Biomedical subjects

J M Hardwick

Publications and source records attributed to J M Hardwick.

At least 55 records · Page 3Linked to original sources

PubMed1996

Bc1-2 protects mice against fatal alphavirus encephalitis.

Virus-induced apoptosis has been well characterized in vitro, but the role of apoptosis in viral pathogenesis is not well understood. The suicide of a cell in response to viral infection is postulated to be an important host defense for the organism, leading to a reduction in its total viral burden. However, virus-induced death of nonregenerating cells in the central nervous system may be detrimental to the host. Therefore, to investigate the role of apoptosis in the pathogenesis of fatal encephalitis, we constructed a recombinant alphavirus chimera that expresses the antiapoptotic gene, bcl-2, in virally infected neural cells. Infection of neonatal mice with the alphavirus chimera expressing human bcl-2 [Sindbis virus (SIN)/bcl-2] resulted in a significantly lower mortality rate (7.5%) as compared with infection with control chimeric viruses containing a chloramphenicol acetyltransferase (CAT) reporter gene (SIN/CAT) (78.1%) or bcl-2 containing a premature stop codon (SIN/bcl-2stop) (72.1%) (P < 0.001). Viral titers were reduced 5-fold 1 day after infection and 10-fold 6 days after infection in the brains of SIN/bcl-2-infected mice as compared to SIN/CAT or SIN/bcl-2stop-infected mice. In situ end labeling to detect apoptotic nuclei demonstrated a reduction in the number of foci of apoptotic cells in the brains of mice infected with SIN/bcl-2 as compared with SIN/bcl-2stop. The reduction in apoptosis was associated with a reduction in the number of foci of cells expressing alphavirus RNA. Thus, the antiapoptotic gene, bcl-2, suppresses viral replication and protects against a lethal viral disease, suggesting an interaction between cellular genetic control of viral replication and cell death.

Alphavirus Infections↗

PubMed1996

Bax-independent inhibition of apoptosis by Bcl-XL.

The Bcl-2-related protein, Bcl-XL, has been shown to block apoptosis induced by a variety of stimuli and to be a stronger protector against apoptosis than Bcl-2 under certain circumstances. Using site-specific mutagenesis, we show here that the amino-acid residues critical for protection of cells by Bcl-XL against Sindbis virus-induced apoptosis are clustered within the Bcl-2-homology regions 1 and 2 (BH1 and BH2 regions). The residues necessary for Bcl-XL function are not identical to those required for Bcl-2 function. Although it has been suggested that heterodimerization between Bcl-XL and Bax is essential for the anti-death activity of Bcl-XL (refs 7,8), our results suggest that the interaction with Bax is not required for Bcl-XL to exert its death-repressing activity. Specific mutations that disrupt the ability of Bcl-XL to interact with Bax or Bak still preserve 70-80% of the anti-death activity of wild-type Bcl-XL.

Amino Acid Sequence↗

PubMed1996

BCL-2 but not its Epstein-Barr virus-encoded homologue, BHRF1, is commonly expressed in posttransplantation lymphoproliferative disorders.

Posttransplantation lymphoproliferative disease (PTLD) is virtually always associated with Epstein-Barr virus (EBV) infection. BCL-2 and other proteins that confer resistance to apoptosis have been implicated in the pathogenesis of a variety of malignancies including lymphomas. One EBV protein, BHRF1, is a homologue of BCL-2, whereas another, the latency membrane protein 1 (LMP-1), upregulates BCL-2 expression in vitro. In the present study, we used immunohistochemistry to study the expression of these viral and cellular proteins as well as a variety of other EBV-encoded proteins in PTLD. BHRF1 was not detected in any PTLD specimen, whereas BCL-2 was shown in 12 of 17 lesions examined. With one exception, all LMP1-positive cases also expressed BCL-2 and the absence of LMP1 was always associated with a lack of BCL-2 expression. The results do not support a role for the EBV homologue of BCL-2 in PTLD, but they do support a role for viral induction of BCL-2 expression.

Amino Acid Sequence↗

PubMed1996

Alphavirus-induced apoptosis in mouse brains correlates with neurovirulence.

Sindbis virus induces apoptotic cell death in cultured cell lines, raising the possibility that apoptosis of infected neurons and other target cells in vivo may contribute to the resulting disease and mortality. To investigate the role of apoptosis in Sindbis virus pathogenesis, infected mouse brains were assayed by the in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling technique and for DNA ladder formation. Infection with recombinant Sindbis virus strain 633 resulted in widespread apoptosis in newborn mouse brains and spinal cords, but few apoptotic cells were observed following infection of 2-week-old animals. This finding correlates with the age-dependent mortality observed in mice. The more neurovirulent virus TE, which differs from 633 by a single amino acid in the E2 glycoprotein, induced significant apoptosis in brains and spinal cords of 2-week-old animals, consistent with its ability to cause fatal disease in older animals. Double-labeling experiments demonstrated that the apoptotic cells were also infected with Sindbis virus. Thus, Sindbis virus-induced apoptosis appears to be a result of virus infection and is likely to reflect pathogenic mechanisms for other viruses.

Age Factors↗

PubMed1996

Anti-apoptotic genes of baculoviruses.

Baculoviruses possess two different classes of genes with anti-apoptptic activity: p35 and iap. The p35 gene product (P35) is able to block apoptosis induced by a variety of stimuli in phylogenetically diverse organisms. P35 has recently been shown to be capable of inhibiting the ICE/ced-3 family of cysteine proteases, a family of enzymes which are implicated in cell death and which exhibit specificity for cleavage at aspartate residues. The products of the iap genes are a distinct class of proteins containing a carboxyl ring finger and tandem duplications of a unique motif known as the BIR motif. Homologues of the baculovirus iap genes have been identified in the human genome. Both classes of baculovirus anti-apoptotic genes will continue to be important tools in defining the pathways involved in apoptosis. Since our demonstration in 1991 that a baculovirus prevents host cells from undergoing apoptosis by expressing a gene known as p35(Clem et al., 1991), the study of baculovirus-induced apoptosis and the anti-apoptotic genes they possess has led to discoveries with far-reaching implications for viral pathogenesis, human disease, and the study of cell death. It is now known that a variety of eukaryotic viruses encode genes which allow them to control cellular apoptosis. Understanding the mechanism(s) by which these viral gene products act provides fundamental insights into the pathways regulating apoptosis. In this review, we discuss the inhibition of apoptosis by baculoviruses, concentrating mainly on the nature and mechanism of action of the two classes of baculovirus genes, p35 and iap, which are able to control apoptosis in a diversity ofeukaryotes.

Journal Article↗

PubMed1995

Thiol agents and Bcl-2 identify an alphavirus-induced apoptotic pathway that requires activation of the transcription factor NF-kappa B.

Oxidative stress has been proposed as a common mediator of apoptotic death. To investigate further the role of oxidants in this process we have studied the effects of antioxidants on Sindbis virus (SV)-induced apoptosis in two cell lines, AT-3 (a prostate carcinoma line) and N18 (a neuroblastoma line). The thiol antioxidant, N-acetylcysteine (NAC), at concentrations above 30 mM, completely abrogates SV-induced apoptosis in AT-3 and N18 cells. The effects of NAC cannot be attributed to inhibition of viral entry or viral replication, changes in extracellular osmolarity or to increases in cellular glutathione levels, nor can they be mimicked by chelators of trace metals, inhibitors of lipid peroxidation or peroxide scavengers. In contrast, other thiol agents including pyrrolidine dithiocarbamate (PDTC, 75 microM) are protective. Because NAC and PDTC are among the most effective inhibitors of the transcription factor NF-kappa B, we examined SV's ability to activate NF-kappa B before the onset of morphologic or biochemical evidence of apoptosis. Within hours of infection, SV induced a robust increase in nuclear NF-kappa B activity in AT-3 and N18 cells; this activation was suppressible by NAC and PDTC. Over-expression of bcl-2 in AT-3 cells, which has been shown to inhibit SV-induced apoptosis, also inhibits SV-induced NF-kappa B activation. To determine if NF-kappa B activation is necessary for SV-induced apoptosis in these cells, we used double stranded oligonucleotides with consensus NF-kappa B sequences as transcription factor decoys (TFDs) to inhibit NF-kappa B binding to native DNA sites. Wild-type, but not mutant, TFDs inhibit SV-induced apoptosis in AT-3 cells. In contrast, TFD inhibition of NF-kappa B nuclear activity in N18 cells did not prevent SV-induced apoptosis. Taken together, these observations define a cell type-specific, transcription factor signaling pathway necessary for SV-induced apoptosis. Understanding the precise mechanism by which Bcl-2 and thiol agents inhibit SV-induced nuclear NF-kappa B activity in AT-3 cells may provide insights into the pluripotent antiapoptotic actions of these agents.

Acetylcysteine↗

PubMed1995

Apoptosis as a cause of death in measles virus-infected cells.

To determine the mechanism of measles virus-induced cell death, we studied the infection of Vero cells and monocytic cell lines with wild-type (Chicago-1) and vaccine (Edmonston) strains of measles virus. DNA fragmentation indicative of apoptosis was apparent by flow cytometry, agarose gel electrophoresis, and electron microscopy. Within syncytia, DNA strand breaks were demonstrated by end labeling with terminal transferase and then by visualization.

Animals↗

PubMed1994

Neurovirulent strains of Alphavirus induce apoptosis in bcl-2-expressing cells: role of a single amino acid change in the E2 glycoprotein.

The isolation and sequence comparison of avirulent and neurovirulent strains of polio virus, alpha virus, herpes virus, immunodeficiency virus, and other viruses have identified genetic changes that are required to cause disease in the nervous system. The molecular mechanisms by which these genetic changes result in neurovirulence are unknown. An avirulent laboratory strain of the Alphavirus Sindbis kills most cultured cell lines not by lethal parasitism, but by inducing apoptosis or programmed cell death. Transfection of cultured cells with the human bcl-2 oncogene can block Sindbis virus-induced apoptosis, resulting in a persistent viral infection resembling that observed in brains of immunodeficient mice. We investigated the possibility that neurovirulent strains of Sindbis virus could overcome the protective effects of bcl-2--a potential mechanism to explain the ability of these strains to cause fatal disease. Strains of Sindbis virus that were lethal for 2- to 4-week-old mice induced apoptotic death in cultured cells despite the presence of bcl-2. Using recombinant viruses, we show that a single amino acid change in the E2 glycoprotein of Sindbis virus confers both neurovirulence and the ability to kill cells expressing bcl-2.

Amino Acid Sequence↗

PubMed1994

The effects of alphavirus infection on neurons.

Sindbis virus is an alphavirus that causes encephalitis in mice. The primary target cells for central nervous system infection are neurons. The outcome of neuronal infection is dependent on the age of the mouse at the time of infection (maturity of the neuron) and the strain of virus used for infection (virulence of the virus). Sindbis virus causes neuronal death by inducing apoptosis. As neurons mature, they become resistant to virus-induced apoptosis, resulting in a persistent infection. Host production of antibody to a viral surface glycoprotein acts to downregulate virus replication in the infected neurons by a noncytolytic mechanism and clears infectious virus from the central nervous system. Specific genetic changes in the virus result in more virulent strains that cause severe disease and sometimes death in mature animals. These same genetic alterations also confer the ability to overcome the resistance of neurons to induction of cell death. Therefore, mature neurons infected with virulent viruses do not recover from infection even in the presence of an adequate immune response.

Aging↗

PubMed1994

Age-dependent susceptibility to fatal encephalitis: alphavirus infection of neurons.

Sindbis virus encephalitis in mice provides a model for studying age-dependent susceptibility to acute viral encephalitis. The AR339 strain of SV causes fatal encephalitis in newborn mice, but weanling mice recover uneventfully. Increased virulence for older mice is associated with a single amino acid change from Gln to His at position 55 of the E2 glycoprotein. Weanling mice with normal immune systems clear infectious virus from neurons through an antibody-mediated mechanism. This does not happen in newborn mice because the infected neurons die soon after they are infected. Death in immature neurons, as well as most other mammalian cells infected with Sindbis virus, occurs by induction of apoptosis. This can be prevented by cellular expression of bcl-2, an inhibitor of apoptosis, which is expressed by mature neurons in culture. We conclude that mature neurons are resistant to induction of apoptosis after infection with SV through expression of cellular inhibitors of apoptosis. This provides the opportunity for antibody to clear virus by a noncytolytic mechanism.

Aging↗

PubMed1994

Persistence of alphaviruses in vertebrate hosts.

Alphaviruses are a group of arthropod-borne, positive-strand RNA viruses that cause acute encephalitis or arthritis. These viruses were previously thought to cause only acute infections in vertebrates, but recent evidence suggests that host immunological and tissue-specific factors may act together to promote the persistence of alphavirus genomes in vivo.

Alphavirus↗

PubMed1994

Visna virus Tat protein: a potent transcription factor with both activator and suppressor domains.

Visna virus is a pathogenic lentivirus of sheep tat is distantly related to the primate lentiviruses, including human immunodeficiency virus type 1. The visna virus genome encodes a small regulatory protein, Tat, which is necessary for efficient viral replication and enhanced viral transcription. To investigate the mechanism of action of the visna Tat protein and to localize the protein domain(s) responsible for transcriptional activation, chimeric proteins containing visna virus Tat sequences fused to the DNA binding domain of the yeast transactivation factor GAL4 (residues 1 to 147) were made. The GAL4-Tat fusion proteins were transfected into cells and tested for the ability to activate the adenovirus E1b promoter via upstream GAL4 DNA binding sites. Full-length GAL4-Tat fusion proteins were weak transactivators in this system, giving only a two- to fourfold increase in transcription in several cell types, including HeLa and sheep choroid plexus cells. In contrast, fusion of the N-terminal region of the Tat protein to GAL4 revealed a potent activation domain. Amino acids 13 to 38 appeared to be the most critical for activation. No other region of the protein showed any activation in the GAL4 system. This N-terminal region of the visna virus Tat protein has a large number of acidic and hydrophobic residues, suggesting that Tat has an acidic activation domain common to many transcriptional transactivators. Mutations in hydrophobic and bulky aromatic residues dramatically reduced the activity of the chimeric protein. Competition experiments suggest that mechanism of the visna virus Tat activation domain may closely resemble that of the herpesvirus activator VP16 and human immunodeficiency virus Tat, a related lentivirus activator, since both significantly reduce the level of visna virus Tat activation. Finally, a domain between residues 39 and 53 was identified in the Tat protein that, in the GAL4 system, negatively regulates activation by Tat.

Adenovirus E1B Proteins↗

PubMed1993

Conversion of lytic to persistent alphavirus infection by the bcl-2 cellular oncogene.

Little is known about virus-host cell interactions that regulate the lytic potential of viruses during productive replication. Sindbis virus (SV), a single-stranded positive-sense RNA virus in the alphavirus genus (family Togaviridae), results in lytic infection in most vertebrate cell lines, but persistent productive infection in post-mitotic neurons. The cellular oncogene bcl-2, which encodes an inner mitochondrial membrane protein of M(r) 26,000 (ref. 2), blocks programmed cell death (apoptosis) in neurons. We therefore investigated whether SV infection induces programmed cell death in non-neuronal cells, and if so, whether virus-induced programmed cell death can be blocked by transfection with bcl-2. We demonstrate that SV infection of baby hamster kidney (BHK-2), mouse neuroblastoma (N18), and rat prostatic adenocarcinoma (AT-3) cells results in programmed cell death, whereas SV infection of bcl-2-transfected AT-3 cells results in long-term persistent productive infection. Thus cellular bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced programmed cell death.

Adenocarcinoma↗

PubMed1992

The Epstein-Barr virus R transactivator (Rta) contains a complex, potent activation domain with properties different from those of VP16.

Rta, encoded by Epstein-Barr virus (EBV), is a potent activator of transcription via enhancer sequences located upstream of several viral genes. To identify the domains of Rta that facilitate transcription by interacting with cellular transcription factors, different segments of Rta were linked to the DNA binding domain of yeast transactivator GAL4 (residues 1 to 147). These GAL4-Rta fusion proteins were tested in transfected cells for their ability to activate the adeno E1b promoter with an upstream GAL4 DNA binding site. The acidic C-terminal domain of Rta (amino acids 520 to 605) was a potent activator but behaved differently from VP16 in dose-response and competition experiments. A subterminal domain of Rta (amino acids 416 to 519) linked to GAL4 had weak activation activity. Deletion of these domains from native Rta showed that the C-terminal domain was required for transactivation, but the subterminal domain was required only in B cells. The C-terminal activation domain of Rta contains a pattern of positionally conserved hydrophobic residues shared with VP16 and other transactivators. Substitution of several conserved hydrophobic amino acids in Rta severely impaired transactivation. The improtance of hydrophobic residues was further substantiated by comparing EBV Rta with that of herpesvirus saimiri, which revealed little sequence similarity except for a few acidic residues and the positionally conserved hydrophobic amino acids. The C-terminal domain of EBV Rta contains three partially overlapping copies of this hydrophobic motif. Mutational analysis indicated that all three copies were required for full activity. However, two of the three copies appeared to be sufficient to produce full activity on a target promoter with multiple binding sites, suggesting that these motifs are functional subdomains that can synergize.

Amino Acid Sequence↗

PubMed1991

Antibody-mediated clearance of alphavirus infection from neurons.

Humoral immunity is important for protection against viral infection and neutralization of extracellular virus, but clearance of virus from infected tissues is thought to be mediated solely by cellular immunity. However, in a SCID mouse model of persistent alphavirus encephalomyelitis, adoptive transfer of hyperimmune serum resulted in clearance of infectious virus and viral RNA from the nervous system, whereas adoptive transfer of sensitized T lymphocytes had no effect on viral replication. Three monoclonal antibodies to two different epitopes on the E2 envelope glycoprotein mediated viral clearance. Treatment of alphavirus-infected primary cultured rat neurons with these monoclonal antibodies to E2 resulted in decreased viral protein synthesis, followed by gradual termination of mature infectious virion production. Thus, antibody can mediate clearance of alphavirus infection from neurons by restricting viral gene expression.

Alphavirus↗

PubMed1990

An enhancer within the divergent promoter of Epstein-Barr virus responds synergistically to the R and Z transactivators.

The EA-R and NotI repeat genes of Epstein-Barr virus (EBV) are oriented head to head and separated by a 1,000-base-pair (bp) divergent promoter region. We have identified functional domains within this divergent promoter which are important for regulation of the rightward EA-R gene. Both the R transactivator (Rta) and the Z transactivator (Zta) increase the abundance of correctly initiated EA-R transcripts. A 258-bp fragment (-114 to -372 from the EA-R cap site) contained the primary Rta and Zta response elements and was capable of transferring Rta and Zta activity to a heterologous promoter in an orientation- and position-independent manner. Rta activated this 258-bp enhancer region in both EBV-positive and EBV-negative cells. However, Zta activity appeared to be dependent on another EBV gene product, since Zta activated the enhancer efficiently (500- to 2,000-fold) in EBV-positive cells but had little or no activity in EBV-negative cells. The combination of Rta and Zta produced a striking synergistic effect on the enhancer in the absence of any additional EBV components, suggesting that the interaction between Zta and Rta accounts for the Zta response observed in EBV-positive cells. An Rta response element was mapped to a domain located 60 bp away from a Zta-binding site within the enhancer. Although Rta activated the enhancer and other early promoters without additional EBV- or B-cell-specific factors, it did not activate the lytic cycle of EBV, in contrast to Zta. Immunofluorescence patterns of Rta and Zta with antipeptide antisera indicated that they have overlapping but different subcellular localizations. Both transactivators were found in the nucleus, but Rta was also found in the cytoplasm.

Animals↗

PubMed1990

The zta transactivator involved in induction of lytic cycle gene expression in Epstein-Barr virus-infected lymphocytes binds to both AP-1 and ZRE sites in target promoter and enhancer regions.

The BZLF1 or zta immediate-early gene of Epstein-Barr virus (EBV) encodes a 33-kilodalton phosphorylated nuclear protein that is a specific transcriptional activator of the EBV lytic cycle when introduced into latently infected B lymphocytes. We have shown previously that the divergent EBV DSL target promoter contains two zta-response regions, one within the minimal promoter and the other in an upstream lymphocyte-dependent enhancer region. In this study, we used footprinting and gel mobility retardation assays to reveal that bacterially synthesized Zta fusion proteins bound directly to six TGTGCAA-like motifs within DSL. Four of the Zta-binding sites lay adjacent to cellular TATA and CAAT factor-binding sites within the minimal promoter, and two mapped within the enhancer region. Single-copy oligonucleotides containing these Zta-binding sites conferred Zta responsiveness to heterologous promoters. In addition, the Zta protein, which possesses a similar basic domain to the conserved DNA-binding region of the c-Fos, c-Jun, GCN4, and CREB protein family, proved to bind directly to the consensus AP-1 site in the collagenase 12-O-tetradecanoylphorbol-13-acetate response element. Cotransfection with zta also trans activated a target reporter gene containing inserted wild-type 12-O-tetradecanoylphorbol-13-acetate response element oligonucleotides. Cellular AP-1 binding activity proved to be low in latently EBV-infected Raji cells but was induced (together with the Zta protein) after activation of the lytic cycle with 12-O-tetradecanoylphorbol-13-acetate. We conclude that EBV may have captured and modified a cellular gene encoding a c-jun-like DNA-binding protein during its evolutionary divergence from other herpesviruses and that this protein is used to specifically redirect transcriptional activity toward expression of EBV lytic-cycle genes in infected cells.

Amino Acid Sequence↗

PubMed1989

Responsiveness of the Epstein-Barr virus NotI repeat promoter to the Z transactivator is mediated in a cell-type-specific manner by two independent signal regions.

Cells latently infected with Epstein-Barr virus (EBV) can be activated to express lytic-cycle polypeptides by the introduction of the EBV-encoded Z transactivator, indicating that this protein has a pivotal role in virus reactivation. We examined the target specificity of the Z transactivator in short-term contransfection assays and found that the most responsive target to Z transactivation was the divergent NotI repeat promoter, located within the EBV BamHI H fragment. In contrast, target plasmids containing the cat gene linked to heterologous viral promoters were not activated by cotransfection with the Z gene. S1 nuclease analysis of RNA from chemically induced B95-8 cells and from Vero cells cotransfected with NotI repeat promoter-CAT and Z showed that Z transactivation increased the level of correctly initiated, stable RNA transcripts. The NotI repeat gene (ntr) gives rise to a highly abundant mRNA species after chemical induction of lytic virus replication, but no protein product had been previously identified. Using monospecific antiserum raised against a synthetic peptide from the BHLF1 open reading frame, we demonstrated that the ntr gene encodes a protein product that is found in nuclear patches colocalizing with nucleoli. A series of deletions introduced into the upstream sequences of the NotI-repeat-promoter revealed two separate Z-response regions. The minimal promoter region between -7 and -155 of the leftward RNA cap site and an upstream region between -644 and -902 were both independently capable of conferring Z responsiveness. However, the minimal region, which was activated by Z cotransfection in Vero cells, was poorly responsive in lymphocytes, whereas the response of the far-upstream region to Z cotransfection was lymphocyte specific. In its human host, EBV infects both epithelial and lymphocyte populations. This dual lifestyle may have led to the evolution of multiple Z-response signals that enable the Z transactivator to interact with both cell-specific promoter and enhancer factors.

Animals↗