Biomedical subjects
Publications and source records attributed to J L McKnight.
Medical systems are tools of limited power to improve health status in modern societies. The associational tools of local communities are now the most important health-giving resources. Health professionals must learn the nature of these tools and understand their relation to and support of them.
Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) is an uncommon but potentially fatal complication of immunosuppression in solid-organ transplant recipients. A semiquantitative DNA polymerase chain reaction assay was developed to amplify a unique 269-bp region of the EBNA-1 gene in peripheral blood lymphocytes (PBL) using the primers described by Telenti et al (J Clin Microbiol 28:2187, 1990). Serial samples were studied from 23 transplant recipients, 12 of whom were diagnosed with PTLD. The majority of transplant recipients who were EBV seropositive at the time of transplant surgery and who did not develop PTLD (5 of 7, 71%) exhibited less than a 10-fold increase in the levels of EBV-infected PBL over the 0.1 to 5 EBV genomes/10(6) PBL observed in immunocompetent EBV seropositive controls. Transplant recipients who were seronegative at the time of transplantation and who underwent a primary EBV infection but did not develop PTLD exhibited a reduced capacity to control viremia because the levels of EBV-infected PBL were up to 400 times greater than the 1.0 to 50 EBV genomes/10(6) PBL observed in individuals undergoing acute infectious mononucleosis (Rocci et al: N Engl J Med 296:132, 1977). However, all transplant recipients who developed PTLD exhibited a marked elevation of EBV-infected PBL independent of their serologic state at the time of transplantation. Six of the 10 transplant recipients with PTLD exhibited > or = 300,000 EBV genomes/10(5) PBL, two exhibited 10,000 to 50,000 EBV-infected genomes/10(5) PBL, and one each exhibited 2,500 and 500 EBV genomes/10(5) PBL. However, the latter two samples were obtained 4 to 5 weeks after the diagnosis of PTLD and may reflect a decrease in viral load resulting from immunomodulation. Marked decreases in the levels of EBV nuclear antigen-1 (EBNA-1), EBNA-2, and EBNA-LP antibodies correlated with the increase in EBV-infected PBL. Hence, a quantitative difference in circulating EBV viral load and EBNA antibody levels is evident between transplant recipients with and without PTLD and may be useful as a noninvasive prognostic marker with which to monitor and/or predict the development of PTLD.
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The reactivation of Epstein-Barr virus from latency requires the transcriptional induction of the viral encoded lytic cycle initiator gene, BZLF1, and a concomitant switch from OriP to OriLyt directed viral DNA replication. To investigate the role of host cell factors in these events, a series of EBV-immortalized clonal lymphoblastoid cell lines (LCL) were derived from the spontaneous outgrowth of peripheral blood lymphocytes from a single EBV-seropositive individual. We show that the state of virus activation among this family of isogenic clonal LCL differs, suggesting that each B-cell clone expresses a different complement of cellular factors that influence the state of viral activation. As a first step in the identification of factors involved in EBV reactivation, nuclear extracts were prepared from tightly latent, spontaneously replicating and latent LCL treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and sodium butyrate. The extracts were used in gel mobility shift analyses to compare DNA-protein complex formation among a series of target DNA sequences, including OriLyt and promoter sequences from BZLF1 and BRLF1. The DNA-protein complex patterns were reproducible and indistinguishable among extracts obtained from the latent LCL, but differed from those observed in extracts obtained from the spontaneously replicating LCL, particularly in regard to the binding of a CREB protein to the BZLF1 promoter. Moreover, extracts prepared from LCL treated with TPA to induce virus reactivation resulted in the formation of complexes that differed from those prepared from the spontaneously replicating LCL. Taken together, these data suggest that B-cell factors govern the state of viral activation and that EBV may be reactivated by more than one pathway.
The 65-kDa herpes simplex virus type 1 encoded alpha trans-induction factor (alpha TIF or VP16) has two important functions: it is required for the efficient transcriptional induction of the alpha or immediate-early genes, and it acts as an essential structural component of the virion. The transcription properties of alpha TIF have been well studied in vitro. The protein is a powerful inducer of RNA polymerase II-directed transcription and, similar to the cellular transcriptional transactivators GAL4 and CGN4, contains separable DNA binding and transactivation domains. In contrast, little is known about the structural function of alpha TIF because this function can be studied only during virus replication and structural mutants are lethal. The in vivo analysis of alpha TIF is further complicated by the likelihood that the transcription and structural functions are not entirely separable. In this study, we take an alternate approach toward the development of alpha TIF mutants and their subsequent characterization. Rather than analyzing the effects of intragenic mutations, we have examined the properties of a mutant virus which expresses an alpha TIF fusion protein containing 61 amino acids of another herpes simplex virus type 1 virion protein, VP13/14, fused to its C terminus. This is the first report which demonstrates that the C-terminus of alpha TIF can tolerate the addition of an adjacent protein domain without compromising its transactivation function in vivo. Moreover, the VP13/14 sequences do not interfere with the protein-protein interactions required for virion targeting and assembly.
Epstein-Barr virus (EBV) is associated with the development of several B cell malignancies including Burkitt's lymphoma (BL), post-transplant lymphoproliferative disease (PTLD), and AIDS-related lymphomas. The latter two diseases result from EBV-driven B cell proliferation in the absence of normal immunosurveillance and as such, represent a heterogenous family of lymphoproliferative disorders. This article reviews studies on EBV gene expression and antibody development in PTLD and introduces recent information on the levels of EBV+ peripheral blood lymphocytes to discuss possible mechanisms of pathogenesis under varying conditions of immunosuppression.
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Epstein-Barr virus (EBV) is associated with the development of two human B-cell malignancies, Burkitt's lymphoma and lymphomas that occur in the immunosuppressed host. The latter category of disease has become important recently as it is seen primarily in organ transplant recipients and individuals with acquired immunodeficiency syndrome. One possible mechanism for lymphoma development involves a reduction in or lack of EBV-specific cytotoxic T-cell recognition. In support of this model are previous observations that the expression of EBV nuclear antigen 2 (EBNA2) and latent membrane protein, two viral antigens associated with major histocompatibility complex class I-restricted T-cell killing, are downregulated in Burkitt's lymphoma and in early passage lymphoblastoid cell lines (LCL) derived from the malignant lesions. To determine whether a similar mechanism could occur in the development of posttransplant lymphoproliferative disorders (PTLD), we compared EBV gene expression among 23 PTLD tumor lesions obtained from 11 solid organ transplant recipients and among LCL derived from 3 of these lesions. In this report, we demonstrate, by Southern blot, Western blot, and immunofluorescence analysis, that (1) the tumor lesions exhibit varying patterns of restricted viral gene expression; (2) LCL derived from these lesions may represent the in vitro selection of cell subpopulations; and (3) immunosuppressed individuals have a markedly reduced antibody response to the latent cycle antigens, EBNA1, EBNA2, and EBNA-LP, but not to the lytic cycle viral capsid antigen when compared with normal immunocompetent controls.
The gene products of herpes simplex virus type 1 UL46 and UL47 enhance the efficiency of alpha TIF (VP16)-mediated alpha gene expression through an unknown mechanism of action. To further characterize the function of the UL46- and UL47-encoded proteins during virus infection, a series of isogenic herpes simplex virus type 1 strain F-derived UL46 and UL47 single-deletion mutants and a UL46/47 double-deletion mutant were constructed and compared with the wild type. Analysis of purified virions obtained from the UL46 deletion mutant showed for the first time that UL46 encoded the viron tegument phosphoproteins VP11 and VP12 (VP11/12). Similar analyses of the UL47 deletion mutants confirmed an earlier report by McLean et al. that UL47 also encoded two virion tegument phosphoproteins, VP13 and VP14 (VP13/14) (G. McLean, F. Rixon, N. Langeland, L. Haarr, and H. Marsden, J. Gen. Virol. 71:2953-2960, 1990). Kinetic analysis demonstrated a delay of approximately 2 h in the appearance of thymidine kinase (TK) activity in all of the UL46 and UL47 single-deletion mutants. In the UL46/47 double-deletion mutant, the delay in TK activity increased twofold, suggesting that the proteins encoded by UL46 and UL47 may act at the same level. Since the delay in TK expression occurred within the first 4 h of infection, the actions of VP11/12 and VP13/14 resulted from their virion association and not from their de novo synthesis as late (beta gamma and gamma) genes. Densitometric analysis of purified virions showed that the levels of VP11/12 and VP13/14 in the virion tegument were near the molar ratios of alpha TIF. On the basis of these observations, we predict that the abilities of UL46 and UL47 to enhance alpha TIF-mediated transcription could result from a stoichiometric association of VP11/12 and VP13/14 with alpha TIF within the infecting virion.
IL-6, a multifunctional cytokine produced by monocytes, fibroblasts, and endothelial cells, promotes the growth of EBV-immortalized B cells in vitro and renders these cells tumorigenic in athymic mice. In the present study, serum/plasma IL-6 bioactivity was found to be abnormally elevated, albeit transiently, in 17 of 18 solid organ transplant recipients with posttransplant lymphoproliferative disease (PTLD), with a mean maximal level of 196.7 U/ml. This represents a 16.4 increase above the normal mean (11.3 U/ml). In contrast, only 3 of 10 solid organ transplant recipients with uncomplicated courses posttransplant had abnormally elevated serum/plasma IL-6 bioactivity (mean maximal level 41.4 U/ml, P = 0.0007). When transferred to single cell culture, the 11 PTLD tissues produced 640 to 1.25 x 10(6) IL-6 U/ml in the culture supernatant, with a mean maximal level of 35,025 IL-6 U/ml. Cell separation experiments demonstrated that the adherent cells, identified as non-B cells, were the principal source of IL-6 production in vitro by PTLD tissue. Control cultures of inflammatory lymphoid tissue negative for lymphoproliferative disease as well as of PBL from patients with acute EBV-induced infectious mononucleosis consistently produced < 10 IL-6 U/ml. Thus, IL-6 is produced at high levels by PTLD tissues and may play a critical role in the pathogenesis of PTLD.
The transcriptional induction of the alpha or immediate-early gene class of herpes simplex virus type 1 effected by the alpha trans-induction factor (alpha TIF, ICP25, VP16, Vmw65) requires an alpha-specific cis-acting site. Increased transcription does not result from the direct, independent binding of alpha TIF, but rather from an alpha TIF-dependent formation of a protein-DNA complex containing, in addition to alpha TIF, at least one host cell factor. One of the host factors is a POU domain protein which recognizes an octamer element in the alpha-specific consensus. There is evidence that alpha TIF may drive the formation of multiple protein-DNA complexes containing a POU protein and additional host factors. Previously, the gene products of UL46 and UL47 have been implicated in modulating the alpha TIF-dependent transcriptional induction of alpha genes. Our current studies have extended these analyses from a transient-expression system to a series of viral deletion mutants. In these studies we demonstrate that neither UL46- nor UL47-encoded gene product, either separately or in combination, is required for viral growth in cell culture. The absence of UL47 reduces by up to 80% the ability of the virus to induce an alpha-regulated thymidine kinase reporter gene resident in 143TK- cells. Autoradiograms of [35S]methionine pulse-labeled infected cell proteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, show that deleting UL46 and/or UL47 has no discernable effect on the synthesis of alpha TIF or alpha TIF-containing proteins. Subsequent Western immunoblot analysis, with rabbit anti-alpha TIF antibodies made to an alpha TIF-Staphylococcus aureus protein A fusion, demonstrated that the accumulation and steady-state levels of alpha TIF or alpha TIF-containing proteins was indistinguishable from that of the thymidine kinase-negative isogenic parental virus, R delta 305.
Two organ transplant recipients who received organs from a common donor and were diagnosed as having an Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disorder were studied to determine the mode of EBV transmission. The results of restriction fragment length polymorphism, polymerase chain reaction, and minisatellite DNA analyses demonstrate that both patients had a common strain of EBV and that this strain was transmitted from the donor's organs to both recipients. Posttransplant lymphoproliferative disorder resulted from the proliferation of EBV-immortalized B lymphocytes of the recipient, not those of the donor.
HSV-1 latency and reactivation were studied in vivo by spontaneous and iontophoresis-induced ocular viral shedding in New Zealand rabbits, Balb/c and A/J mice latently infected with wild-type KOS, and dl x 3.1, a progeny ICP0 deletion mutant. The presence of trigeminal ganglionic latency was demonstrated by the in vitro methods of cocultivation and in situ hybridization. Although the efficiency of ganglionic latency was significantly less (P less than .0001) for dl x 3.1 than for KOS in both mice and rabbits, only dl x 3.1 shed spontaneously in the NZ rabbit. Iontophoresis of adrenergic agents failed to induce reactivation and ocular viral shedding of KOS or dl x 3.1 in mice or rabbits. The establishment of latency and reactivation of KOS and dl x 3.1 was dependent on the host animal. We conclude that host factors as exemplified by host species and host strain differences significantly affected the ability of KOS and dl x 3.1 to establish latency, to reactivate, and to shed spontaneously. ICP0 expression was not required for the establishment or maintenance of latency, nor was it required for the reactivation of latent HSV-1. Furthermore, the biological activity of KOS and dl x 3.1 during latency in vivo did not correlate with latency studies based on in vitro methods.
In herpes simplex virus-infected cells, gene expression is tightly regulated. In this review, we compare the properties of two trans-activating factors which regulate the expression of viral genes. The first, alpha trans-inducing factor (alpha TIF) is a structural component which induces the 5 alpha genes, the first set of genes transcribed after infection. Alpha TIF requires for induction a cis-acting site present in promoter-regulatory domains of all alpha genes. The cis site binds 2 host proteins, alpha H1 and alpha H2-alpha H3. These host proteins have a maximum bound molecular weight of 110,000 and 64,000, respectively. DNase 1 protection assays indicate that alpha H1 protects the entire cis site, whereas alpha H2-alpha H3 binds the 3' domain of the cis site. The methylation interference assays indicate that the contact points of alpha H1 and alpha H2-H3 are at the 5' and 3' termini of the cis site, respectively. Both proteins can bind to the cis site concurrently. Alpha TIF does not bind directly to DNA but was shown to be present in DNA-protein complexes. The binding of alpha TIF to these DNA-protein complexes requires the participation of alpha H1. In contrast to alpha TIF, the product of the alpha 4 gene, a protein 163,000 in apparent molecular weight binds to DNA directly and regulates genes both positively and negatively. The data indicate that alpha 4 protein can bind to at least 2 binding sites differing in nucleotide sequence and which can be present in promoters, across the transcription initiation sites, and in 5' transcribed non-coding sequences. The regulatory functions of the alpha 4 protein may reflect both the nature and location of the binding site. The biological implications of the viral trans-acting proteins are discussed.
In herpes simplex virus 1-infected cells, the transcription of alpha genes, the first set of genes to be expressed, is induced by a virion component, the alpha-trans-induction factor, and requires a cis site. Homologs of the cis site are present in the promoter-regulatory domains of all alpha genes and bind two cellular proteins designated as alpha H1 and alpha H2-alpha H3. We report that alpha-trans-induction factor, synthesized in vitro or present in nuclear extracts of infected cells, forms complexes with viral DNA fragments containing its cis-acting site only in the presence of cellular proteins and only under conditions that also enable the binding of the alpha H1 protein to the DNA. The induction of alpha genes by alpha-trans-induction factor appears, therefore, to be mediated by the interaction of the viral protein with cellular proteins at its cis-acting site.
Herpes simplex viruses encode a structural protein which induces, in trans, expression of alpha genes, the first set of genes to be expressed after infection of permissive cells. This protein, designated as the alpha-trans-inducing factor (alpha-TIF), maps within the BamHI F fragment, and its gene has been sequenced. In the course of mapping the domain of the alpha-TIF gene, it was noted that the intact BamHI fragment was consistently more effective than the complete domain of the alpha-TIF gene in inducing expression of alpha genes. Cotransfections of DNA fragments containing an alpha indicator gene and the alpha-TIF gene with various regions of the BamHI F DNA fragment revealed that the sequences located 3' to the alpha-TIF gene raised the activity of the alpha-TIF gene to nearly the same level as that of the intact BamHI F fragment. The nucleotide sequence and S1 nuclease mapping analyses revealed the presence of two transcribed open reading frames capable of encoding polypeptides with translated molecular weights of 77,357 and 70,527. To determine whether the effect of these sequences in trans on alpha-TIF-mediated induction of alpha genes was due to expression of these genes or competition for transcriptional factors, we constructed plasmids that contained both genes. Into each or both of these genes we inserted, near the translation initiation sites, 14-base-pair linkers carrying translational stop codons (TAG) in all three reading frames. Analyses of these plasmids indicated that the gene encoding the 70,527-molecular-weight polypeptide reduced alpha-TIF-dependent induction of alpha genes, whereas the gene encoding the 77,357-molecular-weight polypeptide increased this activity. Insertion of the stop codons abolished these activities.
This laboratory reported earlier that chimeric genes consisting of the structural sequences of the thymidine kinase (TK) gene fused to the promoter-regulatory domains of late (gamma 2) genes were regulated as bonafide gamma 2 genes when resident in the herpes simplex virus 1 genome but could not be differentiated from beta genes when introduced by transfection and stably integrated into the environment of the host genome (S. Silver and B. Roizman, Mol. Cell. Biol. 5, 518-528, 1985). We report here that beta-TK and the chimeric gamma 2-TK gene transfected into TK- baby hamster kidney (BHKtk-) were induced by alpha 4 and alpha 0 but not by the other alpha genes. Specifically: Both TK genes were induced by cotransfection with DNA fragments carrying an intact alpha 4 or an intact alpha 0 gene, but not by fragments carrying only the promoter-regulatory domain or the structural sequences of the alpha 4 gene or intact alpha 22, alpha 27, and alpha 47 genes. An alpha 4 gene carrying a 2700-bp deletion in its 3' coding sequence also induced both genes, although less efficiently. RNA homologous to the alpha 4 gene recovered from the cytoplasm of cells transfected with either the intact or truncated alpha 4 gene mapped to the bonafide site of transcription initiation of the alpha 4 gene. RNA homologous to the chimeric TK gene extracted from the cytoplasm of cells transfected with both gamma 2-TK and the alpha 4 gene was transcribed from the bonafide gamma 2 gene capping site fused to the TK gene. These results indicate that the alpha 4 gene and the alpha 0 gene are each capable of inducing the expression of both beta and gamma 2 genes resident in the environment of the cellular genome, that the active site responsible for induction is located near the N terminus of the alpha 4 protein, and reinforce the conclusion that gamma 2 genes resident in the environment of the host cell cannot be used to identify the authentic determinants of gamma 2 gene regulation by currently available tests.