Crystal Structure of the Marburg Virus VP35 Oligomerization Domain.

Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (genera Marburgvirus and Ebolavirus, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOV VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV. IMPORTANCE: Marburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the mechanisms governing VP35's functions and inform the design of therapeutics.

Radioprotective effects produced by the condensation of plasmid DNA with avidin and biotinylated gold nanoparticles.

The treatment of aqueous solutions of plasmid DNA with the protein avidin results in significant changes in physical, chemical, and biochemical properties. These effects include increased light scattering, formation of micron-sized particles containing both DNA and protein, and plasmid protection against thermal denaturation, radical attack, and nuclease digestion. All of these changes are consistent with condensation of the plasmid by avidin. Avidin can be displaced from the plasmid at higher ionic strengths. Avidin is not displaced from the plasmid by an excess of a tetra-arginine ligand, nor by the presence of biotin. Therefore, this system offers the opportunity to reversibly bind biotin-labeled species to a condensed DNA-protein complex. An example application is the use of biotinylated gold nanoparticles. This system offers the ability to examine in better detail the chemical mechanisms involved in important radiobiological effects. Examples include protein modulation of radiation damage to DNA, and radiosensitization by gold nanoparticles.

Free terminal amines in DNA-binding peptides alter the product distribution from guanine radicals produced by single electron oxidation.

PURPOSE: Electron deficient guanine radical species are major intermediates produced in DNA by the direct effect of ionizing irradiation. There is evidence that they react with amine groups in closely bound ligands to form covalent crosslinks. Crosslink formation is very poorly characterized in terms of quantitative rate and yield data. We sought to address this issue by using oligo-arginine ligands to model the close association of DNA and its binding proteins in chromatin. MATERIALS AND METHODS: Guanine radicals were prepared in plasmid DNA by single electron oxidation. The product distribution derived from them was assayed by strand break formation after four different post-irradiation incubations. RESULTS: We compared the yields of DNA damage produced in the presence of four ligands in which neither, one, or both of the amino and carboxylate termini were blocked with amides. Free carboxylate groups were unreactive. Significantly higher yields of heat labile sites were observed when the amino terminus was unblocked. The rate of the reaction was characterized by diluting the unblocked amino group with its amide blocked derivative. CONCLUSION: These observations provide a means to develop quantitative estimates for the yields in which these labile sites are formed in chromatin by exposure to ionizing irradiation.

Modeling the influence of histone proteins on the sensitivity of DNA to ionizing radiation.

The DNA-binding proteins that are present in chromatin significantly affect the sensitivity of cells to ionizing radiation and to the radiation chemistry of DNA damage. The interaction between protein and DNA modifies the radiation chemistry of the latter. To model these processes, we have examined the effects of ionizing radiation on the minichromosome form of SV40 (which contains histone proteins arranged in nucleosomes) and also on plasmid DNA in the presence of lysozyme. Although high concentrations of lysozyme can bring about an extensive radioprotection by condensation of the plasmid, at lower levels it still produces significant radioprotective effects under conditions where this associative phase separation does not take place. The presence of histones or of lysozyme decreases the yield of modified guanines produced by ionizing radiation. Comparison with previous observations made with oligopeptides suggests that the mechanism responsible is electron donation to guanyl radicals in the DNA by tryptophan and tyrosine residues in the proteins. However, there was no evidence for DNA-protein crosslink formation.