By immunoaffinity column chromatography, we have purified two RNA polymerase complexes, the transcriptase and replicase, from vesicular stomatitis virus-infected baby hamster kidney cells. The transcriptase is a multiprotein complex, containing the virus-encoded RNA polymerase L and P proteins, and two cellular proteins, translation elongation factor-1␣ and heat-shock protein 60. In addition, the complex contains a submolar amount of cellular mRNA cap guanylyltransferase. The replicase, on the other hand, is a complex containing the viral proteins, L, P, and the nucleocapsid (N), but lacking elongation factor-1␣, heat-shock protein 60, and guanylyltransferase. The transcriptase complex synthesizes capped mRNAs and initiates transcription at the first gene (N) start site, whereas the replicase complex initiates RNA synthesis at the precise 3 end of the genome RNA and synthesizes encapsidated replication products in the presence of the N-P complex. We propose that two RNA polymerase complexes that differ in their content of virally and host-encoded proteins are separately responsible for transcription and replication of vesicular stomatitis virus genome RNA. Ahallmark of all nonsegmented negative-strand (ns)RNA viruses (mononegavirales order) such as rabies, measles, Sendai, parainfluenza, Ebola, and many others is that mature virions contain a virally encoded RNA-dependent RNA polymerase (referred to as transcriptase), which transcribes the negative-sense genome RNA into discrete mRNAs on entry into the cell to initiate infection (1, 2). To replicate the genome RNA, the transcriptase is hypothesized to be modified by an unknown mechanism to form a replicase that synthesizes the full-length positive-strand genome RNA (3, 4); the replicase then synthesizes multiple copies of nsRNA using the positive RNA as template. During each step of the replication reaction, both plusand minus-strand RNAs are concomitantly enwrapped by the newly synthesized nucleocapsid (N) protein (5, 6) to form the ribonucleoprotein (RNP) complex. The composition of the replicase and the process of replication remain an enigma.We have been studying vesicular stomatitis virus (VSV) as a prototypic nonsegmented negative-strand RNA virus to probe the structure and function of transcriptase and the putative replicase to delineate the mechanism of transcription and replication of this class of viruses. VSV, like rabies virus, belongs to the rhabdovirus family and contains a single-strand genome RNA of negative polarity (Ϸ11.2 kb long) tightly associated with the N protein and helically packed within a bullet-shaped shell that is surrounded by the host-cell plasma membrane (3, 4). Two membrane proteins, spike glycoprotein (G) and matrix (M) protein, are located outside and inside of the membrane, respectively. Two proteins are associated with the helical RNP, the RNA polymerase large (L) (241 kDa) and phosphoprotein (P) (29 kDa) (7), which together constitute the active transcriptase holoenzyme complex (3, 4). It is generally believed that the L pro...

Fructosamine oxidases (FAOX) catalyze the oxidative deglycation of low molecular weight fructosamines (Amadori products). These proteins are of interest in developing an enzyme to deglycate proteins implicated in diabetic complications. We report here the crystal structures of FAOX-II from the fungi Aspergillus fumigatus, in free form and in complex with the inhibitor fructosyl-thioacetate, at 1.75 and 1.6 Å resolution, respectively. FAOX-II is a two domain FAD-enzyme with an overall topology that is most similar to that of monomeric sarcosine oxidase. Active site residues Tyr-60, Arg-112 and Lys-368 bind the carboxylic portion of the fructosamine, whereas Glu-280 and Arg-411 bind the fructosyl portion. From structure-guided sequence comparison, Glu-280 was identified as a signature residue for FAOX activity. Two flexible surface loops become ordered upon binding of the inhibitor in a catalytic site that is about 12 Å deep, providing an explanation for the very low activity of FAOX enzymes toward protein-bound fructosamines, which would have difficulty accessing the active site. Structure-based mutagenesis showed that substitution of Glu-280 and Arg-411 eliminates enzyme activity. In contrast, modification of other active site residues or of amino acids in the flexible active site loops has little effect, highlighting these regions as potential targets in designing an enzyme that will accept larger substrates.Fructosamines are formed by condensation of glucose with the amino group of amino acids or proteins. Fructosamines are formed spontaneously, i.e. non-enzymatically, at a rate that depends on temperature, sugar anomerization rate, concentration, and turnover rate of the target proteins. In medicine, protein-bound fructosamines (also named glycated proteins) have attracted much attention since their formation is increased in diabetes and taken to be in part responsible for diabetic complications. Fructosamines are relatively unstable compounds and are precursors for advanced glycation end products (AGEs), 5 some of which cause proteins cross-linking, extracellular matrix stiffening, and activation of the receptor for AGEs (RAGEs) (1, 2). As an example, the fructosamine-derived lysine-arginine cross-link glucosepane is to date the single major cross-link known to accumulate in human collagen in diabetes and aging (3).Several years ago our laboratory initiated a search for deglycating enzymes in soil organisms with the goal of finding enzymes that could deglycate proteins. In doing so we found enzymes with "amadoriase" activity toward low molecular weight substrates in soil samples, first in Pseudomonas sp. (4) and later in Aspergillus fumigatus (5-7). The latter turned out to have similar properties with the enzyme first published by Horiuchi et al. (8) under the name fructose amino acid oxidase (EC 1.5.3). Considerable work has since been published on these enzymes, which we are referring to in this work under the generic name fructosamine oxidases (FAOX).In addition to FAOX enzymes, two different families o...

Genome segment 9 of the 11-segment RNA genomes of three cytoplasmic polyhedrosis virus (CPV) isolates from Antheraea mylitta (AmCPV), Antheraea assamensis (AaCPV) and Antheraea proylei (ApCPV) were converted to cDNA, cloned and sequenced. In each case, this genome segment consists of 1473 nucleotides with one long ORF of 1035 bp and encodes a protein of 345 amino acids, termed NSP38, with a molecular mass of 38 kDa. Secondary structure prediction showed the presence of nine α-helices in the central and terminal domains with localized similarity to RNAbinding motifs of bluetongue virus and infectious bursal disease virus RNA polymerases. Nucleotide sequences were 99n6 % identical between these three strains of CPVs, but no similarity was found to any other nucleotide or protein sequence in public databases. The ORF from AmCPV cDNA was expressed as a His-tagged fusion protein in E. coli and polyclonal antibody was raised against the purified protein.

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