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With the aim of deriving a definitive phylogenetic tree for as many mammalian and avian herpesvirus species as possible, alignments were made of amino acid sequences from eight conserved and ubiquitously present genes of herpesviruses, with 48 virus species each represented by at least one gene. Phylogenetic trees for both single-gene and concatenated alignments were evaluated thoroughly by maximum-likelihood methods, with each of the three herpesvirus subfamilies (the Alpha-, Beta-, and Gammaherpesvirinae) examined independently. Composite trees were constructed starting with the top-scoring tree based on the broadest set of genes and supplemented by addition of virus species from trees based on narrower gene sets, to give finally a 46-species tree; branching order for three regions within the tree remained unresolved. Sublineages of the Alpha-and Betaherpesvirinae showed extensive cospeciation with host lineages by criteria of congruence in branching patterns and consistency in extent of divergence. The Gammaherpesvirinae presented a more complex picture, with both higher and lower substitution rates in different sublineages. The final tree obtained represents the most detailed view to date of phylogenetic relationships in any family of large-genome viruses.The Herpesviridae are a numerous family of large DNA viruses which have as their natural hosts humans, other mammals and vertebrates, and in one described case, an invertebrate (11, 16). The genomes of herpesviruses of mammals and birds clearly evince descent from a common ancestor, but with a great range of variation in terms of nucleotide substitution, gene content, and genomic arrangement (15). The Herpesviridae have been divided into three subfamilies, the Alpha-, Beta-, and Gammaherpesvirinae, initially from their distinct biological properties and latterly more precisely on the basis of their genomic attributes (16). Over the last two decades an extensive body of herpesvirus DNA sequence data has been built up, from single-gene analyses to studies of whole genomes (in the range 120 to 240 kbp). Phylogenetic studies using herpesvirus sequences have been undertaken, demonstrating clear division into the three subfamilies and, in some sublineages, patterns of divergence consistent with cospeciation of virus and host (7,9,13,14). Herpesviruses of fish (2, 3), amphibians (4), and invertebrates (A. J. Davison, personal communication) are only remotely related to the mammalian and avian viruses, while certain turtle viruses (the only reptile herpesviruses for which some sequence is known) probably group with the mammalian and avian viruses (18).We describe in this report a major update of herpesvirus phylogenetic analysis, using the greatly increased number of gene sequences now available from a wide range of mammalian and avian herpesviruses, and enabled by advances both in processing power of modern computers and in methods for analysis of relationships among gene sequences. We aimed to produce by good current practice a single phylogenetic tree that would...

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Nucleocapsid (N) proteins from representative viruses of three genera within the Paramyxoviridae were expressed in insect cells using recombinant baculoviruses. RNA-containing structures, which appear morphologically identical to viral nucleocapsids, were isolated and subsequently imaged under a transmission electron microscope. Analysis of these images revealed marked differences in nucleocapsid morphology among the genera investigated, most notably between viruses of the Paramyxovirinae and the Pneumovirinae subfamilies. Helical pitch measurements were made, revealing that measles virus (MV, a Morbillivirus within the subfamily Paramyxovirinae) N protein produces helices that adopt multiple conformations with varying degrees of flexibility, while that of the Rubulavirus simian virus type 5 (SV5, subfamily Paramyxovirinae) produces more rigid structures with a less heterogeneous pitch distribution. Nucleocapsids produced by respiratory syncytial virus (RSV, subfamily Pneumovirinae) appear significantly narrower than those of MV and SV5 and have a longer pitch than the most extended form of MV. In addition to helical nucleocapsids, ring structures were also produced, image analysis of which has demonstrated that rings assembled from MV N protein consist of 13 subunits. This is consistent with previous reports that Sendai virus nucleocapsids have 13n07 subunits per turn. It was determined, however, that SV5 subnucleocapsid rings have 14 subunits, while rings derived from the radically different RSV nucleocapsid have been found to contain predominantly 10 subunits.

the asymmetry and then the width of the rocking curve by a simple rotation of the sample; (ii) the use of very inclined reflecting planes in a tilted and symmetric geometry enables a decrease in the thermal load on monochromators, since the trace of the incident beam on the surface of the crystal is then much larger than in the case of symmetric reflections on the surface (Macrander et al., 1992 [Giacovazzo, Guagliardi, Ravelli & Siliqi (1994). Z. Kristallogr. 209,[136][137][138][139][140][141][142]. Indeed, the amount of information available leads to a signal-to-noise ratio close to unity; consequently, the correct solution, even if attained, cannot be recognized among the trial solutions. Attention is here focused onto the case in which diffraction data of one isomorphous derivative are additionally available. It is shown that in such a case direct ab initio solution of protein structures is feasible. Tests based on calculated diffraction data suggest the procedure to follow for a possible success.

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able difference in the cobalt environment between the two states and that the conformation observed in the crystal structure is also representative for the aqueous solution.Very pronounced differences exist in the near-edge region between cobalt(II). cobalt(III) and alkyl (e.g. methyl) cobalamins. For several Sporomusa Ovata proteins canying a p-cresolylcobamide cofactor, strong indications could be derived from the EXAFS and near-edge regions of the X-ray absorption spectrum that the cofactor occurs in the methylated form under oxidative ambient conditions. MS02.07.03 HOW COENZY!VIE B12RADICALS ARE GEN-ERATED: METHYLMALONYL-COA MUTASE AT 2A RESOLUTION. P.R. Evans and F. Mancia-MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK This structure shows how the enzyme catalyses the formation of the adenosyl radical from coenzyme B 12· MethylmalonylCoA mutase is a member of a class of enzymes that bind the cobalt-containing Y-deoxyadenosyl-cobalamin cofactor (coenzyme B u) and catalyse 1.2 intramolecular remTangements in which a hydrogen atom is exchanged with a group on an adjacent carbon. Methylmalonyl-CoA mutase catalyses the interconversion between (2R)-methylmalonyl-CoA and succinyl-CoA. Such reactions involve radical intermediates: the initial radical arises from the homolysis of the unique Co-C bond of coenzyme B 12 , amongst the very few metal-carbon bond known in nature. A long-standing puzzle has been how the protein weakens this Co-C bond towards homolytic cleavage.Methylmalonyl-CoA mutase is the only adenosylcobalamindependent enzyme present in both animals and microrganisms. In the bacterium Propionibacterium slzermanii it is a key enzyme in the fermentation to propionate, whilst in mammalian liver it is responsible for the conversion of odd-chain fatty acids and branched-chain amino acids to succinyl-CoA for further degradation. The Pshennanii enzyme is an u.~ heterodimer of l50kD total molecular weight with one active site per dimer. We have solved the structure of the ternm·y complex between the recombinant protein expressed in E.coli. coenzyme B 1 2, and the partial substrate desulpho-CoA (coenzyme A with the final sulphur atom replaced by a hydrogen).Each subunit has essentially a two domain architecture. In the catalytic u chain, the B 1 2 is sandwiched between a C-terminal t1avodoxin a/~ domain and anN-terminal ~/a TIM bmTel. A conserved histidine from the t1avodoxin-like domain provides axial coordination to the cobalt atom in a very similm· way to that seen in methionine svnthase. The histidine-cobalt distance is verv long (2.5A compmed to 1.95-2.2A in free cobalamins), suggesti~g that the enzyme positions the histidine in order to weaken the Co-C of the cofactor and favour the forn1ation of the initial radical The substrate is bound through a hole along the axis of the barrel. pointing into a deeply buried active site on the 5'-deoxyadenosyl or catalytic side of the B 12·MS02.07.04 EXAFS ANALYSES OF MANGAI\'ESE EN-ZYMES James E. Penner-Hahn I, Timothy L. Stemmler!, Pamela J....