Potato virus A (PVA) particles were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the amino acids of the coat protein was determined to assess their in situ steric accessibility. This method revealed that the N-terminal 15 amino acids of the PVA coat protein and a region comprising amino acids 27 to 50 are the most accessible at the particle surface to labeling with tritium atoms. A model of the spatial arrangement of the PVA coat protein polypeptide chain within the virus particle was derived from the experimental data obtained by tritium bombardment combined with predictions of secondary-structure elements and the principles of packing α-helices and β-structures in proteins. The model predicts three regions of tertiary structure: (i) the surface-exposed N-terminal region, comprising an unstructured N terminus of 8 amino acids and two β-strands, (ii) a C-terminal region including two α-helices, as well as three β-strands that form a two-layer structure called an abCd unit, and (iii) a central region comprising a bundle of four α-helices in a fold similar to that found in tobacco mosaic virus coat protein. This is the first model of the three-dimensional structure of a potyvirus coat protein.

Influenza A virus matrix protein M1 is one of the most important and abundant proteins in the virus particles broadly involved in essential processes of the viral life cycle. The absence of high-resolution data on the full-length M1 makes the structural investigation of the intact protein particularly important. We employed synchrotron small-angle X-ray scattering (SAXS), analytical ultracentrifugation and atomic force microscopy (AFM) to study the structure of M1 at acidic pH. The low-resolution structural models built from the SAXS data reveal a structurally anisotropic M1 molecule consisting of a compact NM-fragment and an extended and partially flexible C-terminal domain. The M1 monomers co-exist in solution with a small fraction of large clusters that have a layered architecture similar to that observed in the authentic influenza virions. AFM analysis on a lipid-like negatively charged surface reveals that M1 forms ordered stripes correlating well with the clusters observed by SAXS. The free NM-domain is monomeric in acidic solution with the overall structure similar to that observed in previously determined crystal structures. The NM-domain does not spontaneously self assemble supporting the key role of the C-terminus of M1 in the formation of supramolecular structures. Our results suggest that the flexibility of the C-terminus is an essential feature, which may be responsible for the multi-functionality of the entire protein. In particular, this flexibility could allow M1 to structurally organise the viral membrane to maintain the integrity and the shape of the intact influenza virus.

A method of tritium introduction into different types of organic molecules that is based on the interaction of atomic tritium with solid organic target is described. Tritium atoms are formed on the hot W-wire, which is heated by the electric current. Such an approach is called “tritium thermal activation method”. Here we summarize the results of labeling globular proteins (lysozyme, human and bovine serum albumins); derivatives of pantothenic acid and amino acids; ionic surfactants (sodium dodecylsulfate and alkyltrimethylammonium bromides) and nonionic high-molecular weight surfactants – pluronics. For the first time it is observed that if the target-compound is fixed and its radicals are stable the specific radioactivity of the labeled product can be drastically increased (up to 400 times) when the target temperature is ca. 295 K compared with the results obtained at 77 K. The influence of labeling parameters as tritium gas pressure, exposure time and W-wire temperature was tested for each target temperature that results in the optimum labeling conditions with high specific radioactivity and chemical yield of the resulting compound.

Intact influenza A virions were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the matrix protein M1 was analyzed to determine the in situ accessibility of its tryptic fragments. These data were combined with the previously reported x-ray crystal structure of the M1 fragment 2–158 [Sha, B. & Luo, M. (1997) Nat. Struct. Biol. 4, 239–244] and the predicted topology of the C domain (159–252) to propose a model of M1 arrangement in the virus particle.