Biomedical subjects

J A Kerry

Publications and source records attributed to J A Kerry.

15 recordsLinked to original sources

PubMed1999

Transcriptional regulation of the human cytomegalovirus US11 early gene.

The human cytomegalovirus (HCMV) US11 early gene encodes a protein involved in the down-regulation of major histocompatibility complex class I cell surface expression in HCMV-infected cells. Consequently, this gene is thought to play an important role in HCMV evasion of immune recognition. In this study, we examined the transcriptional regulation of US11 gene expression. Analysis of deletions within the US11 promoter suggests that two sequence elements are important for activation by the viral immediate-early (IE) proteins. Deletion of a CREB site located at -83 relative to the cap site resulted in a reduction in promoter activity to 50% of the wild-type level. Deletion of an additional ATF site immediately upstream of the TATA box resulted in abrogation of responsiveness to the IE proteins. To confirm the role of the CREB and ATF sites within the US11 promoter, mutagenesis of these two sites, both individually and in combination, was carried out. Results indicate that both the CREB element and the ATF site were required for full promoter activity, with the ATF site critical for US11 promoter activation. The loss of transcriptional activation correlated with a loss of cellular proteins binding to the mutated US11 promoter elements. In combination with the viral IE proteins, the HCMV tegument protein pp71 (UL82) was found to up-regulate the US11 promoter by six- to sevenfold in transient assays. These results suggest that pp71 may contribute to the activation of the US11 promoter at early times after infection. Up-regulation by pp71 required the presence of the CREB and ATF sites within the US11 promoter for full activation. The role of the ATF and CREB elements in regulating US11 gene expression during viral infection was then assessed. The US11 gene is not required for replication of HCMV in tissue culture. This property was exploited to generate US11 promoter mutants regulating expression of the endogenous US11 gene in the natural genomic context. We generated recombinant HCMV that contained the US11 promoter with mutations in either the CREB or ATF element or both regulating the expression of the endogenous US11 gene. Northern blot analysis of infected cell mRNA revealed that mutation of the CREB element reduced US11 mRNA expression to approximately 25% of that of the wild-type promoter, with identical kinetics of expression. Mutation of the ATF site alone reduced US11 mRNA levels to 6% of that of the wild-type promoter, with mRNA detectable only at 8 h after infection. Mutation of both the CREB and ATF elements in the US11 promoter reduced US11 gene expression to undetectable levels. These results demonstrate that the CREB and ATF sites cooperate to regulate the US11 promoter in HCMV-infected cells.

Base Sequence↗

PubMed1998

The human interferon-inducible protein, IFI 16, is a repressor of transcription.

IFI 16 is a member of a family of interferon-inducible proteins, including the human MNDA (myeloid nuclear differentiation antigen), the recently identified AIM-2 (absent in melanoma), and the homologous murine molecules, p202, p204, and D3. IFI 16 contains a domain at the amino terminus capable of binding double-stranded DNA and a bipartite nuclear localization signal. No molecular or biological function has been assigned to any of the human family members, although a role in transcription regulation has been proposed. In the present study, we show IFI 16 fused to the GAL4 DNA binding domain can function as a transcriptional repressor. IFI 16-mediated repression is not dependent on the position or distance of IFI 16 binding, relative to the site of transcription initiation, and it can significantly repress when only one GAL4 DNA element is present in the promoter. We mapped the transcriptional repression domains to the 200 amino acid repeat regions common to all human and mouse family members. We also demonstrate that wild type IFI 16 can repress transcription of a reporter gene containing the minimal promoter region of the human cytomegalovirus UL54 gene. Thus, IFI 16 is a transcriptional repressor, with a modular structure typical of many known transcription regulators.

Base Sequence↗

PubMed1997

Translational regulation of the human cytomegalovirus pp28 (UL99) late gene.

The pp28 (UL99) gene of human cytomegalovirus is expressed as a true late gene, in that DNA synthesis is absolutely required for mRNA expression. Our previous studies demonstrated that pp28 promoter sequences from position -40 to +106 are sufficient for late gene expression in the context of the viral genome (C. P. Kohler, J. A. Kerry, M. Carter, V. P. Muzithras, T. R. Jones, and R. M. Stenberg, J. Virol. 68:6589-6597, 1994). To extend these studies, we have examined the sequences in the downstream leader region of the pp28 gene for their role in late gene expression. Deletion of sequences from position -6 to +46 (deltaSS) results in a threefold increase in gene expression in transient assays. In contrast, deletion of sequences from position +46 to +88 (deltaA) has little effect on gene expression. These results indicate that the sequences from position -6 to +46 may repress gene expression. To further analyze this region, site-directed mutagenesis was performed. Mutation of residues from either position +1 to +6 (SS1) or position +12 to +17 (SS2) duplicated the effect of the deltaSS deletion mutant, indicating that sequences from position +1 to +17 were important for the inhibitory effect. To assess the biological significance of these events, a recombinant virus construct containing the deltaSS mutant promoter regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene was generated. Analysis of this virus (RV delta SSCAT) revealed that deletion of sequences from position -6 to +46 does not alter the kinetic class of this promoter. However, the ratio of CAT protein to CAT mRNA levels in RV delta SSCAT-infected cells was 8- to 12-fold higher than that observed in the parental RV24/26CAT-infected cells. These results imply that the leader sequences within the pp28 gene can regulate the translation of this late gene.

Base Sequence↗

PubMed1997

The role of ATF in regulating the human cytomegalovirus DNA polymerase (UL54) promoter during viral infection.

Previous analysis of the human cytomegalovirus (HCMV) DNA polymerase (UL54) early gene promoter demonstrated that transcriptional activation of this gene is dependent upon the interaction of cellular transcription factors with viral transactivators (J. A. Kerry, M. A. Priddy, T. Y. Jervey, C. P. Kohler, T. L. Staley, C. D. Vanson, T. R. Jones, A. C. Iskenderian, D. G. Anders, and R. M. Stenberg, J. Virol. 70:373-382, 1996). A sequence element, IR1, was shown to be the primary regulatory element of this promoter in transient assays. However, assessment of this element in the context of the viral genome revealed IR1-independent activation at late times after infection. To extend these studies, we aim to identify additional sequence elements involved in the activation of the UL54 promoter. Our present studies demonstrate that the level of binding of proteins to the ATF site in the UL54 promoter is enhanced by viral infection. Furthermore this increase is sensitive to treatment with phosphonoacetic acid (PAA), a DNA synthesis inhibitor. These data suggest that the increase in the level of ATF binding activity is regulated, either directly or indirectly, by HCMV late gene expression. By using specific antibodies, we determined that ATF-1 was a major component of the proteins binding to the UL54 ATF site at late times. In addition, we have demonstrated direct binding of recombinant ATF-1 to the UL54 ATF site. To assess the biological significance of these events, a recombinant virus construct was generated that contained the UL54 promoter with a mutation in the ATF site regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene inserted between open reading frames US9 and US10. Analysis of this virus (RVATFmCAT) revealed that mutation of the ATF site does not alter the kinetics of UL54 promoter activation. However, levels of CAT mRNA and activity were reduced by 5- to 10-fold compared to those of the wild-type promoter at all stages of infection. These findings indicate that ATF-1 can regulate the levels of UL54 promoter activity at both early and late times. Furthermore, these results imply that HCMV can regulate the activity of cellular factors involved in early gene regulation.

Activating Transcription Factor 1↗

PubMed1996

Multiple regulatory events influence human cytomegalovirus DNA polymerase (UL54) expression during viral infection.

The human cytomegalovirus (HCMV) DNA polymerase gene (UL54; also called pol) is a prototypical early gene in that expression is mandatory for viral DNA replication. Recently, we have identified the major regulatory element in the UL54 promoter responsive to the major immediate early (MIE) proteins (UL122 and UL123) (J.A. Kerry, M.A. Priddy, and R. M. Stenberg, J. Virol. 68:4167-4176, 1994). Mutation of this element, inverted repeat sequence 1 (IR1), abrogates binding of cellular proteins to the UL54 promoter and reduces promoter activity in response to viral proteins in transient-transfection assays. To extend our studies on the UL54 promoter, we aimed to examine the role of IR1 in UL54 regulation throughout the course of infection. These studies show that viral proteins in addition to the MIE proteins can activate the UL54 promoter. Proteins from UL112-113 and IRS1/TRS1, recently identified as essential loci for transient complementation of HCMV oriLyt-dependent DNA replication, were found to function as transactivators of the UL54 promoter in association with MIE proteins. UL112-113 enhanced UL54 promoter activation by MIE proteins three- to fourfold. Constitutive expression of UL112-113 demonstrated that the MIE protein dependence of UL112-113 transactivational activity was not related to activation of cognate promoter sequences, suggesting that UL112-113 proteins function in cooperation with the MIE proteins. Mutation of IR1 was found to abrogate stimulation of the UL54 promoter by UL112-113, suggesting that this element is also involved in UL112-113 stimulatory activity. These results demonstrate that additional viral proteins influence UL54 promoter expression in transient-transfection assays via the IR1 element. To confirm the biological relevance of IR1 in regulating UL54 promoter activity during viral infection, a recombinant virus construct containing the UL54 promoter with a mutated IR1 element regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene (RVIRmCAT) was generated. Analysis of RVIRmCAT revealed that mutation of IR1 dramatically reduces UL54 promoter activity at early times after infection. However, at late times after infection CAT expression by RVIRmCAT, as assessed by RNA and protein levels, was approximately equivalent to expression by wild-type RVpolCAT. These data demonstrate IR1-independent regulation of the UL54 promoter at late times after infection. Together these results show that multiple regulatory events affect UL54 promoter expression during the course of infection.

Base Sequence↗

PubMed1995

Isolation and characterization of a low-abundance splice variant from the human cytomegalovirus major immediate-early gene region.

The major immediate-early (IE) gene region of human cytomegalovirus (HCMV) encodes several proteins as a result of differential RNA splicing events. By expression vector cloning of HCMV IE mRNA, we isolated and characterized a cDNA for a novel splice variant from the major IE gene region. The RNA product is a derivative of the IE55 mRNA and contains an additional splice from nucleotides 170,635 to 170,307 in the IE2 gene region (UL122), resulting in a 1.4-kb mRNA. The predicted open reading frame codes for a 164-amino-acid protein with a calculated molecular mass of 18 kDa (IE18). Mung bean nuclease analysis and PCR were used to characterize expression of IE18 mRNA in HCMV-infected cells. While the 1.4-kb mRNA was detected in infected human fibroblasts in the presence of a protein synthesis inhibitor, it was not detectable during a normal infection. However, the 1.4-kb mRNA was readily detected in infected human monocyte-derived macrophages at IE times. These results suggest that the novel IE18 mRNA exhibits cell type-specific expression indicating differential regulation of the major IE gene region in different permissive cell types.

Amino Acid Sequence↗

PubMed1995

Cytomegalovirus genes: their structure and function.

During the past several years, studies have indicated that cytomegalovirus (CMV) genes can be grouped into two broad categories; those essential for replication in cell culture and those dispensible for virus replication. The latter group of genes are likely to be important for pathogenesis and host-virus interactions. As the field progresses, the need to utilize and establish biological systems capable of addressing gene function during a natural infection of cells in culture, or in the infected animal, is becoming more apparent. Herein, we describe the current status of some of those systems, what has been learned and where these studies may lead. Specifically, we address studies that assess mechanisms of gene activation and function in biologically relevant systems. These include (i) the identification of genes dispensible for replication in cell culture, (ii) the use of dispensible regions of the CMV genome to manipulate genetic information for assessing gene function and activation, and (iii) the identification of a related group of essential loci important for replication of human CMV (HCMV) DNA and what is presently known of the function of those genes during HCMV infection.

Cytomegalovirus↗

PubMed1994

Use of recombinant virus to assess human cytomegalovirus early and late promoters in the context of the viral genome.

We have developed a system to study human cytomegalovirus (HCMV) cis-acting promoter elements within the context of the viral genome. A recombinant HCMV (RV134) containing a marker gene (beta-glucuronidase) was used to insert HCMV promoter-chloramphenicol acetyltransferase gene constructs into the viral genome between open reading frames US9 and US10. Using this system, we have studied the promoters for the early DNA polymerase gene (UL54), the early-late lower matrix phosphoprotein gene (pp65, UL83), and the true late 28-kDa structural phosphoprotein gene (pp28, UL99). Transient-expression assays demonstrated that the pp65 and pp28 promoters are activated earlier and to higher levels than typically observed with the endogenous gene. In contrast, insertion of these promoters into the viral genome resulted in kinetics which mimicked that of the endogenous genes. In addition, we have also tested a variant of the pp28 promoter (d24/26CAT) which is deleted from -609 to -41. This promoter behaved similarly to the wild-type pp28 promoter, indicating that sequences from -40 to +106 are sufficient for conferring true late kinetics. Taken together, these data demonstrate that the viral genome affords a level of regulation on HCMV gene expression that has been previously unrealized. Therefore, these experiments provide a model system for the analysis of cis-acting promoter regulatory elements in the context of the viral genome.

Blotting, Southern↗

PubMed1994

Identification of sequence elements in the human cytomegalovirus DNA polymerase gene promoter required for activation by viral gene products.

To determine the mechanisms involved in the regulation of human cytomegalovirus early gene expression, we have examined the gene that encodes the viral DNA polymerase (UL54, pol). Our previous studies demonstrated that sequences required for activation of the pol promoter by immediate-early proteins are contained within a region from -128 to +20 and that cellular proteins can bind to this activation domain. In this study, we demonstrate by competition analysis that binding of cellular proteins to pol is associated with an 18-bp region containing a single copy of a novel inverted repeat, IR1. Time course analysis indicated that viral infection increased the level of protein binding to IR1, concurrent with the activation of the pol promoter. Mutation of the IR1 element abrogated binding of cellular factors to the pol promoter and reduced by threefold the activation by immediate-early proteins. Similarly, mutation of IR1 rendered the promoter poorly responsive to activation by viral infection. Mutation of additional sequence elements in the pol promoter had little effect, indicating that IR1 plays the major role in pol promoter regulation. These studies demonstrate that the interaction between cellular factors and IR1 is important for the regulation of expression of the polymerase gene by viral proteins.

Base Sequence↗

PubMed1993

Amino acid substitutions alter the tissue distribution of murine interferon-alpha 1.

Novel analogs created by site-directed mutagenesis of murine interferon-alpha 1 (IFN-alpha 1) were used to examine the effect of alterations in structure and biological activity of murine IFN-alpha 1 on tissue distribution in mice. The analogs were biosynthetically labeled with [35S]methionine using a cell-free transcription-translation system and injected intravenously into adult male BALB/c mice. Levels of murine IFN-alpha 1 (dpm/gram wet weight) were highest in the liver, spleen, kidney, and lung, lower in the heart, and quite low in testis, brain, skin, and muscle. The tissue distribution of the analogs differed from that of murine IFN-alpha 1. In general, analogs with reduced antiviral activity showed reduced uptake by the spleen and lung. The amount in the kidney of the analog R33E, which has no detectable antiviral activity in vitro, was substantially higher than that of native IFN, suggesting a greater rate of excretion of this analog. An analog of human IFN-alpha 4, which had increased antiviral activity on murine cells, showed increased uptake in the liver, spleen, and lung. These findings, together with the results of a previous study using autoradiography (Johns et al., 1990, Cancer Res. 50, 4718-4723) indicate that nonspecific uptake by parenchymal cells in the liver, spleen, and lung is unaffected by changes in antiviral activity, while specific, receptor-mediated localization of IFN in regions rich in macrophages is reduced in accordance with the reduction in antiviral activity.

Amino Acids↗

PubMed1990

Pharmacokinetics, tissue distribution, and cell localization of [35S]methionine-labeled recombinant human and murine alpha interferons in mice.

The pharmacokinetics, tissue distribution, cell localization, and penetration into tumor xenografts of recombinant [35S]methionine-labeled human alpha interferon (HuIFN-alpha) and murine alpha interferon (MuIFN-alpha) were examined in mice. Both interferons (IFNs) were removed from the blood in a rapid biphasic manner; HuIFN-alpha was cleared faster than MuIFN-alpha. Tissues were analyzed for radioactivity and over 90% of the IFNs was accounted for. The IFNs were detected predominantly in liver, kidney, gastrointestinal tract, pancreas, spleen, and lung. The levels of MuIFN-alpha compared with HuIFN-alpha were greater in the liver, spleen, and lung and less in the kidney, pancreas, and gastrointestinal tract. Heart, brain, testes, thymus, lymph nodes, fat, skin, and skeletal muscle contained much lower but measurable levels of both IFNs. There was penetration of HuIFN-alpha into tumor xenografts. The pharmacokinetics of IFN-alpha were independent of the strain of mouse, BALB/c or CBA, immune deprivation, or the presence of a tumor xenograft. Autoradiography of tissue sections from mice given injections of HuIFN-alpha or MuIFN-alpha indicated focal radioactivity in proximal convoluted tubules in the kidney and diffuse radioactivity in the liver, gastrointestinal tract, and pancrease. MuIFN-alpha, but not HuIFN-alpha, showed intense localization in cells in hepatic sinusoids, marginal zones in the spleen, and pulmonary alveolar walls, suggesting uptake by cells of the monocyte/macrophage lineage in these sites. The study shows the utility of biosynthetic labeling for pharmacokinetic studies of cytokines, clear differences in tissue distribution of IFN-alpha according to its species of origin, and targeting of homologous IFN-alpha to cells of the monocytic lineage.

Animals↗

PubMed1988

Conserved amino acid residues arginine 33 and tyrosine 123 are critical for the antiviral activity of murine interferon-alpha 1.

Analogues of murine interferon-alpha 1 were produced by site directed mutagenesis and expressed in vitro. Replacement of the conserved residue arginine 33 by glutamic acid resulted in an interferon with no detectable antiviral activity. Substitution of tyrosine residue 123 shows dramatic reductions in the antiviral activity. These analogues demonstrate the importance of the conserved residues arginine 33 and tyrosine 123 in maintaining the antiviral activity of murine interferon-alpha 1.

Amino Acid Sequence↗

PubMed1986

Sheep brain pyridoxal kinase: fluorescence spectroscopy of the dimeric enzyme.

Pyridoxal kinase (ATP:pyridoxal 5-phosphotransferase, EC 2.7.1.35) has been purified 9000-fold from sheep brain by affinity chromatography. The enzyme of 80,000 molecular weight is made up of two identical-size subunits. The interaction of the inhibitor N-dansyl-1,8-diaminooctane with the nucleotide site of the kinase was examined by means of steady and nanosecond fluorescence spectroscopy. N-Dansyl-1,8-diaminooctane is a competitive inhibitor with respect to ATP at saturating concentrations of pyridoxal. It binds to the nucleotide site of the enzyme with Kd = 2.2 microM. Bound N-dansyl-1,8-diaminooctane is shielded from collisional encounters with the external quencher acrylamide. The collisional rate constant for bound N-dansyl-1,8-diaminooctane (Kq = 1.4 X 10(8) M-1 X s-1) is 10-times lower than the value obtained for the free chromophore. Nanosecond emission anisotropy measurements yield a rotational correlation time of 42 ns for the inhibitor complexes to the kinase. Both steady and nanosecond fluorescence results are consistent with a model in which the inhibitor bound to the nucleotide site is immobilized by amino acids located at the catalytic site.

Animals↗

PubMed1986

Brain pyridoxal kinase. Purification and characterization.

Pyridoxal kinase has been purified 9000-fold from sheep brain. The purification procedure involves ammonium sulphate fractionation, DEAE-cellulose chromatography, affinity chromatography and Sephadex G-100 gel filtration. The final chromatography step yields a homogeneous preparation of high specific activity with a pI of 5. The molecular mass of the native enzyme was estimated to be approximately 80 kDa by 10-25% gradient polyacrylamide gel electrophoresis and Sephadex G-200 gel filtration. The subunit molecular mass was determined by sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis to be 40 kDa compared with a series of molecular mass standards. This indicates that pyridoxal kinase is a dimeric enzyme. Further results obtained from electron microscopy, using a negative staining technique, provide evidence that pyridoxal kinase exists as a dispherical subunit structure.

Animals↗

PubMed1986

Purification and characterization of pyridoxal kinase from human erythrocytes.

Pyridoxal kinase has been purified 50,000-fold from human erythrocytes. The purification procedure included dextran-induced aggregation of red blood cells, ammonium sulphate fractionation of the haemolysate, DEAE-cellulose chromatography, hydroxyapatite chromatography. Sephadex G-100 gel filtration and omega-aminooctyl agarose chromatography. The enzyme preparation migrated as a single protein and activity band on analytical gel electrophoresis. Determination of the Michaelis constants for pyridoxal, pyridoxine and pyridoxamine using a new assay gave comparable values of 33 microM, 16 microM and 6.2 microM respectively. Various amines were shown as competitive inhibitors of pyridoxal kinase with respect to ATP. The inhibition order was: N-dansyl-1,8-diaminooctane greater than 1,8-diaminooctane greater than 1,6-diaminohexane greater than 1,4-diaminobutane greater than gamma-aminobutyric acid, whereas octane, hexane and butane were not inhibitors. Results suggest that the amino groups on the above inhibitors are essential for competitive inhibition at saturating concentrations of pyridoxal. It was also observed that increasing the chain length of the hydrophobic backbone of these competitive inhibitors can facilitate its action.

Catalysis↗