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G L Smith

Publications and source records attributed to G L Smith.

At least 19 recordsLinked to original sources
PubMed1996

A mechanism for the inhibition of fever by a virus.

Poxviruses encode proteins that block the activity of cytokines. Here we show that the study of such virulence factors can contribute to our understanding of not only virus pathogenesis but also the physiological role of cytokines. Fever is a nonspecific response to infection that contributes to host defense. Several cytokines induce an elevation of body temperature when injected into animals, but in naturally occurring fever it has been difficult to show that any cytokine has a critical role. We describe the first example of the suppression of fever by a virus and the molecular mechanism leading to it. Several vaccinia virus strains including smallpox vaccines express soluble interleukin 1 (IL-1) receptors, which bind IL-1 beta but not IL-1 alpha. These viruses prevent the febrile response in infected mice, whereas strains that naturally or through genetic engineering lack the receptor induce fever. Repair of the defective IL-1 beta inhibitor in the smallpox vaccine Copenhagen, a more virulent virus than the widely used vaccine strains Wyeth and Lister, suppresses fever and attenuates the disease. The vaccinia-induced fever was inhibited with antibodies to IL-1 beta. These findings provide strong evidence that IL-1 beta, and not other cytokines, is the major endogenous pyrogen in a poxvirus infection.

Animals↗
PubMed1996

Ligation of double-stranded and single-stranded [oligo(dT)] DNA by vaccinia virus DNA ligase.

Vaccinia virus DNA ligase has been expressed in Escherichia coli, purified, and biochemically characterized. The enzyme ligates double-stranded (ds) DNA substrates with either cohesive or blunt-end termini and the latter reaction is stimulated by PEG. Vaccinia virus DNA ligase can also ligate oligo(dT) when annealed to either a poly(dA) or a poly(rA) backbone and, remarkably, free oligo(dT). This ligation of a single-stranded (ss) substrate is unique among eukaryotic DNA ligases. The enzyme requires high ATP concentrations with a Km for the overall ligation of a ssDNA substrate of 0.8 mM. The salt, divalent cation, temperature, and pH requirements of the enzyme for the optimal ligation of ss and ds substrate are described.

Adenosine Triphosphate↗
PubMed1996

Comparative effects of inorganic phosphate and oxalate on uptake and release of Ca2+ by the sarcoplasmic reticulum in saponin skinned rat cardiac trabeculae.

1. Ventricular trabeculae from the right ventricle of rat heart were suspended in a 6 microliters bath and "skinned' with saponin (50 mg ml-1). Preparations were perfused with solutions mimicking the intracellular milieu and the [Ca2+] within the bath was monitored continuously using fura-2. 2. Application of 20 mM caffeine released Ca2+ from the sarcoplasmic reticulum (SR), resulting in a transient increase in the fura-2 fluorescence ratio. Caffeine-induced Ca2+ transients were smaller in the presence of 30 or 60 mM inorganic phosphate (Pi). This depressive effect of Pi on SR function was reversed by 10 mM creatine phosphate (CP). Caffeine-induced Ca2+ transients were also reduced in the presence of 10 mM oxalate, although this effect was not reversed by CP. 3. When perfusion was stopped in the presence of 30 or 60 mM Pi, the [Ca2+] within the bath remained constant. However, when the flow was stopped in the presence of 60 mM Pi and 10 mM CP, a prolonged decrease in [Ca2+] occurred, consistent with precipitation of calcium phosphate within the SR. A similar decrease in [Ca2+] was observed when perfusion was stopped in the presence of 2 or 20 mM oxalate, in the absence or presence of CP. 4. The SR was Ca2+ depleted by withdrawal of ATP and exposure to 20 mM caffeine. Perfusion was then stopped and ATP reapplied, resulting in a maintained decrease in [Ca2+] within the bath, due to SR Ca2+ uptake. Net Ca2+ uptake was markedly reduced in the presence of 30 mM Pi. In contrast, 20 mM oxalate increased Ca2+ uptake and the [Ca2+] within the bath continued to fall over 2-3 min. 5. Introduction of Pi released Ca2+ from the SR. Ryanodine (100 microM) abolished caffeine-induced Ca2+ release while Pi-induced Ca2+ release was unaffected. Pi-induced Ca2+ release was reduced in the constant presence of 20 mM caffeine or 10 mM CP and was abolished completely by disruption of the SR membrane with Triton X-100. Pi-induced Ca2+ release occurred after abolition of SR Ca2+ uptake by ATP withdrawal. 6. These results suggest that the Pi-induced decrease in releasable Ca2+ does not result from precipitation of calcium phosphate within the SR lumen. Pi inhibits net SR Ca2+ uptake, but this appears to result from activation of a ryanodine-insensitive Ca2+ efflux pathway rather then inhibition of Ca2+ uptake. Possible mechanisms are considered, including reversal of the SR Ca2+ pump.

Animals↗
PubMed1996

Soluble interferon-gamma receptors encoded by poxviruses.

Poxviruses encode a broad range of proteins that counteract the formidable attack of the immune response initiated in the host after infection, among which are proteins that mimic the extracellular binding domain of host cytokine receptors and are secreted from virus-infected cells. A soluble interferon-gamma receptor (IFN-gamma R) is produced early after infection and efficiently blocks the binding of IFN-gamma to cellular receptors, thus inhibiting both the anti-viral and immune functions of IFN-gamma. An IFN-gamma R is highly conserved among members of the poxvirus family, suggesting a major role in viral pathogenesis. The highly species-specific nature of the IFN system enables questions concerning the evolutionary relationship between poxviruses and their hosts to be addressed. The IFN-gamma R encoded by myxoma virus, a natural pathogen of rabbits, is specific for rabbit IFN-gamma. However, the IFN-gamma R encoded by orthopoxviruses (vaccinia, cowpox, camelpox, ectromelia) shows a novel, broad species specificity suggesting that these viruses have evolved in several species. The implications for the unknown origin and natural host(s) of vaccinia virus are discussed.

Animals↗
PubMed1996

Receptors for gamma-interferon encoded by poxviruses: implications for the unknown origin of vaccinia virus.

Poxviruses encode soluble interferon-gamma receptors (IFN-gamma Rs) that inhibit IFN-gamma activity and play a major role in virus pathogenesis. In contrast to the highly species specific cellular homologues, the vaccinia IFN-gamma R has novel broad species specificity. This has implications for the unknown origin and natural host(s) of vaccinia virus, the vaccine used for smallpox eradication.

Antigens, CD↗
PubMed1996

Virus proteins that bind cytokines, chemokines or interferons.

Some viruses express proteins to evade non-specific host defences such as complement, interferons and the inflammatory response. Recent notable discoveries are the broad species specificity of orthopoxvirus interferon receptors, herpesvirus and poxvirus proteins related to chemokine receptors and proteins that bind more than one cytokine, interferon or chemokine.

Animals↗
PubMed1996

Predictive validity of the suicide probability scale among adolescents in group home treatment.

OBJECTIVE: To determine the predictive validity of the Suicide Probability Scale (SPS). METHOD: Prospective design, predicting subsequent suicide attempts, suicide verbalizations, and minor self-destructive behaviors from SPS scores at admission to a group home in a sample of 855 adolescents. RESULTS: SPS scores significantly predicted all three suicide-relevant measures. Only 48% of subsequent attempts could have been predicted from SPS criteria that would have put 21% of newly admitted adolescents in the at-risk group. Although the prediction is far from perfect, it can prevent some suicide attempts when combined with effective suicide precautions. CONCLUSIONS: This is the first known evidence of predictive validity of any measure of suicide risk in adolescents. Its predictive ability may have been reduced by interventions designed to reduce suicide risk. Despite the large number of false-positives and modest predictive validity, a large body of literature indicates that quantitative predictions perform better than clinical judgments in predicting psychological outcomes. However, the SPS measures only a subset of factors predictive of suicide, including suicidal ideation, hopelessness, and social isolation. Risk factors not assessed by the SPS include previous suicide attempts, conduct disorder, substance abuse, and reasons for living. These additional risk factors may be important to improve upon the predictive validity of the SPS.

Adolescent↗
PubMed1996

Stimulation of vaccinia virion DNA helicase I8R, but not A18R, by a vaccinia core protein L4R, an ssDNA binding protein.

Four DNA-dependent ATPases were purified from vaccinia virions and tested for DNA helicase activity on two dsDNA substrates. ATPases D6R and D11L were inactive on both substrates, A18R unwound the substrate with a short 20 bp duplex region and 18R unwound both substrates. In addition, the 18R protein was stimulated to unwind longer DNA duplexes by a 25 kDa protein purified from vaccinia virions, representing the cleaved product of the L4R gene, an ssDNA binding protein. Purified recombinant 25 kDa L4R protein also stimulated 18R DNA helicase activity and maximum activity was observed only when there were < 13 nucleotides of DNA per molecule of L4R protein. The DNA helicase activity of the A18R protein was not stimulated by either recombinant 25 kDa L4R protein or by an E. coli ssDNA binding protein.

Amino Acid Sequence↗
PubMed1996

Vaccinia virus glycoprotein A34R is required for infectivity of extracellular enveloped virus.

The vaccinia virus strain Western Reserve (WR) A34R gene encodes a C-type lectin-like glycoprotein, gp22-24, that is present in the outer membrane of extracellular enveloped virus (EEV) with type II membrane topology (S.A. Duncan and G.L. Smith, J. Virol. 66:1610-1621, 1992). Here we that a WR A34R deletion mutant (WR delta A34R) released 19- to 24-fold more EEV from infected cells than did WR virus, but the specific infectivity of the released virions was reduced 5- to 6-fold. Rupture of the WR delta A34R EEV outer envelope by freeze-thawing increased virus infectivity by five- to sixfold, because of the release of infectious intracellular mature virus. All other known EEV-specific proteins are incorporated into WR delta A34R EEV, and thus the loss of gp22-24 is solely responsible for the reduction of EEV specific infectivity. The WR delta A34R virus is highly attenuated in vivo compared with WR or a revertant virus in which the A34R gene was reinserted into WR delta A34R. This attenuation is consistent with the known important role of EEV in virus dissemination and virulence. Vaccinia virus strain International Health Department-J (IHD-J) produces large amounts of EEV and forms comets because of an amino acid substitution within the A34R protein (R. Blasco, R. Sisler, and B. Moss, J. Virol. 67:3319-3325, 1993), but despite this, IHD-J EEV has a specific infectivity equivalent to that of WR EEV. Substitution of the IHD-J A34R gene into the WR strain induced comet formation and greater release of EEV, while coexpression of both genes did not; hence, the WR phenotype is dominant. All orthopoxviruses tested express the A34R protein, but most viruses, including variola virus, have the WR rather than the IHD-J A34R genotype. The A34R protein affects plaque formation, EEV release, EEV infectivity, and virus virulence.

Animals↗
PubMed1996

Vaccinia virion protein I8R has both DNA and RNA helicase activities: implications for vaccinia virus transcription.

A nucleic acid-dependent ATPase was purified from vaccinia virions and shown to have both DNA:DNA and RNA:RNA helicase activities. This is only the third helicase to be identified that can unwind both DNA and RNA duplexes. The DNA helicase activity copurified with nucleoside triphosphate phosphohydrolase II (NPHII), an RNA helicase encoded by gene I8R (S. Shuman, Proc. Natl. Acad. Sci. USA 89:10935-10939, 1992). Immunodepletion with two antisera to NPHII and analysis of recombinant NPHII protein (C. H. Gross and S. Shuman, J. Virol. 69:4727-4736, 1995) confirmed that the DNA helicase activity was encoded by the I8R gene. The I8R DNA helicase unwound DNA in a 3'-to-5' direction only, unwound duplexes of 35 bp but not 45 bp, and could be stimulated to unwind longer duplexes by the Escherichia coli single-stranded DNA-binding protein. DNA helicase activity was not stimulated by salt and was sensitive to 100 mM NaCl or KCl. The I8R protein has amino acid similarity to human RNA helicase A and to nuclear DNA helicase II, a bovine DNA and RNA helicase. On the basis of the phenotype of I8R temperature-sensitive mutants, it was suggested that the I8R protein is not required for DNA replication but might aid in the extrusion of early mRNA from the virus core. The DNA helicase activity of the I8R protein allows another interpretation of the mutant phenotype, namely, that the I8R DNA helicase activity is required for initiation of early transcription from within vaccinia virions.

Acid Anhydride Hydrolases↗
PubMed1996

Overexpression of the vaccinia virus A38L integral membrane protein promotes Ca2+ influx into infected cells.

The vaccinia virus Western Reserve A38L protein is a hydrophobic integral membrane glycoprotein with amino acid similarity to mammalian integrin-associated protein. The protein has an N-terminal immunoglobulin superfamily domain, followed by five membrane-spanning domains and a short cytoplasmic tail. Deletion of the protein reduces virus plaque size but does not affect virus virulence (J. E. Parkinson, C. M. Sanderson, and G. L. Smith, Virology, in press). In this study, we have used a recombinant vaccinia virus in which the A38L gene may be inducibly overexpressed by addition of isopropyl-beta-D-thiogalactopyranoside (IPTG), to demonstrate that overexpression of the vaccinia virus A38L gene produces drastic changes in the morphology, permeability, and adhesion of infected cells. In particular, A38L overexpression caused swelling of cells, marginalization of nuclear chromatin, and vacuolization of the endoplasmic reticulum, features characteristic of cell necrosis. By 18 h postinfection, cells become permeable and lytic as defined by the free entry of propidium iodide and loss of the cytoplasmic enzyme lactate dehydrogenase. Chelation of extracellular Ca2+ 22 h postinfection inhibited further release of lactate dehydrogenase, showing that Ca2+ influx was required for A38L-induced lysis. Direct measurement of 45Ca2+ influx showed that the rate of Ca2+ uptake was directly related to the period of A38L induction. The A38L protein, therefore, promotes the formation of pores within the plasma membrane of cells, and these pores facilitate Ca2+ entry and induce necrosis. Addition of rifampin inhibited virus assembly but not the ability of A38L to induce necrosis, indicating that pore formation is independent of viral morphogenesis. Finally, overexpression of the A38L protein resulted in a reduced plaque size and a threefold decrease in production of infective particles in vitro. The A38L protein represents the first example of a virus protein which directly or indirectly promotes the influx of extracellular Ca2+.

Calcium↗
PubMed1996

Vaccinia virions lacking core protein VP8 are deficient in early transcription.

When synthesis of the 25-kDa vaccinia virus core protein VP8 is repressed, mature virus particles of normal appearance are produced to approximately 80% of wild-type levels but these particles are over 100-fold less infectious than wild-type particles (D. Wilcock and G. L. Smith, Virology 202:294-304, 1994). Here we show that virions which lack VP8 can bind to and enter cells but the levels of steady-state RNA are greatly reduced in comparison with those for wild-type infections. In vitro assays using permeabilized virions demonstrated that VP8-deficient virions had drastically reduced rates of transcription (RNA synthesis was decreased by 80 to 96%) and that the extrusion of RNA transcripts from these virions was also decreased. Low concentrations of sodium deoxycholate extracted proteins more efficiently from VP8-deficient virions than from wild-type virions. The increased fragility of VP8-deficient virions and their slower RNA extrusion rates suggest that VP8 may be required for the correct formation of the core. Virions which lack VP8 were shown to contain a full complement of transcription enzymes, and soluble extracts from these virions were active in transcription assays using either single-stranded M13 DNA or exogenous plasmid template containing a vaccinia virus early promoter. Thus, the defect in transcription is due not to a lack of specific transcriptional enzymes within virions but rather to the inability of these enzymes to efficiently transcribe the DNA genome packaged within VP8-deficient virions. These results suggest that VP8 is required for the correct packaging of the viral DNA genome and/or for the efficient transcription of packaged virion DNA, which has a higher degree of structural complexity than plasmid templates. Possible roles for VP8 in these processes are discussed.

Animals↗
PubMed1996

Radiation exposure of orthopaedic trainees: quantifying the risk.

Interest in radiation protection issues has recently grown within the medical profession. Several investigators have examined the radiation exposure of orthopaedic surgeons using X-rays during trauma surgery, and found that doses received are within acceptable limits. These studies however, have been performed over short time-periods or have been confined to single procedures only. In this paper, we report the results of monitoring of the total exposure across all procedures of a group of orthopaedic surgeons over a 4-month period. Doses received were all below the International Commission on Radiological Protection (ICRP) recommended limits. While these results are reassuring, they do not represent grounds for complacency. Continued vigilance will be essential as work practices continue to change. In orthopaedics, in contrast with other specialties, the limiting dose is that to the hands. In view of this finding, extremity dosimetry for surgeons regularly using X-rays in theatre should be considered.

Gloves, Protective↗
PubMed1995

The vaccinia virus A38L gene product is a 33-kDa integral membrane glycoprotein.

The vaccinia virus gene A38L encodes a highly hydrophobic protein with amino acid similarity to mammalian integrin-associated protein (IAP). In this report we have identified the A38L protein of strain Western Reserve (WR), defined its membrane topology, and analyzed its role in virus production and virulence. An antiserum raised against an A38L peptide identified the A38L gene product as a 33-kDa protein which is expressed at low levels during virus infection. A serum from a rabbit previously infected with WR virus recognized the A38L protein, thus confirming that the A38L gene is expressed in vivo. Using a coupled in vitro-translation/membrane-translocation system the 33-kDa protein was shown to be a membrane-associated and glycosylated form of a 29-kDa polypeptide precursor. The membrane topology of the A38L protein was defined by its glycosylation and protease sensitivity when associated with microsomal membranes. The N-terminal immunoglobulin-like variable domain was protected from exogenous protease and was therefore in the lumen of the vesicle, whereas the C-terminus was sensitive and therefore cytoplasmic. A38L deletion and revertant viruses were constructed and were used to study the involvement of A38L in virus assembly, release, and virulence. Deletion of the A38L gene caused a slight reduction in virus plaque size but did not affect the production of intracellular mature virus or extracellular enveloped virus particles in tissue culture cells nor the virulence of the virus in the murine intranasal model. The A38L protein therefore possesses similar sequence and membrane topology to the mammalian IAP protein but is not required for virus particle production or virulence.

Amino Acid Sequence↗
PubMed1995

Vaccinia virus encodes a soluble type I interferon receptor of novel structure and broad species specificity.

Vaccinia virus (VV) and other orthopoxviruses express a soluble type I interferon (IFN) receptor that for VV strain Western Reserve is encoded by gene B18R. The 60-65 kDa glycoprotein is related to the interleukin-1 receptors and is a member of the immunoglobulin superfamily, unlike other type I IFN receptors, which belong to the class II cytokine receptor family. The receptor has high affinity (KD, 174 pM) for human IFN alpha and, unlike other type I IFN receptors, has broad species specificity, binding to human, rabbit, bovine, rat, and mouse type I IFNs. This may have aided VV replication in multiple host species during evolution. A VV B18R deletion mutant is attenuated in a murine intranasal model. This type I IFN receptor represents the fourth VV protein that interferes with IFN and the fourth soluble cytokine receptor expressed by poxviruses.

Animals↗