Site-specific protease activity of the carboxyl-terminal domain of Semliki Forest virus replicase protein nsP2.

The virus-specific components (nsP1-nsP4) of Semliki Forest virus RNA polymerase are synthesized as a large polyprotein (P1234), which is cleaved by a virus-encoded protease. Based on mutagenesis studies, nsP2 has been implicated as the protease moiety of P1234. Here, we show that purified nsP2 (799 amino acids) and its C-terminal domain Pro39 (amino acids 459-799) specifically process P1234 and its cleavage intermediates. Analysis of cleavage products of in vitro synthesized P12, P23, and P34 revealed cleavages at sites 1/2, 2/3, and 3/4. The cleavage regions of P1/2, P2/3, and P3/4 were expressed as thioredoxin fusion proteins (Trx12, Trx23, and Trx34), containing approximately 20 amino acids on each side of the cleavage sites. After exposure of these purified fusion proteins to nsP2 or Pro39, the reaction products were analyzed by SDS-polyacrylamide gel electrophoresis, mass spectrometry, and amino-terminal sequencing. The expected amino termini of nsP2, nsP3, and nsP4 were detected. The cleavage at 3/4 site was most efficient, whereas cleavage at 1/2 site required 5000-fold more of Pro39, and 2/3 site was almost resistant to cleavage. The activity of Pro39 was inhibited by N-ethylmaleimide, Zn(2+), and Cu(2+), but not by EDTA, phenylmethylsulfonyl fluoride, or pepstatin, in accordance with the thiol proteinase nature of nsP2.

Cloning and expression of a new site-specific methyltransferase M.SscL1I from Staphylococcus sp. L1.

The gene of the new site-specific methyltransferase M.SscL1I belonging to the same modification-restriction system as the previously described by us site-specific endonuclease SscL1I has been cloned from the natural strain Staphylococcus sp. L1. A plasmid to express the methylase gene under control of the T7 phage-specific promotor has been constructed. Conditions were found to express the recombinant methylase M.SscL1I and to purify it to near homogeneity. It is shown that the methylase modifies the adenine base in the recognition site 5;-GANTC-3;.

Identification of a novel function of the alphavirus capping apparatus. RNA 5'-triphosphatase activity of Nsp2.

Both genomic and subgenomic RNAs of the Alphavirus have m(7)G(5')ppp(5')N (cap0 structure) at their 5' end. Previously it has been shown that Alphavirus-specific nonstructural protein Nsp1 has guanine-7N-methyltransferase and guanylyltransferase activities needed in the synthesis of the cap structure. During normal cap synthesis the 5' gamma-phosphate of the nascent viral RNA chain is removed by a specific RNA 5'-triphosphatase before condensation with GMP, delivered by the guanylyltransferase. Using a novel RNA triphosphatase assay, we show here that nonstructural protein Nsp2 (799 amino acids) of Semliki Forest virus specifically cleaves the gamma,beta-triphosphate bond at the 5' end of RNA. The same activity was demonstrated for Nsp2 of Sindbis virus, as well as for the amino-terminal fragment of Semliki Forest virus Nsp2-N (residues 1-470). The carboxyl-terminal part of Semliki Forest virus Nsp2-C (residues 471-799) had no RNA triphosphatase activity. Replacement of Lys-192 by Asn in the nucleotide-binding site completely abolished RNA triphosphatase and nucleoside triphosphatase activities of Semliki Forest virus Nsp2 and Nsp2-N. Here we provide biochemical characterization of the newly found function of Nsp2 and discuss the unique properties of the entire Alphavirus-capping apparatus.

Site-specific endonuclease SscL1 I from strain Staphylococcus species L1.

A site-specific endonuclease SscL1 I preparation has been isolated and purified to near homogeneity from the strain Staphylococcus sp. L1 without admixtures of other nuclease activity. DNA cleavage proceeds according to the scheme: 5'-G down arrow ANTC-3' 3'-CTNA up arrow G-5', and thus the isolated enzyme is an isoschizomer of restriction endonuclease HinfI and belongs to the second class of restriction endonucleases. SscL1 I works over a broad range of temperature and pH. The enzyme is characterized by high stability during storage.

Management of extensive burns in children.

2723 children were treated in the Burn Department of the Pediatric Clinical Hospital No. 9, Moscow, during 3 years (1986-1988), among them 1465 children being under 3 years of age. Since 1985, the Department has been using the beds of "Clinitron" type, 79 children (3 months-14 years of age) with extensive burns having been treated on the Clinitron beds with air-fluidized pillows. During the treatment, the children received the usual therapy accepted in the Department. The use of the air-pillows beds promoted rapid drying of the wounds, accelerated epithelialization of the superficial burns, reduced the periods of the wound preparation for autodermoplasty for deep burns, prevented rejection and lysis of the replanted grafts.