Serological interrelationships in the turnip yellow mosaic virus group

The serological interrelationships between eleven viruses in the turnip yellow mosaic virus group (tymoviruses) were studied with 286 antisera from 48 rabbits. Antisera from different rabbits and from different bleedings of the same rabbit showed considerable differences in their homologous and heterologous reactivities. A classification based on average serological differentiation indices is proposed. There is a continuous range of serological relationships in the tymovirus group and subdivision into a turnip yellow mosaic virus and an Andean potato latent virus subgroup does not seem justified.     

Uncoating of turnip yellow mosaic virus RNA in vivo

Following mechanical inoculation of leaves with turnip yellow mosaic virus (TYMV), a significant proportion of the retained inoculum is uncoated within 45 sec, and the process is more or less complete after 2 min. At least 80-90% of the uncoating takes place in the epidermis. The application of virus to the intact leaf is essential for uncoating to occur. The uncoating process is not confined to plants which are known hosts for TYMV. The process gives rise to empty shells and low molecular weight protein. The empty shells probably lose a pentamer or hexamer of protein when the RNA is released. On a per cell basis the number of virus particles uncoated can be very large-approximately 106 particles per cell. The data suggest that at high inoculum concentrations most of the released RNA is inactivated on or within the epidermis.     

Translational studies with turnip yellow mosaic virus RNAs isolated from major and minor virus particles

The major and minor nucleoprotein components in turnip yellow mosaic virus (TYMV) preparations have been fractionated by four cycles of CsCl gradient equilibrium centrifugations. RNAs isolated from the various fractions were translated in a rabbit reticulocyte cell-free system. Three classes of RNAs were studied: genomic RNA with an Mr of 2.0 x 10(6), the subgenomic coat protein messenger with an (r) of 2.4 x 10(5) (H. Guilley and J. P. Briand, 1978, Cell15, 113-122) and RNA molecules of intermediate sizes. Genomic RNA is found in the major TYMV components. These particles do not contain the coat protein messenger. The latter messenger has mainly been detected in two types of minor components, those which have an RNA content exceeding that of the major TYMV virions and particles which contain the coat protein messenger as their sole RNA constituent. RNAs of intermediate sizes are found in minor components less dense than the major particles; the full-length translational products of these RNAs and of genomic RNA overlap with one another and share a common NH2 terminus. It is concluded that a number of intermediate-sized RNAs and the genomic RNA have a common site for initiation of translation located close to their 5'-termini.     

Behaviour of turnip yellow mosaic virus nucleoproteins under alkaline conditions

A modified CsCl density gradient procedure is described by which purified preparations of turnip yellow mosaic virus (TYMV) can be fractionated to give 12 nucleoprotein components. When the B1 nucleoprotein is subjected to pH 11.5-11.6 in 1 M KCl (conditions under which artificial empty protein shells are formed), the apparent radius (r) of the virus increases from about 14.6 to 15.2 nm within 30 sec. The increase in r is most probably due to swelling of the particles. Escape of the RNA from these particles is completed in 3-10 min, while RNA degradation continues for at least 30 min. On return to pH 7.0, r returns to about 14.6 nm. When any of the minor nucleoprotein fractions containing less than the full complement of RNA are subjected to the above conditions, the RNA does not escape from the particle. Experiments with the B0 components also showed that these particles did not give an increase in r, although the RNA was degraded to about the same extent as that of the B1 nucleoprotein.     

Characterization of the virus encoded subunit of turnip yellow mosaic virus RNA replicase

An antiserum raised against TYMV-RNA encoded protein P115 partially inhibits TYMV RNA replicase activity, demonstrating that this protein is involved in TYMV RNA synthesis. The detection of protein P115 by an antibody linked polymerase assay demonstrates that protein P115 is indeed a subunit of the TYMV RNA replicase, the enzyme known to synthesize viral RNA in infected Chinese cabbage. The use of translation products of other tymoviruses indicates that the serological relationship between the virus-encoded replicase subunits of these viruses and protein P115 is very weak at the best.     

The RNA of turnip yellow mosaic virus exhibits icosahedral order

Difference electron density maps, based on structure factor amplitudes and experimental phases from crystals of wild-type turnip yellow mosaic virus and those of empty capsids prepared by freeze-thawing, show a large portion of the encapsidated RNA to have an icosahedral distribution. Four unique segments of base-paired, double-helical RNA, one to two turns in length, lie between 33-A and 101-A radius and are organized about either 2-fold or 5-fold icosahedral axes. In addition, single-stranded loops of RNA invade the pentameric and hexameric capsomeres where they contact the interior capsid surface. The remaining RNA, not seen in electron density maps, must serve as connecting links between these secondary structural elements and is likely icosahedrally disordered. The distribution of RNA observed crystallographically appears to be in agreement with models based on biochemical data and secondary structural analyses.     

Mechanism for release of RNA from turnip yellow mosaic virus at high pH

The formation of empty protein shells from intact turnip yellow mosaic virus (TYMV) following treatment at alkaline pH has been investigated. The loss of viral RNA is accompanied by the release of a small amount of protein which has sedimentation properties expected for aggregates of about five to eight coat protein subunits. These results imply that the release of RNA at elevated pH occurs concomitantly with the removal of a discrete amount of protein from the virus shell. The protein released may be associated with the viral RNA since its sedimentation properties are altered following treatment with pancreatic ribonuclease.