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The mechanism of supercoil release was investigated by studying single-turnover experiments performed with supercoiled plasmid DNA under conditions in which topoisomerase cleaves at a single site per DNA molecule. Viccinia type I topoisomerase was shown to follow a free rotation mechanism for removing supercoils from DNA. An average of five supercoils removed per cleavage event was observed.

Vaccinia virus is a complex DNA virus that exhibits significant genetic and physical autonomy from the host cell. Most if not all of the functions involved in replication and transcription of the 192-kb genome are virally encoded. Although significant progress has been made in identifying trans-acting factors involved in DNA synthesis, the mechanism of genome replication has remained poorly understood. The genome is a linear duplex with covalently closed hairpin termini, and it has been presumed that sequences and/or structures within these termini are important for the initiation of genome replication. In this report we describe the construction of minichromosomes containing a central plasmid insert flanked by hairpin termini derived from the viral genome and their use as replication templates. When replication of these minichromosomes was compared with a control substrate containing synthetic hairpin termini, specificity for viral telomeres was apparent. Inclusion of [greater than or equal to]200 bp from the viral telomere was sufficient to confer optimal replication efficiency, whereas 65-bp telomeres were not effective. Chimeric 200-bp telomeres containing the 65-bp terminal element and 135 bp of ectopic sequence also failed to confer efficient replication, providing additional evidence that telomere function is sequence-specific. Replication of these exogenous templates was dependent upon the viral replication machinery, was temporally coincident with viral replication, and generated covalently closed minichromosome products. These data provide compelling evidence for specificity in template recognition and utilization in vaccinia virus-infected cells.

Dual specificity protein phosphatases (DSPs) regulate mitogenic signal transduction and control the cell cycle. Here, the crystal structure of a human DSP, vaccinia H1-related phosphatase (or VHR), was determined at 2.1 angstrom resolution. A shallow active site pocket in VHR allows for the hydrolysis of phosphorylated serine, threonine, or tyrosine protein residues, whereas the deeper active site of protein tyrosine phosphatases (PTPs) restricts substrate specificity to only phosphotyrosine. Positively charged crevices near the active site may explain the enzyme's preference for substrates with two phosphorylated residues. The VHR structure defines a conserved structural scaffold for both DSPs and PTPs. A 'recognition region,' connecting helix a1 to strand p1, may determine differences in substrate specificity between VHR, the PTPs, and other DSPs., The protein encoded by the H1 open reading frame in vaccinia virus was identified as the first dual specificity protein phosphatase (DSP), capable of hydrolyzing phosphate monoesters from peptides containing [...]

The 1.85 angstrom X-ray crystal structure of the AS11 mutant of VP39 bifunctional vaccinia virus protein complexed with its S-adenosylmethionine (AdoMet) cofactor is presented. VP39 acts as both an mRNA cap-specific RNA 2'-O-methyltransferase and a poly(A) polymerase processivity factor. Despite structural similarities to prokaryotic and eukaryotic methyltransferases, VP39 has a novel underlying architecture for an RNA-binding protein.

Polyadenylylated RNAs (POLADS) were cloned to evaluate selective cell protein synthesis inhibitors. Polyadenylylated viral or host mRNAs, host tRNA and small nuclear RNA can function as POLADS in vaccinia virus (VV) infection. The polyadenylylation process is attributed to the VV poly(A) polymerase that is generated by the virus core and penetrates the cytoplasm in replication.

A stringent specificity for adenosine triphosphate and an ability to discriminate between nicked and gapped DNAs at the substrate binding step were noted in an investigation of the specificity and fidelity of the viral enzyme vaccinia DNA ligase using purified recombinant ligase and synthetic duplex DNA substrates containing a single strand discontinuity. Findings reveal that the ligase was fairly tolerant of mismatches at a nick involving the 5' phosphate donor terminus, but was blocked strongly by mismatches at the 3' OH acceptor terminus, particularly by purine-purine mispairs.

Kinetic studies of the catalysis of site-specific DNA cleavage by Vaccinia DNA topoisomerase I as a function of pH indicates attack of the active site to be catalyzed by base followed by acid catalysis. A change in conformation state takes place, as revealed by variations in pH responses of internal equilibrium and resealing. Topoisomerization proceeds through this conformational change.

Vaccinia virus gene, a viral prolifin in the Escherichia coli, exhibits a binding property with the phosphatidylinositol 4,5-biphosphate and phosphatidylinositol 4-monophosphate in large unilamellar vesicles and mucelles. The prolifin does not bind to phosphatidylcholine and phosphatidylserine. The characterization of these interactions reveals that the process of binding of the prolifin is very slow, the rate constants ranging from 1 to 4 X 10 to the power 4 per mole per second.

Vaccinia virus F10L gene encodes vaccinia protein kinase 2 (VPK2) with a 50-kDa polypeptide. The polypeptide phosphorylates alpha-casein, seryl residues and a casein kinase I-specific peptide substrate. VPK2 gene disruption using a positive selectable marker indicates that VPK2 is required for the activity of the virus. VPK2 gene mediates protein phosphorylation to modulate transcription elements.

The characterization of a Shope fibroma virus (SFV) genome and its significant homology to uracil DNA glycosylase gene were analyzed. Experiments involved the construction of SFV uracil DNA glycosylase expression vector, in vitro expression of this glycosylase and ethidium bromide fluorescence assaying of glycosylase activity. The capability of poxvirus for repairing DNA was demonstrated.

The structure and assembly of vaccinia virus was characterized. The study protocol involved phospholipid analysis of the two distinct infectious forms, the intracellular naked virus (INV) and the extracellular enveloped virus, as well as immunoelectron microscopy using antibodies against different subcellular compartments. The results showed that the vaccinia virus becomes enwrapped by a membrane cisterna from the intermediate compartment between the endoplasmic reticulum and the Golgi apparatus, thus acquiring a double membrane in one step. This disproves the notion of de novo biogenesis of lipid envelpes by INVs.

A recombinant DNA packaging method to generate chimeric vaccinia viruses was developed. The procedure involved the direct insertion of foreign DNA within the vaccinia genome and subsequent packaging using fowlpox helper virus in either primary chicken embryo fibroblasts or a mammalian cell line. The procedure was used to identifydifferent chimeric viruses. The procedure yielded chimeras which could not havebeen obtained using classical procedures. This procedure will allow the cloningof DNA fragments without the need for prior subcloning in prokaryotic systems.

An experiment was conducted to show that proprotein convertase 2 (PC) and PC3 are loclaized in the pancreas to the islets of Langerhans and that both proteases selectively process specific cleavage sites in proinsulin when coexpressed with rat proinsulin 1 by means of vaccinia virus vectors. These results suggest that proinsulin is converted to insulin in the maturing secretory granules and that PC2 and PC3 represent a core of endoproteases necessary for prohormone maturation.

The vaccinia virus E3L gene was shown to encode both the dsRNA-binding protein p25 and specific inhibitory activity for the dsRNA-dependent protein kinase. The E3L products lysate in vitro and act as inhibitor of the P1/eIF-2alpha kinase when expressed in COS cells. These results suggest that the products of the E3Lgene are necessary and sufficient for potent inhibitory activity of the dsRNA- dependent protein kinase.

The vaccinia virus gene encoding for mRNA (nucleoside-O-2')-methyltransferase was identified and isolated in an attempt to investigate the role of nucleoside-O-2' methylation of the mRNA 5' cap structure in genetic translation. Results reveal that the gene encoding forvaccinia mRNA methyltransferase is also the same gene that encodes for the VP39subunit of poly(A) polymerase. Studies further show that cap-specific methyltransferase activity is present in heterodimeric and monomeric VP39 indicating that these proteins are capable of mediating both methyltransferase and poly(A) polymerase stimulatory activity in vaccinia cells.

VACCINIA VIRUS WAS SUCCESSFULLY USED FOR THE PREVENTION of smallpox and the eradication of variola virus, the causative agent of that disease. The protective effect of vaccination was reported by [...]

Many candidate antigens of malaria vaccines have limited immunological recognition. One exception is Pfs25, a cysteine-rich, 25-kilodalton sexual stage surface protein of Plasmodium falciparum. Pfs25 is a target of monoclonal [...]

The case of inadvertent contact vaccinia transmission from a mother to her breastfeeding infant through direct skin-to-skin and skin-to-mucous membrane contact is examined. There is a need for special precautions to prevent secondary transfer of contact vaccinia to breastfeeding mothers.