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Vaccinia virus (VACV) outbreaks, first reported in Brazil in 1999, affect dairy cattle and humans in rural areas (1). Although studies have shown evidence of VACV circulation among several mammal [...]

Eczema vaccinatum (EV) is a complication of smallpox vaccination occurring in patients with atopic dermatitis. In affected individuals, vaccinia virus (VV) spreads through the skin, resulting in large primary lesions and satellite lesions, and infects internal organs. BALB/c mice inoculated with VV at sites of Th2-biased allergic skin inflammation elicited by epicutaneous ovalbumin (OVA) sensitization exhibited larger primary lesions that were erosive, more satellite lesions, and higher viral loads in skin and internal organs than mice inoculated in saline-exposed skin, unsensitized skin, or skin sites with Th1-dominant inflammation. VV inoculation in OVA-sensitized skin induced marked local expression of IL-17 transcripts and massive neutrophil infiltration compared to VV inoculation in saline-exposed skin. Treatment with anti-IL-17 decreased the size of primary lesions, numbers of satellite lesions, and viral loads. Addition of IL-17 promoted VV replication in skin explants. These results suggest that IL-17 may be a potential therapeutic target in EV. atopic dermatitis | eczema vaccinatum

We previously reported that the replication-competent vaccinia virus (VACV) GLV-1h68 shows remarkable oncolytic activity and efficacy in different animal models as a single treatment modality and also in combination with chemotherapy [Yu YA, et al. (2009) Mol Cancer Ther 8:141-151]. Here, we report the construction of 3 VACV strains encoding GLAF-1, a previously undescribed engineered single-chain antibody (scAb). This unique scAb is transcribed from 3 vaccinia promoters (synthetic early, early/late, and late) and directed against both human and murine VEGFs. The expression of GLAF-1 was demonstrated in cell cultures. Also, the replication efficiency of all GLAF-l-expressing VACV strains in cell culture was similar to that of the parental GLV-1h68 virus. Successful tumor-specific delivery and continued production of functional scAb derived from individual VACV strains were obtained in tumor xenografts following a single intravenous injection of the virus. The VACV strains expressing the scAb exhibited significantly enhanced therapeutic efficacy in comparison to treatment of human tumor xenografts with the parental virus GLV-1h68. This enhanced efficacy was comparable to the concomitant treatment of tumors with a one-time i.v. injection of GLV-1h68 and multiple i.p. injections of Avastin. Taken together, the VACV-mediated delivery and production of immunotherapeutic anti-VEGF scAb in colonized tumors may open the way for a unique therapy concept: tumor-specific, locally amplified drug therapy in humans. Avastin | GLV-1h68 | tumor colonization | angiogenesis | amplified drug therapy

Unlike most DNA viruses, poxviruses replicate in the cytoplasm of host cells. They encode enzymes needed for genome replication and transcription, including their own thymidine and thymidylate kinases. Some herpes viruses encode only 1 enzyme catalyzing both reactions, a peculiarity used for prodrug activation to obtain maximum specificity. We have solved the crystal structures of vaccinia virus thymidylate kinase bound to TDP or brivudin monophosphate. Although the viral and human enzymes have similar sequences (42% identity), they differ in their homodimeric association and active-site geometry. The vaccinia TMP kinase dimer arrangement is orthogonal and not antiparallel as in human enzyme. This different monomer orientation is related to the presence of a canal connecting the edge of the dimer interface to the TMP base binding pocket. Consequently, the pox enzyme accommodates nucleotides with bulkier bases, like brivudin monophosphate and dGMP; these are efficiently phosphorylated and stabilize the enzyme. The brivudin monophosphate-bound structure explains the structural basis for this specificity, opening the way to the rational development of specific antipox agents that may also be suitable for poxvirus TMP kinase gene-based chemotherapy of cancer. BVdU | dihalovinyl-dU | dimerization mode | TMP kinase | vaccinia virus

F-box proteins direct the degradation of an extensive range of proteins via the ubiquitin-proteasome system. Members of this large family of proteins are typically bipartite. They recruit specific substrates through a substrate-binding domain and, via the F-box, link these to core components of a major class of ubiquitin ligases (SCF1). F-box proteins thus determine the specificity of SCF1-mediated ubiquitination. F-box-like motifs were recently detected in poxvirus ankyrin repeat (ANK) proteins but clear compositional differences to typical F-box proteins raise questions regarding the classification and function of the motif. Here we show that all five ANK proteins of a representative poxvirus, Orf virus, interact in vivo with core components of the SCF1 ubiquitin ligase complex. Interaction is dependent on the poxviral F-box-like motif and the adaptor subunit of the complex (SKP1). The viral protein does not block enzymatic activity of the complex. These observations identify the poxviral motif as a functional F-box. They also identify a new class of F-box that in contrast to cellular counterparts is truncated, has an extreme C-terminal location and is paired with an ANK protein-binding domain. ANK proteins constitute the largest family of poxviral proteins but their function and the significance of their abundance have remained an enigma. We propose that poxviruses use these unique ANK/F-box proteins to dictate target specificity to SCF1 ubiquitin ligases and thereby exploit the cell's ubiquitin-proteasome machinery. E3 ligase | Orf virus | proteasome | viral pathogenesis | Vaccinia virus

Poxviruses are large enveloped viruses that replicate in the cytoplasm of vertebrate or invertebrate cells. At least six virus-encoded proteins are required for synthesis and processing of the double-stranded DNA genome of vaccinia virus, the prototype member of the family. One of these proteins, D5, is an NTPase that contains an N-terminal archaeoeukaryotic primase domain and a C-terminal superfamily III helicase domain. Here we report that individual conserved aspartic acid residues in the predicted primase active site were required for in vivo complementation of infectious virus formation as well as genome and plasmid replication. Furthermore, purified recombinant D5 protein synthesized oligoribonucleotides in vitro. Incorporation of label from [[alpha]-.sup.32]CTP or [[alpha]-.sup.32P]UTP into a RNase-sensitive and DNase-resistant product was demonstrated by using single-stranded circular bacteriophage DNA templates and depended on ATP or GTP and a divalent cation. Mutagenesis studies showed that the primase and NTPase activities of the recombinant D5 protein could be independently inactivated. Highly conserved orthologs of D5 are present in all poxviruses that have been sequenced, and more diverged orthologs are found in members of all other families of nucleocytoplasmic large DNA viruses. These viral primases may have roles in initiation of DNA replication or lagging-strand synthesis and represent potential therapeutic targets. DNA replication | nucleocytoplasmic large DNA viruses | RNA primer | vaccinia virus

The effects of intercalating PAH-DNA adducts on the religation reaction of vaccinia DNA topoisomerase are examined. It is found that intercalating benzo[c]phenanthrene and benzo[a]pyrene adducts act as roadblocks to ExoIII digestion but differ in their longevity of inhibition.

A study in which the N7 hydrogen bond acceptor groups of each guanine and adenine within the consensus sequence are replaced with unnatural 7-deaza bases is described. The double modifications have shown equal destabilizing effects in the transition state and covalent complex leading to slower rates of cleavage and less damaging effects on ligation.

Poxviruses have evolved elaborate mechanisms for cell entry, assembly, and exocytosis. Recently, four vaccinia virus membrane proteins, namely A21, A28, H2 and L5, were reported to be necessary for cell entry and virus-induced cell-cell fusion but not for virion morphogenesis or attachment of virus particles to cells. Using immunoaffinity purification followed by mass spectrometry, we now show that these four proteins as well as four additional previously uncharacterized putative membrane proteins (A16, G3, G9, and J5) form a stable complex. These proteins fall into two groups: A21, A28, G3, H2, and L5 have an N-terminal transmembrane domain, 0-2 intramolecular disulfide bonds, and no sequence similarity, whereas A16, G9, and J5 have a C-terminal transmembrane domain and 4-10 predicted disulfide bonds and are homologous. Studies with conditional-lethal null mutants indicated that the viral membrane was crucial for assembly of the complex and that the absence of individual polypeptide components profoundly decreased complex formation or stability, suggesting a complicated interaction network. Analysis of purified virions, however, demonstrated that the polypeptides of the complex trafficked independently to the viral membrane even under conditions in which the complex itself could not be isolated. All eight proteins comprising the entry-fusion complex are conserved in all poxviruses, suggesting that they have nonredundant functions and that the basic entry mechanism evolved before the division between vertebrate and invertebrate poxvirus species. mass spectrometry | transmembrane proteins | vaccinia virus | viral envelope proteins | virus entry

Passatempo virus was isolated during a zoonotic outbreak. Biologic features and molecular characterization of hemagglutinin, thymidine kinase, and vaccinia growth factor genes suggested a vaccinia virus infection, which strengthens the [...]

During morphogenesis, poxviruses undergo a remarkable transition from spherical immature forms to brick-shaped infectious particles lacking helical or icosahedral symmetry. In this study, we show that the transitory honeycomb lattice coating the lipoprotein membrane of immature vaccinia virus particles is formed from trimers of a 62-kD protein encoded by the viral D13L gene. Deep-etch electron microscopy demonstrated that anti-D13 antibodies bound to the external protein coat and that lattice fragments were in affinity-purified D13 preparations. Soluble D13 appeared mostly trimeric by gel electrophoresis and ultracentrifugation, which is consistent with structural requirements for a honeycomb. In the presence or absence of other virion proteins, a mutated D13 with one amino acid substitution formed stacks of membrane-unassociated flat sheets that closely resembled the curved honeycombs of immature virions except for the absence of pentagonal facets. A homologous domain that is present in D13 and capsid proteins of certain other lipid-containing viruses support the idea that the developmental stages of poxviruses reflect their evolution from an icosahedral ancestor.

The vaccinia virus (VV) E3L protein is essential for virulence and has anti-apoptotic activity. In mice, Z-DNA-binding activity of the N-terminal domain of E3L (Z[alpha]) is necessary for viral lethality. Here, we report that inhibition of hygromycin-B-induced apoptosis in HeLa cells depends on Z-DNA binding of the E3L Z[alpha] domain. Z-DNA-binding domains of other proteins are equally effective in blocking apoptosis. Using a transient reporter assay, we demonstrate transactivation of human IL-6, nuclear factor of activated T cells (NF-AT), and p53 genes by E3L. This activation also requires Z-DNA binding of the N-terminal domain of E3L. Overall, this work suggests that the important role of E3L in VV pathogenesis involves modulating expression of host cellular genes at the transcriptional level and inhibiting apoptosis of host cells through Z-DNA binding. apoptosis | interleukin-6 | p53 | poxvirus

The combination of cryo-microscopy and electron tomographic reconstruction has allowed us to determine the structure of one of the more complex viruses, intracellular mature vaccinia virus, at a resolution of 4-6 nm. The tomographic reconstruction allows us to dissect the different structural components of the viral particle, avoiding projection artifacts derived from previous microscopic observations. A surface-rendering representation revealed brick-shaped viral particles with slightly rounded edges and dimensions of [approximately equal to] 360 x 270 x 250 nm. The outer layer was consistent with a lipid membrane (5-6 nm thick), below which usually two lateral bodies were found, built up by a heterogeneous material without apparent ordering or repetitive features. The internal core presented an inner cavity with electron dense coils of presumptive DNA-protein complexes, together with areas of very low density. The core was surrounded by two layers comprising an overall thickness of [approximately equal to] 18-19 nm; the inner layer was consistent with a lipid membrane. The outer layer was discontinuous, formed by a periodic palisade built by the side interaction of T-shaped protein spikes that were anchored in the lower membrane and were arranged into small hexagonal crystallites. It was possible to detect a few pore-like structures that communicated the inner side of the core with the region outside the layer built by the T-shaped spike palisade. cryo-EM | virus structure | 3D reconstruction

A novel multivalent cytotoxic T-lymphocyte peptide construct derived from the tax protein of HTLV-1 separated by arginine spacers that elicited high cellular responses against individual epitopes simultaneously in human leukocyte antigen-A*0201 transgenic mice. A positive corelation was found between processing rates and the immunogenecity of the intended epitopes.

We show that three cytoplasmic thiol oxidoreductases encoded by vaccinia virus comprise a complete pathway for formation of disulfide bonds in intracellular virion membrane proteins. The pathway was defined by analyzing conditional lethal mutants and effects of cysteine to serine substitutions and by trapping disulfide-bonded heterodimer intermediates for each consecutive step. The upstream component, E10R, belongs to the ERV1/ALR family of FAD-containing sulfhydryl oxidases that use oxygen as the electron acceptor. The second component, A2.5L, is a small a-helical protein with a CxxxC motif that forms a stable disulfide-linked heterodimer with E10R and a transient disulfide-linked complex with the third component, G4L. The latter is a thioredoxin-like protein that directly oxidizes thiols of L1R, a structural component of the virion membrane with three stable disulfide bonds, and of the related protein F9L. These five proteins are conserved in all poxviruses, suggesting that the pathway is an ancestral mechanism for direct thiol-disulfide interchanges between proteins even in an unfavorable reducing environment.

Research has been conducted on the reversible DNA phosphodiester bond cleavage. The synthesis of the DNA substrates that carry single nonbridging Rp- or Sp-phosphorothioate modification is analyzed.

Experiments have been conducted on nucleosides and phosphates to demonstrate their contributions to site affinity and transesterification. Cumulative functional analysis of the vaccinia topoisomerase-DNA interface is presented.

Vaccinia virus early, intermediate, and late stage genes are sequentially transcribed by the viral RNA polymerase within the cytoplasm of infected cells. We found that the 34- and 45-kDa polypeptides encoded by vaccinia virus ORFs A8R and A23R, respectively, were necessary to reconstitute transcription of a template with an intermediate stage promoter. Coexpression of the A8R and A23R genes in Escherichia coli was required for in vitro activity. In addition, the two polypeptides copurified, indicating their association as protein subunits of a vaccinia virus intermediate transcription factor. This factor, which we named VITF-3, complemented three vital proteins - namely, the RNA polymerase, capping enzyme, and a 30-kDa protein called VITF-1 that is also a subunit of the RNA polymerase - and an unidentified cell factor called VITF-2. Expression of the A8R and A23R genes occurred between 1 and 5 h after vaccinia virus infection and was not prevented by an inhibitor of DNA replication, consistent with a role for VITF-3 in specifically regulating intermediate transcription in vivo. The vaccinia virus A8R and A23R genes are highly conserved among vertebrate poxviruses, but no other viral or cellular homologs were identified.

Vaccinia uses actin-based motility for virion movement in host cells, but the specific protein components have yet to be defined. A cardinal feature of Listeria and Shigella actin-based motility is the involvement of vasodilator-stimulated phosphoprotein (VASP). This essential adapter recognizes and binds to actin-based motility 1 (ABM-1) consensus sequences [(D/E)FPPPPX(D/E), X = P or T] contained in Listeria Acta and in the p90 host-cell vinculin fragment generated by Shigella infection. VASP, in turn, provides the ABM-2 sequences [XPPPPP, X = G, P, L, S, A] for binding profilin, an actin-regulatory protein that stimulates actin filament assembly. Immunolocalization using rabbit anti-VASP antibody revealed that VASP concentrates behind motile virions in HeLa cells. Profilin was also present in these actin-rich rocket tails, and microinjection of 10 [[micro]molar] (intracellular) ABM-2 peptide [(GPPPPP).sub.3] blocked vaccinia actin-based motility. Vinculin did not colocalize with VASP on motile virions and remained in focal adhesion contacts; however, another ABM-1-containing host protein, zyxin, was concentrated at the rear of motile virions. We also examined time-dependent changes in the location of these cytoskeletal proteins during vaccinia infection. VASP and zyxin were redistributed dramatically several hours before the formation of actin rocket tails, concentrating in the viral factories of the perinuclear cytoplasm. Our findings underscore the universal involvement of ABM-1 and ABM-2 docking sites in actin-based motility of Listeria, Shigella, and now vaccinia.

The utility of modified vaccinia virus Ankara (MVA) as a vector for eliciting AIDS virus-specific cytotoxic T lymphocytes (CTL) was explored in the simian immunodeficiency virus (SIV)/rhesus monkey model. After two intramuscular immunizations with recombinant MVA-[SIV.sub.SM] gag pol, the monkeys developed a Gag epitope-specific CTL response readily detected in peripheral blood lymphocytes by using a functional killing assay. Moreover, those immunizations also elicited a population of CDS+ T lymphocytes in the peripheral blood that bound a specific major histocompatibility complex class I/peptide tetramer. These Gag epitope-specific CD8+ T lymphocytes also were demonstrated by using both functional and tetramer-binding assays in lymph nodes of the immunized monkeys. These observations suggest that MVA may prove a useful vector for an HIV-1 vaccine. They also suggest that tetramer staining may be a useful technology for monitoring CTL generation in vaccine trials in nonhuman primates and in humans.

Research was conducted to examine the interaction between VP39 and its capped RNA substrate via the characterization of two sites on the protein surface and the identification of a third site. VP39 is a vaccinia-encoded mRNA cap-specific 2'-O-methyltransferase. Results revealed the pH sensitivity of RNA-protein contacts. In addition, regions of the RNA substrate were found to bind to a previously unknown site on the VP39 surface.

A termination protein in vaccinia virus that catalyzes ATP hydrolysis and is closely associated with a polymerase-elongation complex has been identified as nucleoside triphosphate phosphohydrolase-I (NPH-I). It is a virus-encoded DNA-dependent ATPase of the DExH-box family. NPH-I has roles in transcription, one with VTF/CE to catalyze release of UUUUUNU-containing nascent RNA from the elongation complex and one alone as a polymerase elongation factor to make readthrough of intrinsic pause sites easier. Vaccinia NPH-I is the energy-coupling factor for eukaryotic transcription termination for mRNA.

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.

A study has been conducted to examine the role of the cytoskeleton during viral infection of the vaccinia virus using mutant and drug studies. The results showed that the vaccinia virus forms a intracellular envelop capable of inducing the formation of actin tails that are similar to those seen in Listeria and Shigella viral infections. The virus extends outwards using the actin projections to contact and infect neighboring cells. Studies show that this mechanism is similar to that used by bacterial pathogens.

Experiments were conducted, wherein vaccinia virus A-type inclusion protein (VV ATI) was cleaved with cyanogen bromide and determined that the myristoyl moiety was present in the C-terminal half of the protein. It is implied that the VV ATI protein is myristoylated in a sequence-independent manner and result suggests that there may be an alternative type of myristoylation pathway in mammalian cells.

Holliday junctions are intermediates in genetic recombination and consist of four strands of DNA that flank a branch point. Their sequences have two-fold (homologous) sequence symmetry in natural systems.

Vaccinia virus (VV) egress has been studied using confocal, video, and electron microscopy. Previously, intracellular-enveloped virus (IEV) particles were proposed to induce the polymerization of actin tails, which propel IEV particles to the cell surface. However, data presented support an alternative model in which microtubules transport virions to the cell surface and actin tails form beneath cell-associated enveloped virus (CEV) particles at the cell surface. Thus, VV is unique in using both microtubules and actin filaments for egress. The following data support this proposal. (a) Microscopy detected actin tails at the surface but not the center of cells. (b) VV mutants lacking the A33R, A34R, or A36R proteins are unable to induce actin tail formation but produce CEV and extracellular-enveloped virus. (c) CEV formation is inhibited by nocodazole but not cytochalasin D or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP1). (d) IEV particles tagged with the enhanced green fluorescent protein fused to the VV B5R protein moved inside cells at 60 [micro]m/min. This movement was stop-start, was along defined pathways, and was inhibited reversibly by nocodazole. This velocity was 20-fold greater than VV movement on actin tails and consonant with the rate of movement of organelles along microtubules.

IL-2 and -15 belong to the four [alpha]-helix bundle family of cytokines and display a spectrum of overlapping immune functions because of shared signal transducing receptor components of the IL-2 receptor complex. However, recent evidence suggests a nonredundant unique role for IL-15 in the establishment and perhaps maintenance of peripheral natural killer (NK) cell populations in vivo. To explore the contribution of locally released IL-15 on peripheral NK-cell-mediated innate immune responses, we generated a recombinant vaccinia virus that expresses IL-15 and evaluated the course of vaccinial disease in athymic nude mice. Coexpression of IL-15 resulted in the attenuation of virulence of vaccinia virus, and mice inoculated with [10.sup.5] plaque-forming units or less resolved the infection successfully. In contrast, mice inoculated with a similar dose of the control vaccinia virus failed to eliminate the virus and died of generalized vaccinial disease. Enhanced expression of IL-12 and IFN-[gamma] as well as induction of chemokines were evident in the mice inoculated with IL-15-expressing vaccinia virus in addition to an increase in NK cells in the spleen. However, in this model system, the degree of attenuation in viral virulence attained with coexpression of IL-15 was much less than that achieved with coexpression of IL-2, suggesting that the peripheral NK-cell-mediated events are more responsive to IL-2 than to IL-15.

Poxviruses employ many strategies to evade and neutralize the host immune response. In this study, we have identified two vaccinia virus ORFs, termed A46R and A52R, that share amino acid sequence similarity with the Toll/IL-1 receptor (TLR) domain, a motif that defines the IL-1/Toll-like receptor (TLR) superfamily of receptors, which have a key role in innate immunity and inflammation. When expressed in mammalian cells, the protein products of both ORFs were shown to interfere specifically with IL-1 signal transduction. A46R partially inhibited IL-1-mediated activation of the transcription factor NF[Kappa]B, and A52R potently blocked both IL-1- and TLR4-mediated NF[Kappa]B activation. MyD88 is a TlR domain-containing adapter molecule known to have a central role in both IL-1 and TLR4 signaling. A52R mimicked the dominant-negative effect of a truncated version of MyD88 on IL-1, TLR4, and IL-18 signaling but had no effect on MyD88-independent signaling pathways. Therefore, A46R and A52R are likely to represent a mechanism used by vaccinia virus of suppressing TIR domain-dependent intracellular signaling.