the synthesis predominantly of two polypeptides of MW 27 x 10(3) and 79 x 10(3) while the third major BMMV-specific polypeptide (MW 40 x 10(3), putative CP) seemed to be encoded by a shorter messenger RNA. The 'cap' analogue, m7GDP, partially inhibited BMMV RNA in vitro translation, suggesting that at least part of the BMMV-specific RNAs are capped. Oligo (dT)-cellulose column chromatography data suggested that poly(A)-tracts are absent from the BMMV genome. The data obtained confirm the previous classification of BMMV within the carmovirus group.

Constraints on the assembly of spherical virus particles.

Examination of protein structure shows that it is not possible to deform protein domains to the extent required by the Caspar-Klug quasi-symmetry surface lattices for the description of viral capsids (D. L. D. Caspar and A. Klug (1962). Cold Spring Harbor Symp. Quant. Biol. 27, 1-24). However, flexibility in proteins can be achieved by a number of ligand-induced events. One type of alteration is that of quaternary structural changes in oligomers. This strategy has been used by southern bean mosaic virus and tomato bushy stunt virus where dimers attain two different states in the assembled capsid. Alterations of subunit interactions can be induced by association with RNA, cations, or other oligomeric units and, hence, successful assembly results from a stepwise aggregation. The nature of the oligomers (dimers, trimers, or pentamers) must be the underlying reason for the occurrence of the Caspar-Klug lattices and the organization into icosahedra. An analysis of the surface lattices shows which types of oligomers will be necessary for assembly.

Mechanisms underlying systemic invasion of pea plants by pea enation mosaic virus.

Leaves of Pisum sativum L.cv. 'Perfected Wales' were inoculated with pea enation mosaic virus, at the 3rd and 4th nodes as early as size permitted. Systemic virus spread was then measured over a period of approximately 9 days by assaying the infectivity of 1-cm stem pieces above the 4th and below the 3rd node. The presence of virus antigen and double-stranded (ds) RNA in these stem pieces was measured by radioimmunoassay over the same period. Relative infectivity and amounts of dsRNA in infected tissue were lowest approximately 3 days after inoculation and sharply increased thereafter. A transient plasmodesmatal abnormality was observed 4-6 days after inoculation. The transient nature of the abnormality, which was preceded (24 h) by low infectivity and dsRNA concentrations, may indicate a plant defense reaction that is subsequently overcome. The rise in dsRNA concentration, starting the 4th day after inoculation, coincided with the increase of vesicular material (cytopathological structures) in the phloem of infected plants. Vesicular material was found in sieve elements that were mature at the time of inoculation. Their presence in this tissue can only be explained by transport of the vesicles from infected sites.