E.M.J. Jaspars

A functional equivalence of top component a RNA and coat protein in the initiation of infection by alfalfa mosaic virus

Abstract The nucleoproteins of alfalfa mosaic virus (AMV) were purified by zonal gradient centrifugation, and their homogeneity and RNA contents were established by gel electrophoresis. Each component was shown to contain one type of RNA of a specific length. Combinations of purified preparations were infectious only when the bottom component (B), middle component (M), and the top component b (Tb) nucleoproteins were present. However, no infectivity was found in a mixture of the three corresponding RNAs. Infection could be started by adding to this mixture a fourth RNA, the top component a RNA (Ta-RNA). The fact that a mixture of B, M, and Tb nucleoprotein is infectious while the corresponding nucleic acid mixture is not, is explained by the finding that the latter becomes infectious upon addition of low amounts of AMV coat protein. Evidence is presented that the activity of AMV coat protein is not due to protection of the RNAs against degrading enzymes. The function of Ta-RNA and its translation product—the coat protein—in the onset of infection is discussed.

The uncoating of alfalfa mosaic virus by its own RNA

Abstract Upon addition of alfalfa mosaic virus (AMV)-RNA to AMV particles the latter are rapidly changed into highly RNase-sensitive, slow-sedimenting structures. These structures still contain intact RNA but they band at a higher density in CsCl gradients than virus particles and lack the typical bacilliform virus structure. The disintegration of the particle structure is thought to be due to the free RNA combining with some of the protein subunits. The presence of protein subunits on RNA molecules was demonstrated by means of biological assay. The reaction is dependent on the concentrations of virus and RNA, and on the time of incubation. When particles of two other viruses were brought in contact with their own RNA or with AMV-RNA no reaction was observed. Addition of other RNAs to AMV particles also had no effect. Because of the specificity of the reaction, we postulate the existence of sites with a high affinity for AMV coat protein on the AMV-RNAs. These sites may be related to the biological role of the coat protein. The fact that the AMV structure becomes disturbed upon addition of its own RNA confirms the idea that in this virus particle the protein-protein interactions are of minor importance.

Purification and characterization of brome mosaic virus RNA-dependent RNA polymerase

RNA-dependent RNA polymerase (RdRp) was solubilized from cellular membranes of brome mosaic virus (BMV)-infected barley. The solubilized enzyme was subsequently purified by glycerol gradient centrifugation and DEAE ion-exchange chromatography. The purified enzyme proved to be highly stable and both dependent on and specific for BMV RNAs. The enzyme is inhibited by high template RNA concentrations. This inhibition indicates feedback regulation of minus-strand synthesis. The nonstructural viral protein P1 was found to be a component of the RdRp complex (R. Quadt, H.J.M. Verbeek, and E.M.J. Jaspars, 1988, Virology 165, 256-261). Using antibodies directed against a C-terminal peptide of P1 a complex of seven 125I-labeled proteins was precipitated. This indicates that the P1 protein is associated with at least six proteins in the infected cell.

Analysis of the protein composition of alfalfa mosaic virus RNA-dependent RNA polymerase

RNA-dependent RNA polymerase (RdRp) was solubilized and purified from cellular membranes isolated from alfalfa mosaic virus (AIMV)-infected tobacco by employing a procedure recently described for brome mosaic virus RdRp [R. Quadt and E.M.J. Jaspars, 1990, Virology 178, 189-194]. The purified AIMV RdRp is completely dependent on added template RNAs and exhibits a high degree of template specificity. Analysis of the protein composition of AIMV RdRp showed that AIMV-encoded proteins P1 and P2 and the coat protein (CP) are present in the active enzyme complex. Minus-strand synthesis by the AIMV RdRp is inhibited by AIMV CP. Native double-stranded AIMV RNAs are utilized as template for viral RNA synthesis by AIMV RdRp indicating that a helicase activity is present in the purified AIMV RdRp preparation.

The biological function and mutual dependence of bottom component and top component a of alfalfa mosaic virus

Abstract Purified bottom component RNA from one strain of alfalfa mosaic virus and purified top component a RNA from a different strain are not infectious or only minimally infectious when inoculated alone, but when a mixture of the two RNAs is inoculated, enhanced infectivity is found. The progenies of single lesions produced from combined inocula were isolated. These isolates show biological and physical properties from both initial strains. Their serological reactions and biological properties exclude the possibility that these isolates are mixtures. The bottom component RNA and the top component a were isolated from one of these isolates and combined with top component a and bottom component RNA, respectively, from the strains to which they belonged originally. Isolates obtained from these combinations are indistinguishable from the initial strains. Apparently the RNAs are not altered when they are replicated in the presence of an RNA from a different strain. These results indicate that at least two components are necessary for an infection with alfalfa mosaic virus. These components are mutually dependent.

Enhancement of infectivity by combination of two ribonucleic acid components from alfalfa mosaic virus

RNA was extracted from alfalfa mosaic virus 425. Infectivity was determined in density gradient fractions and in all possible combinations of two fractions. Certain combinations were remarkably infectious. This led to the purification through two cycles of sucrose gradient centrifugation of a 27 S and a 14 S RNA, from bottom component and top component a, respectively. The purified bottom component RNA had a low infectivity; when purified top component a RNA was added, infectivity increased considerably. Possible explanations for this effect are discussed.

Coat protein blocks the in vitro transcription of the virion RNAs of alfalfa mosaic virus

Using a method described by Miller and Hall [(1983) Virology 125, 236‐241] for brome mosaic virus, we succeeded in obtaining preparations of an alfalfa mosaic virus specified RNA polymerase that is totally dependent on added RNA. The enzyme makes full‐size transcripts on each of the four virion RNAs. Transcription is inhibited by small amounts of coat protein subunits. This is in accordance with a model proposed by Nassuth and Bol [(1983) Virology 124, 75‐85] which says that in a late stage of the infection cycle the coat protein is a factor that regulates the minus‐strand RNA synthesis in a negative sense.

Alfalfa mosaic virus hybrids constructed by exchanging nucleoprotein components

Abstract From two strains of alfalfa mosaic virus (strain 425 and yellow spot mosaic virus) which differ in component composition, serotype, symptoms induced in tobacco and bean, and sensitivity to cycloheximide, the three nucleoprotein components required for infectivity were purified, all possible combinations were made, and the progeny consisting of stable hybrids were characterized. This study resulted in the localization of the markers on the different component RNAs. Top component b RNA determines the serotype, the kind of symptoms on tobacco, the relative proportions of the components, and the sensitivity to cycloheximide. Middle component RNA determines the kind of symptoms on bean. No markers have been found on the bottom component RNA.

Evidence that alfalfa mosaic virus infection starts with three RNA-protein complexes

The tripartite genome of alfalfa mosaic virus (AMV) needs to be activated by its coat protein. To establish whether coat protein exerts its role by interacting structurally with one, two, or all three AMV-RNA species, the infectivity of mixtures of RNA-protein complexes with free RNAs were studied (Smit and Jaspars, Virology 104, 454-461, 1980). These studies were not fully conclusive since some redistribution of coat protein does occur as soon as RNAs are brought into contact with RNA-protein complexes. This problem was overcome by the use of ts mutants of AMV. Free ts coat protein subunits were not able to activate the wt genomic RNAs at 30 degrees in tobacco. Once complexed to the genomic RNAs at 0 degrees , the biological activity of the ts coat protein remained when assayed at the nonpermissive temperature. Apparently, the ts coat protein is inactivated during attempted redistribution at 30 degrees . Studies of mixtures of RNA-protein complexes and free RNA show that coat protein has to be present on at least two of the three RNA species. This result in combination with previous results (Smit and Jaspars, 1980) warrants the conclusion that the alfalfa mosaic virus infection starts with three RNA-protein complexes.

Plant Covirus Systems: Three-Component Systems

Recent developments have brought together viruses which were not previously supposed to bear any relationship except that they were simple RNA-containing plant viruses. These viruses are the spherical bromoviruses, once thought to have a very small genome (Bockstahler and Kaesberg, 1961); the small spherical cucumber mosaic virus and its relatives, which, in contrast to bromoviruses, are transmitted by aphids; the aphid-transmitted alfalfa mosaic virus, which is one of the very few small viruses with bacilliform particles; and a number of structurally not very well studied viruses among which are tobacco streak virus and citrus leaf rugose virus, which seem to have in common the possession of spherical virions of different size.