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Biomedical subjects

P Ghazal

Publications and source records attributed to P Ghazal.

At least 37 records · Page 2Linked to original sources
PubMed2001

Immune checkpoints in viral latency.

The dynamics of the relationship between the immune system and latent viruses are highly complex. Latent viruses not only avoid elimination by the host's primary immune response, they also remain with the host for life in the presence of strong acquired immunity, often exhibiting periodic reactivation and recurrence from the latent state. The continual battle between reemergent infectious virus and immunological memory cells provides an essential virus-host regulatory loop in latency. In this review, we speculate on the critical importance of immune interference mechanisms by viruses contributing to the regulatory loop in viral homeostasis of latency. Central to the notion of viral homeostasis, we further invoke the concept of threshold limits in naive and memory states of immunity to account for the failure of the host to completely eradicate these intracellular parasites.

HIV↗
PubMed2000

Rapid antibody responses by low-dose, single-step, dendritic cell-targeted immunization.

We have compared the kinetics of antibody responses in conventional and dendritic cell-targeted immunization by using a model antigen in mice. Targeting was achieved by linking the reporter antigen (polyclonal goat anti-hamster antibody) to N418, a hamster mAb that binds to the CD11c molecule on the surface of murine dendritic cells. Intradermal injection of submicrogram quantities of goat anti-hamster antibody complexed to mAb N418 elicited goat antibody-specific serum IgG in mice. Antigen-specific IgG titers were detectable by day 5, with titers that ranged from 1:1000 to 1:100,000 by day 7. In contrast, when the goat antigen was injected alone or in the presence of a hamster antibody control to form nontargeted complexes, goat-specific serum IgG was undetectable at day 7. Additional control experiments showed that the interaction between the model antigen and mAb N418 is required for amplification of the serum antibody response. These studies demonstrate that a single-step, facilitated-delivery of small amounts of protein antigen to dendritic cells in vivo can give very rapid and high antibody responses. The approach may be particularly useful for vaccination immediately before or just after exposure to a pathogen and may enhance the utility of subunit antigens as immunogens.

Animals↗
PubMed2000

Annexation of the interchromosomal space during viral infection.

The nucleus is known to be compartmentalized into units of function, but the processes leading to the spatial organization of chromosomes and nuclear compartments are not yet well defined. Here we report direct quantitative analysis of the global structural perturbations of interphase chromosome and interchromosome domain distribution caused by infection with herpes simplex virus-1 (HSV-1). Our results show that the peripheral displacement of host chromosomes that correlates with expansion of the viral replication compartment (VRC) is coupled to a twofold increase in nuclear volume. Live cell dynamic measurements suggest that viral compartment formation is driven by the functional activity of viral components and underscore the significance of spatial regulation of nuclear activities.

Cell Nucleus↗
PubMed2000

Global analysis of herpes simplex virus type 1 transcription using an oligonucleotide-based DNA microarray.

More than 100 transcripts of various abundances and kinetic classes are expressed during phases of productive and latent infections by herpes simplex virus (HSV) type 1. To carry out rapid global analysis of variations in such patterns as a function of perturbation of viral regulatory genes and cell differentiation, we have made DNA microchips containing sets of 75-mer oligonucleotides specific for individual viral transcripts. About half of these are unique for single transcripts, while others function for overlapping ones. We have also included probes for 57 human genes known to be involved in some aspect of stress response. The chips efficiently detect all viral transcripts, and analysis of those abundant under various conditions of infection demonstrates excellent correlation with known kinetics of mRNA accumulation. Further, quantitative sensitivity is high. We have further applied global analysis of transcription to an investigation of mRNA populations in cells infected with a mutant virus in which the essential immediate-early alpha27 (U(L)54) gene has been functionally deleted. Transcripts expressed at 6 h following infection with this mutant can be classified into three groups: those whose abundance is augmented (mainly immediate-early transcripts) or unaltered, those whose abundance is somewhat reduced, and those where there is a significant reduction in transcript levels. These do not conform to any particular kinetic class. Interestingly, levels of many cellular transcripts surveyed are increased. The high proportion of such transcripts suggests that the alpha27 gene plays a major role in the early decline in cellular gene expression so characteristic of HSV infection.

Animals↗
PubMed2000

The major immediate-early gene ie3 of mouse cytomegalovirus is essential for viral growth.

The significance of the major immediate-early gene ie3 of mouse cytomegalovirus (MCMV) and that of the corresponding ie2 gene of human cytomegalovirus to viral replication are not known. To investigate the function of the MCMV IE3 regulatory protein, we generated two different MCMV recombinants that contained a large deletion in the IE3 open reading frame (ORF). The mutant genomes were constructed by the bacterial artificial chromosome mutagenesis technique, and MCMV ie3 deletion mutants were reconstituted on a mouse fibroblast cell line that expresses the MCMV major immediate-early genes. The ie3 deletion mutants failed to replicate on normal mouse fibroblasts even when a high multiplicity of infection was used. The replication defect was rescued when the IE3 protein was provided in trans by a complementing cell line. A revertant virus in which the IE3 ORF was restored was able to replicate with wild-type kinetics in normal mouse fibroblasts, providing evidence that the defective growth phenotype of the ie3 mutants was due to disruption of the ie3 gene. To characterize the point of restriction in viral replication that is controlled by ie3, we analyzed the pattern of expression of selective early (beta) and late (gamma) genes. While we could detect transcripts for the immediate-early gene ie1 in cells infected with the ie3 mutants, we failed to detect transcripts for representative beta and gamma genes. These data demonstrate that the MCMV transactivator IE3 plays an indispensable role during viral replication in tissue culture, implicating a similar role for the human CMV ie2 gene product. To our knowledge, the ie3 deletion mutants represent the first MCMV recombinants isolated that contain a disruption of an essential gene.

3T3 Cells↗
PubMed2000

Identification of a boundary domain adjacent to the potent human cytomegalovirus enhancer that represses transcription of the divergent UL127 promoter.

Transcriptional repression within a complex modular promoter may play a key role in determining the action of enhancer elements. In human cytomegalovirus, the major immediate-early promoter (MIEP) locus contains a highly potent and complex modular enhancer. Evidence is presented suggesting that sequences of the MIEP between nucleotide positions -556 and -673 function to prevent transcription activation by enhancer elements from the UL127 open reading frame divergent promoter. Transient transfection assays of reporter plasmids revealed repressor sequences located between nucleotides -556 and -638. The ability of these sequences to confer repression in the context of an infection was shown using recombinant viruses generated from a bacterial artificial chromosome containing an infectious human cytomegalovirus genome. In addition to repressor sequences between -556 and -638, infection experiments using recombinant virus mutants indicated that sequences between -638 and -673 also contribute to repression of the UL127 promoter. On the basis of in vitro transcription and transient transfection assays, we further show that interposed viral repressor sequences completely inhibit enhancer-mediated activation of not only the homologous but also heterologous promoters. These and other experiments suggest that repression involves an interaction of host-encoded regulatory factors with defined promoter sequences that have the property of proximally interfering with upstream enhancer elements in a chromatin-independent manner. Altogether, our findings establish the presence of a boundary domain that efficiently blocks enhancer-promoter interactions, thus explaining how the enhancer can work to selectively activate the MIEP.

Base Sequence↗
PubMed2000

Principles of homeostasis in governing virus activation and latency.

The goal of our work is to understand, from the molecular to the organismal level, the principles that drive and sustain lifelong infection by viruses. These infectious agents live in a dynamic equilibrium (homeostasis) with their hosts in which both immune and nonimmune pathways contribute to viral homeostasis. Disruption of these pathways can have dramatic consequences on pathogenesis. Immune responses to infection provide a vital countermeasure by the host but are nonsterilizing. They effect an essential and primary control mechanism for viral growth. Essential nonimmune pathways for effecting control of a viral life cycle relate to the obligate dependency of the virus on its host. For these reasons, we view infections as a highly dynamic interplay that takes place between the pathogen and host. This, in many cases, leads to the establishment of an incurable lifelong infection that remains benign but can become life threatening once key homeostatic pathways are disrupted. We discuss these issues in the context of our studies using cytomegalovirus as a clinically relevant pathogen.

Animals↗
PubMed1999

Impaired immunoproteasome assembly and immune responses in PA28-/- mice.

In vitro PA28 binds and activates proteasomes. It is shown here that mice with a disrupted PA28b gene lack PA28a and PA28b polypeptides, demonstrating that PA28 functions as a hetero-oligomer in vivo. Processing of antigenic epitopes derived from exogenous or endogenous antigens is altered in PA28-/- mice. Cytotoxic T lymphocyte responses are impaired, and assembly of immunoproteasomes is greatly inhibited in mice lacking PA28. These results show that PA28 is necessary for immunoproteasome assembly and is required for efficient antigen processing, thus demonstrating the importance of PA28-mediated proteasome function in immune responses.

Animals↗
PubMed1999

Soluble-polymer supported synthesis of a prostanoid library: identification of antiviral activity.

[formula: see text] The prostaglandins are potent natural products taking part in many biological processes. The "convergent generation of diversity" from a "toolbox" of prostanoid components, augmented with additional polymer-supported transformations, can enable construction of valuable libraries. A parallel-pool strategy was used to assemble a small library of prostanoids. The inhibition of a herpes-family virus demonstrated the potential for new drug discovery.

3T3 Cells↗
PubMed1999

Anti-repression of RNA polymerase II transcription by pyrrole-imidazole polyamides.

Pyrrole-imidazole polyamides are ligands that bind in the minor groove of DNA with high affinity and sequence selectivity. Molecules of this class have been shown to disrupt specific transcription factor-DNA interactions and to inhibit basal and activated transcription from various RNA polymerase II and III promoters. A set of eight-ring hairpin-motif pyrrole-imidazole polyamides has been designed to bind within the binding site for the human cytomegalovirus (CMV) UL122 immediate early protein 2 (IE86). IE86 represses transcription of the CMV major immediate early promoter (MIEP) through its cognate cis recognition sequence (crs) located between the TATA box and the transcription initiation site. The designed polyamides bind to their target DNA sequence with nanomolar affinities and with a high degree of sequence selectivity. The polyamides effectively block binding of IE86 protein to the crs in DNase I footprinting experiments. A mismatch polyamide, containing a single imidazole to pyrrole substitution, and also a polyamide binding to a site located 14 base pairs upstream of the repressor binding site, do not prevent IE86 binding to the crs. IE86-mediated transcriptional repression in vitro is relieved by a match polyamide but not by a mismatch polyamide. Transcription from a DNA template harboring a mutation in the crs is not affected either by IE86 protein or by the match polyamides. These results demonstrate that this new class of small molecules, the pyrrole-imidazole polyamides, are not only effective inhibitors of basal and activated transcription, but also can be used to activate transcription by blocking the DNA-binding activity of a repressor protein.

Binding Sites↗
PubMed1999

Murine cytomegalovirus infection down-regulates MHC class II expression on macrophages by induction of IL-10.

Herpesviruses utilize many strategies for weakening the host immune response. For CMV, this includes avoidance of NK clearance and inhibition of MHC class I and class II presentation pathways. In this study, we report that mouse CMV (MCMV) specifically causes a premature and transient activation of host IL-10 very early in the course of infection, resulting in a dramatic and selective reduction in MHC class II surface expression. The expression of IL-10 is normally late in the immune response to a pathogen, serving to dampen the response by suppression of the production of inflammatory cytokines. In infection of macrophages, we show that MCMV induces the production of IL-10, leading to an early and selective reduction in the expression of MHC class II on the surface of the cells. Inhibition of MHC class II expression was not observed in the presence of neutralizing Abs to IL-10 or in macrophages from IL-10-deficient mice. Moreover, MCMV-infected IL-10-deficient mice developed an early and significantly more robust macrophage MHC class II induction than normal mice. Altogether, our results demonstrate that viral induction of an IL-10 autocrine pathway plays an essential early role in selectively reducing MHC class II expression on the surface of APC prior to stimulation by IFN-gamma.

Acute Disease↗
PubMed1999

Defective interleukin (IL)-18-mediated natural killer and T helper cell type 1 responses in IL-1 receptor-associated kinase (IRAK)-deficient mice.

Interleukin (IL)-18 is functionally similar to IL-12 in mediating T helper cell type 1 (Th1) response and natural killer (NK) cell activity but is related to IL-1 in protein structure and signaling, including recruitment of IL-1 receptor-associated kinase (IRAK) to the receptor and activation of c-Jun NH2-terminal kinase (JNK) and nuclear factor (NF)-kappaB. The role of IRAK in IL-18-induced responses was studied in IRAK-deficient mice. Significant defects in JNK induction and partial impairment in NF-kappaB activation were found in IRAK-deficient Th1 cells, resulting in a dramatic decrease in interferon (IFN)-gamma mRNA expression. In vivo Th1 response to Propionibacterium acnes and lipopolysaccharide in IFN-gamma production and induction of NK cytotoxicity by IL-18 were severely impaired in IRAK-deficient mice. IFN-gamma production by activated NK cells in an acute murine cytomegalovirus infection was significantly reduced despite normal induction of NK cytotoxicity. These results demonstrate that IRAK plays an important role in IL-18-induced signaling and function.

Animals↗
PubMed1999

DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression.

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (alpha), early (beta), early-late (gamma1), and late (gamma2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5' noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for beta, gamma1, and gamma2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.

Base Sequence↗
PubMed1998

Recognition properties of a sequence-specific DNA binding antibody.

A sequence-specific DNA-binding antibody was previously generated by incorporating a 17 amino acid alpha-helix from the DNA-binding domain of the transcription factor TFEB into the HCDR3 site of a recombinant human Fab fragment. The recombinant DNA-binding antibody, called Fab-E box, binds the TFEB recognition sequence CACGTG (an E box site) with a 5-10-fold lower affinity than TFEB. Here, we have determined the precise kinetics of interaction of Fab-E box with DNA and show that the lower affinity of Fab-E box relative to TFEB for E box DNA is due to a higher dissociation rate. DNase I protection assays show Fab-E box physically interacts with one half-site of the E box. Additional DNA target sites of Fab-E box were identified by DNase I protection assays. A compilation of these binding sites indicates that the recognition elements for Fab-E box binding include a half-site of the E box, CAW, with an 8 bp consensus sequence identified as YNYYCAWW. Thus, the DNA determinants for Fab-E box recognition extend beyond one-half site of the E box sequence, with preferences for pyrimidines and A+T-rich sequences in the 5' and 3' outer regions of the binding site, respectively. Apparent dissociation constants of Fab-E box for a subset of these target DNA sequences are 5-10-fold greater than the DNA-binding affinity of the antibody with the E box site. Therefore, these results identify important DNA specificity determinants for high-affinity binding by Fab-E box.

Antibody Specificity↗
PubMed1998

Enhancer requirement for murine cytomegalovirus growth and genetic complementation by the human cytomegalovirus enhancer.

The cytomegalovirus (CMV) enhancer is a highly complex regulatory region containing multiple elements that interact with a variety of host-encoded transcription factors. Many of these sequence elements are conserved among the different species strains of CMV, although the arrangement of the various elements and overall sequence composition of the CMV enhancers differ remarkably. To delineate the importance of this region to a productive infection and to explore the possibility of generating a murine CMV (MCMV) under the control of human CMV (HCMV) genetic elements, the MCMV enhancer was resected and replaced either with nonregulatory sequences or with paralogous sequences from HCMV. The effects of these various deletions and substitutions on viral growth in transfected or infected tissue-culture cells were evaluated. We found that mutations in MCMV that eliminate or substitute for the enhancer with nonregulatory sequences showed a severe deficiency in virus synthesis. This growth defect is effectively complemented by the homologous MCMV enhancer as well as the HCMV enhancer. In the latter case, the chimeric viruses (hybrid MCMV strains) containing the molecularly shuffled human enhancer exhibit infectious kinetics similar to that of parental wild-type and wild-type revertant MCMV. These results also show that open reading frames m124, m124.1, and m125 located within the enhancer region are nonessential for growth of MCMV in cells. Most importantly, we conclude that the enhancer of MCMV is required for optimal infection and that its diverged human counterpart can advantageously replace its role in promoting viral infectivity.

3T3 Cells↗
PubMed1998

Ligand induction of retinoic acid receptors alters an acute infection by murine cytomegalovirus.

Here we report that administration of retinoids can alter the outcome of an acute murine cytomegalovirus (MCMV) infection. We show that a crucial viral control element, the major immediate-early enhancer, can be activated by retinoic acid (RA) via multiple RA-responsive elements (DR2) that bind retinoid X receptor-retinoic acid receptor (RAR) heterodimers with apparent dissociation constants ranging from 15 to 33 nM. Viral growth is dramatically increased upon RA treatment of infected tissue culture cells. Using synthetic retinoid receptor-specific agonists and antagonists, we provide evidence that RAR activation in cells is required for mediating the response of MCMV to RA. Oral administration of RA to infected immunocompetent mice selectively exacerbates an infection by MCMV, while cotreatment with an RAR antagonist protects against the adverse effects of RA on MCMV infection. In conclusion, these chemical genetic experiments provide evidence that an RAR-mediated pathway can modulate in vitro and in vivo infections by MCMV.

3T3 Cells↗