Turnip yellow mosaic virus (TY MV) RNA escapes from viral capsids after freezing and thawing the virus, and the remaining capsids look very similar to natural capsids in the electron microscope after negative staining [KatouzianSafadi, M., Favre, A., and Haenni, A. L. (1980) Eur. J . Biochem. 112,478 -4861. In order to understand how an RNA of 2 x lo6 D a (33 % virus by weight) can escape from a compact protein shell we have compared artificial capsids formed after freezing TYMV and natural capsids produced in vivo in infected plants. We have used various physicochemical techniques including analytical ultracentrifugation, X-ray scattering, X-ray diffraction and orientation in a magnetic field. From the combination of these results we conclude that the escape of the RNA is accompanied by the formation of a hole in the capsid surface. The size of such a hole is estimated to 5 -9 coat protein subunits.The uncoated turnip yellow mosaic virus, induced in vitro by freezing and thawing, has been investigated in preceding work [I]. TYMV is an icosahedral virus containing a very high proportion of nucleic acid (33 % of the total weight). synthesis, as well as its ability to be aminoacylated by valine at the 'tRNA-like' structure at its 3' end.These results raise a new question: how can such a large piece of RNA escape from so compact a capsid as that of TYMV? Various possibilities can be suggested : (a) disruption of the viral capsid; (b) a rapid and significant modification of the capsid during the treatment, followed by return to the initial form; (c) eventual formation of a hole in the surface of the capsid by the loss of some coat protein subunits.Observation by electron microscopy after negative staining of frozen and thawed TYMV clearly eliminates the first hypothesis. Protein subunits remained associated in capsids and the artificial capsids formed by freezing (artificial capsids/freeze), show the same structural features as natural empty capsids. The choice of natural empty capsids (natural capsids) as a reference is justified by the following two pieces of information. (a) Matthews' experiments [lo] established clearly that in vivo natural capsids are formed directly from coat protein subunits; it is not clear whether they are precursors of the virions or a by-product of viral morphogenesis, but they are certainly not the result of uncoating of the virus. (b) Using Xray diffraction of single crystals Klug et al. [2] demonstrated that the architecture of natural capsids resembles perfectly that of TY MV.We compared artificial capsids/freeze and natural capsids using various physicochemical techniques, such as analytical ultracentrifugation, X-ray scattering and X-ray diffraction. We demonstrate that artificial capsids formed after RNA release contain a hole due to the lack of 5-9 protein subunits.

Protein MC1 is the major chromosomal protein in methanosarcinaceae. Using photochemical crosslinking on 5-bromouracil-substituted DNA, we identified the region of the protein that interacts with it. This region is located in the C-terminal part of the polypeptide chain, and the crosslinked amino-acids are in the region 74-86. Tryptophan 74 is one of the amino-acids crosslinked to DNA.

The uncoating of turnip yellow mosaic virus in vitro induced by freezing and thawing has been investigated using a variety of biochemical techniques including the aminoacylation capacity of the viral RNA and the ability of the RNA to stimulate protein synthesis, as well as physico‐chemical techniques such as sucrose gradient centrifugation and electron microscopy by negative staining. In particular a fluorescence test has been developed that can serve as a routine method to quantify the RNA liberated during the freeze‐thaw process. Escape of the viral RNA is a highly cooperative phenomenon: it depends critically on the virus concentration during freezing and thawing. Increasing the ionic strength or including foreign proteins diminish the escape of the RNA. The RNA is not damaged by this treatment and its liberation occurs without disruption of the viral capsid.