Rapamycin ameliorates the CTLA4-Ig-mediated defect in CD8(+) T cell immunity during gammaherpesvirus infection.

Latent viral infections are a major concern among immunosuppressed transplant patients. During clinical trials with belatacept, a CTLA4-Ig fusion protein, patients showed an increased risk of Epstein-Barr virus-associated posttransplant lymphoproliferative disorder, thought to be due to a deficient primary CD8(+) T cell response to the virus. Using a murine model of latent viral infection, we observed that rapamycin treatment alone led to a significant increase in virus-specific CD8(+) T cells, as well as increased functionality of these cells, including the ability to make multiple cytokines, while CTLA4-Ig treatment alone significantly dampened the response and inhibited the generation of polyfunctional antigen-specific CD8(+) T cells. However, the addition of rapamycin to the CTLA4-Ig regimen was able to quantitatively and qualitatively restore the antigen-specific CD8(+) T cell response to the virus. This improvement was physiologically relevant, in that CTLA4-Ig treated animals exhibited a greater viral burden following infection that was reduced to levels observed in untreated immunocompetent animals by the addition of rapamycin. These results reveal that modulation of T cell differentiation though inhibition of mTOR signaling can restore virus-specific immune competence even in the absence of CD28 costimulation, and have implications for improving protective immunity in transplant recipients.

Does EBV alter the pathogenesis of malaria?

Plasmodium falciparum infections have been implicated in immune deficiencies resulting in ineffective control of Epstein-Barr virus, thereby increasing the risk of endemic Burkitt lymphoma in children. However, the impact of Epstein-Barr virus infections on the development of immunity to P. falciparum has not been studied in depth. In this review, we examine novel findings from animal co-infection models and human immuno-epidemiologic studies to speculate on the impact of acute gammaherpesvirus co-infection on malarial disease severity. Children are often concurrently or sequentially infected with multiple pathogens, and this has implications for understanding the development of protective immunity as well as in the evaluation of vaccine efficacy.

Mutations in the tyrosine phosphatase CD45 gene in a child with severe combined immunodeficiency disease.

The hematopoietic-specific transmembrane protein tyrosine phosphatase CD45 functions to regulate Src kinases required for T- and B-cell antigen receptor signal transduction. So far, there have been no reports to our knowledge of a human deficiency in a tyrosine-specific phosphatase. Here, we identified a male patient with a deficiency in CD45 due to a large deletion at one allele and a point mutation at the other. The point mutation resulted in the alteration of intervening sequence 13 donor splice site. The patient presented at 2 months of age with severe combined immunodeficiency disease. The population of peripheral blood T lymphocytes was greatly diminished and unresponsive to mitogen stimulation. Despite normal B-lymphocyte numbers, serum immunoglobulin levels decreased with age. Thus, CD45 deficiency in humans results in T- and B-lymphocyte dysfunction.

Murine gamma-herpesvirus 68 causes severe large-vessel arteritis in mice lacking interferon-gamma responsiveness: a new model for virus-induced vascular disease.

Fundamental issues remain unresolved regarding the possible contribution of viruses to vascular pathology, as well as the role of the immune system in regulating these processes. Here we demonstrate that infection of mice with gamma-herpesvirus 68 (gammaHV68) provides a novel model for addressing these issues. Interferon-gamma receptor-deficient (IFNgammaR-/-) mice died weeks to months after gammaHV68 infection from a severe large-vessel panarteritis. GammaHV68-infected B cell-deficient and normal weanling mice exhibited milder large-vessel arteritis. Immunohistochemical analyses demonstrated gammaHV68 antigen in arteritic lesions and revealed a striking tropism of gammaHV68 for smooth muscle cells. These studies demonstrate that IFN-gamma is essential for control of chronic vascular pathology induced by gammaHV68 and suggest gamma-herpesviruses as candidate etiologic agents for human vasculitis.

IFN-gamma action in the media of the great elastic arteries, a novel immunoprivileged site.

Infection of medial smooth muscle cells with gamma-herpesvirus 68 (gammaHV68) causes severe chronic vasculitis that is restricted to the great elastic arteries. We show here that persistence of disease in the great elastic arteries is (a) due to inefficient clearance of viral infection from this site compared with other organs or other vascular sites, and (b) associated with failure of T cells and macrophages to enter the virus-infected elastic media. These findings demonstrate immunoprivilege of the media of the great elastic arteries. We found that IFN-gamma acted on somatic cells during acute infection to prevent the establishment of medial infection and on hematopoietic cells to determine the severity of disease in this site. The immunoprivileged elastic media may provide a site for persistence of pathogens or self antigens leading to chronic vascular disease, a process regulated by IFN-gamma actions on both somatic and hematopoietic cells. These concepts have significant implications for understanding immune responses contributing to or controlling chronic inflammatory diseases of the great vessels.

Unraveling immunity to gamma-herpesviruses: a new model for understanding the role of immunity in chronic virus infection.

Murine gamma-herpesvirus 68 (gammaHV68) infection is a new model for understanding how immunity and chronic gamma-herpesvirus infection inter-relate. gammaHV68 is closely related to the human Epstein-Barr virus and Kaposi's sarcoma herpesvirus and is associated with tumors, vasculitis of the great elastic arteries and splenic fibrosis. Advances in the past year have provided an even stronger foundation for believing that gammaHV68 infection of normal and mutant mice will become the pre-eminent animal model for understanding gamma-herpesvirus pathogenesis and immunity. gammaHV68 latency has been characterized employing new assays for quantitating cells carrying the gammaHV68 genome and cells that reactivate gammaHV68 and for detecting the presence of preformed infectious virus in tissues. These advances have fostered the first steps towards a molecular definition of gammaHV68 latency. It appears that gammaHV68 shares latency programs with human gamma-herpesviruses - including the loci for gene 73, v-bcl-2 and the viral homolog of the G-protein coupled receptor. This provides candidate antigens for analysis of the role of T and B cells in regulating latency. Multiple cellular reservoirs for gammaHV68 latency were uncovered with the demonstration that gammaHV68 latently infects macrophages in addition to B cells. A critical role for B cells in regulating the nature of gammaHV68 latency was discovered and the mechanism was shown to be via alteration of the efficiency of reactivation. Studies of the response of CD4(+) and CD8(+) cells during acute and chronic gammaHV68 were performed. These new studies provide key building blocks for further development of this novel and interesting model system.

Host-cell-determined methylation of specific Epstein-Barr virus promoters regulates the choice between distinct viral latency programs.

Epstein-Barr virus (EBV) is capable of adopting three distinct forms of latency: the type III latency program, in which six EBV-encoded nuclear antigens (EBNAs) are expressed, and the type I and type II latency programs, in which only a single viral nuclear protein, EBNA1, is produced. Several groups have reported heavy CpG methylation of the EBV genome in Burkitt's lymphoma cell lines which maintain type I latency, and loss of viral genome methylation in tumor cell lines has been correlated with a switch to type III latency. Here, evidence that the type III latency program must be inactivated by methylation to allow EBV to enter the type I or type II restricted latency program is provided. The data demonstrates that the EBNA1 gene promoter, Qp, active in types I and II latency, is encompassed by a CpG island which is protected from methylation. CpG methylation inactivates the type III latency program and consequently allows the type I or II latency program to operate by alleviating EBNA1-mediated repression of Qp. Methylation of the type III latency EBNA gene promoter, Cp, appears to be essential to prevent type III latency, since EBNA1 is expressed in all latently infected cells and, as shown here, is the only viral antigen required for activation of Cp. EBV is thus a pathogen which subverts host-cell-determined methylation to regulate distinct genetic programs.

Constitutive activation of Epstein-Barr virus (EBV) nuclear antigen 1 gene transcription by IRF1 and IRF2 during restricted EBV latency.

The Epstein-Barr virus (EBV) EBNA1 gene promoter active in the type I program of restricted viral latency was recently identified and shown to reside in the viral BamHI Q fragment. This promoter, Qp, is active in a wide variety of cell lines and has an architecture reminiscent of eukaryotic housekeeping gene promoters (B. C. Schaefer, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 92:10565-10569, 1995; B. C. Schaefer, J. L. Strominger, and S. H. Speck, Mol. Cell. Biol. 17:364-377, 1997). Here we demonstrate by deletion analysis that the important cis-acting elements regulating Qp are clustered in a relatively small region (ca. 80 bp) surrounding the site of transcription initiation. Immediately upstream of the site of initiation is a region which is protected from DNase I digestion by crude nuclear extracts. Electrophoretic mobility shift analyses (EMSA) employing probes spanning this region demonstrated the presence of two major protein complexes. Deletion analysis of Qp demonstrated that at least one of these complexes plays an important role in Qp activity. Evidence that interferon response factor 2 (IRF2) is a major constituent of the most prominent EMSA complex and that IRF1 may be a minor component of this complex is presented. Transfections into IRF1-/-, IRF2-/-, and IRF1,2-/- fibroblasts demonstrated that absence of both IRF1 and IRF2 reduced Qp activity to approximately the same extent as mutation of the IRF-binding site in Qp, strongly implicating IRF2, and perhaps IRF1, in the regulation of Qp activity. Notably, transcription from Qp was not inducible by either alpha or gamma interferon in EBV-negative B cells but rather was shown to be constitutively activated by IRF1 and IRF2. This observation suggests that IRF1 and IRF2 have a previously unrecognized role as constitutive activators of specific genes. Additionally, data presented indicate that a protein complex containing the nonhistone architectural protein HMG-I(Y) binds to the region identified as the major transcription initiation site for Qp. This observation raises the possibility that HMG-I(Y)-induced DNA bending plays a role in the initiation of transcription from Qp.

DNA-binding-defective mutants of the Epstein-Barr virus lytic switch activator Zta transactivate with altered specificities.

The Epstein-Barr virus BRLF1 and BZLF1 genes are the first viral genes transcribed upon induction of the viral lytic cycle. The protein products of both genes (referred to here as Rta and Zta, respectively) activate expression of other viral genes, thereby initiating the lytic cascade. Among the viral antigens expressed upon induction of the lytic cycle, however, Zta is unique in its ability to disrupt viral latency; expression of the BZLF1 gene is both necessary and sufficient for triggering the viral lytic cascade. We have previously shown that Zta can activate its own promoter (Zp), through binding to two Zta recognition sequences (ZIIIA and ZIIIB). Here we describe mutant Zta proteins that do not bind DNA (referred to as Zta DNA-binding mutants [Zdbm]) but retain the ability to transactivate Zp. Consistent with the inability of these mutants to bind DNA, transactivation of Zp by Zdbm is not dependent on the Zta recognition sequences. Instead, transactivation by Zdbm is dependent upon promoter elements that bind cellular factors. An examination of other viral and cellular promoters identified promoters that are weakly responsive or unresponsive to Zdbm. An analysis of a panel of artificial promoters containing one copy of various promoter elements demonstrated a specificity for Zdbm activation that is distinct from that of Zta. These results suggest that non-DNA-binding forms of some transactivators retain the ability to transactivate specific target promoters without direct binding to DNA.

Host and viral genetics of chronic infection: a mouse model of gamma-herpesvirus pathogenesis.

A general association of human and primate lymphotropic herpesviruses (gamma-herpesviruses) with the development of lymphomas, as well as other tumors, especially in immunocompromised hosts, has been well documented. The lack of relevant small animal models for human gamma-herpesviruses has impeded progress in understanding the role of these viruses in the development of chronic disease. Recent research characterizing infection of inbred strains of mice with a murine gamma-herpesvirus, gamma-herpesvirus 68 (gammaHV68), is providing insights into viral and host factors involved in the establishment and control of chronic gamma-herpesvirus infection.

Reactivation of Epstein-Barr virus: regulation and function of the BZLF1 gene.

The switch from latent infection to virus replication in Epstein-Barr virus (EBV)-infected B cells is initiated by expression of the viral BZLF1 gene. Recent studies have identified the key cellular transcription factors involved in regulating this switch in viral programs and the signal transduction pathways to which they respond. Understanding this switch may facilitate development of strategies to interfere with EBV infection.

Role of phospholipid in the low affinity reactions between cytochrome c and cytochrome oxidase.

The steady-state oxidation of ferrocytochrome c by cytochrome oxidase monitored spectrophotometrically showed that: (1) the kinetics were strictly biphasic with purified enzyme, while mitochondrial membrane-bound enzyme exhibited multiphasic kinetics with extended low affinity phases; (2) the TNmax for the highest affinity phase was as slow as 5-10 electron X s-1 for both preparations, while for the low affinity phases it was about 45 electron X s-1 for the purified enzyme and 150 electron X s-1 for the mitochondrial membrane-bound enzyme; (3) reconstitution of purified enzyme into acidic phospholipid vesicles partially repleted the extended low affinity phases, while reconstitution into uncharged vesicles had no effect.

Helminth infection reactivates latent γ-herpesvirus via cytokine competition at a viral promoter.

Mammals are coinfected by multiple pathogens that interact through unknown mechanisms. We found that helminth infection, characterized by the induction of the cytokine interleukin-4 (IL-4) and the activation of the transcription factor Stat6, reactivated murine γ-herpesvirus infection in vivo. IL-4 promoted viral replication and blocked the antiviral effects of interferon-γ (IFNγ) by inducing Stat6 binding to the promoter for an important viral transcriptional transactivator. IL-4 also reactivated human Kaposi's sarcoma-associated herpesvirus from latency in cultured cells. Exogenous IL-4 plus blockade of IFNγ reactivated latent murine γ-herpesvirus infection in vivo, suggesting a "two-signal" model for viral reactivation. Thus, chronic herpesvirus infection, a component of the mammalian virome, is regulated by the counterpoised actions of multiple cytokines on viral promoters that have evolved to sense host immune status.

Identification of phorbol ester response elements in the promoter of Epstein-Barr virus putative lytic switch gene BZLF1.

The product of the Epstein-Barr virus BZLF1 gene encodes a protein which is related to c-fos, it has been shown to bind specifically to a consensus AP-1 site, and its expression in latently Epstein-Barr virus-infected lymphocytes is sufficient to trigger the viral lytic cycle. We identified several elements within the BZLF1 promoter (Zp) which are responsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of the viral lytic cycle. These elements fall into two classes based on the factors which bind to these sequences and their resulting functional behavior. Four of the elements are homologous (ZI elements) and share homology to a protein-binding domain in the promoter region of the coordinately expressed BRLF1 gene. When cloned upstream of heterologous promoters, the ZI elements function as silencers which exhibit TPA-inducible enhancer activity. A distinct TPA-responsive element (ZII) is located near the TATA box and shares homology with the AP-1-binding site in the c-jun promoter. A synthetic oligonucleotide with a sequence corresponding to the ZII element effectively competes for binding of nuclear factors to the c-jun AP-1 site. Furthermore, we found that a complex of c-jun and c-fos bound to the ZII domain.

Autoregulation of Epstein-Barr virus putative lytic switch gene BZLF1.

Expression of the Epstein-Barr virus (EBV) BZLF1 gene in latently infected lymphocytes is sufficient to trigger the viral lytic cycle. As shown in the accompanying report (E. Flemington and S.H. Speck, J. Virol. 64:1217-1226, 1990), the promoter for the BZLF1 gene (Zp) contains two distinct types of elements (ZI and ZII [an AP-1-like domain]) which are responsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of the viral lytic cycle. Although Zp can be activated with TPA in an EBV-negative Burkitt's lymphoma cell line (Ramos), its activity is considerably lower than in EBV-positive cell lines which can be induced with TPA. Here we show that the protein product of the BZLF1 gene (ZEBRA) can transactivate its own promoter by a mechanism which involves direct binding to a region distinct from the ZI and ZII element. Moreover, we show that this region is composed of two distinct ZEBRA-binding-transactivation domains. Interestingly, these two domains are not homologous, and while one domain (ZIIIA) is similar to previously described ZEBRA-binding domains, the second (ZIIIB) is a higher-affinity site which bears no detectable homology to the consensus ZEBRA recognition sequence. We also show that transactivation is independent of the otherwise essential ZII domain, suggesting that ZEBRA binding may functionally replace or supercede the need for a functional ZII domain. This observation supports a model for activation of the lytic cycle whereby synthesis of a critical level of ZEBRA signals commitment to BZLF1 transcription and initiation of the lytic cascade.

Evidence for coiled-coil dimer formation by an Epstein-Barr virus transactivator that lacks a heptad repeat of leucine residues.

Two regions of the Epstein-Barr virus (EBV) BZLF1 gene product, ZEBRA, share sequence homology with c-Fos, one of which corresponds to the DNA binding domain of c-Fos. ZEBRA does not, however, contain the heptad repeat of leucines present in the dimerization domains of leucine zipper proteins. Here it is shown that ZEBRA binds its recognition sites as a homodimer and that the region adjacent to the basic DNA binding domain is essential for dimerization. This region contains a 4-3 repeat of predominantly hydrophobic residues, which is precisely in register with the hydrophobic heptad repeat present in the leucine zipper proteins with respect to the basic DNA binding domain. A mutational analysis of ZEBRA supports a model for dimerization involving a coiled-coil interaction. These results indicate that a heptad repeat of leucines is not a structural requirement for formation of coiled-coil dimers by transcription factors.

Epstein-Barr virus BZLF1 trans activator induces the promoter of a cellular cognate gene, c-fos.

The Epstein-Barr virus BZLF1 gene product ZEBRA is a DNA-binding protein that is partially homologous to c-Fos, binds specifically to AP-1 sites, and can induce the lytic cycle in latently infected B lymphocytes. Induction of the viral lytic cycle can also be achieved by treatment with the phorbol ester 12-O-tetrade-canoylphorbol-13-acetate, a reagent which activates gene expression in part through AP-1 (Jun/Fos). In this article the interrelationship between ZEBRA and AP-1 is extended by the demonstration that ZEBRA can induce c-Fos expression through AP-1 and "AP-1-like" sites present in the c-fos promoter. Induction of c-Fos may be necessary for the expression of other viral lytic genes and perhaps cellular genes whose products are required for viral replication.

Characterization of the deletion and rearrangement in the BamHI C region of the X50-7 Epstein-Barr virus genome, a mutant viral strain which exhibits constitutive BamHI W promoter activity.

Epstein-Barr virus infection of peripheral B lymphocytes predominantly results in a latent infection, with a concomitant growth transformation of the infected cells. These cells express six nuclear antigens (EBNAs) and three membrane antigens. Transcription of all the EBNA genes is driven by one of two promoters, Cp or Wp, located near the left end of the viral genome, and the activities of these promoters are mutually exclusive. We have previously shown that Wp is exclusively used during the initial stages of B-cell immortalization, followed by a switch to Cp usage. However, several cell lines which appear to have failed to switch from Wp to Cp usage and which exhibit constitutive Wp activity have been identified. In two cases, we have shown that this failure to switch is the result of a deletion of approximately 3.5 kb, spanning Cp. In this paper, we characterize the deletion in one of these cell lines, X50-7, and demonstrate not only that the viral genome in this cell line has sustained a deletion in the region of Cp, but also that there has been a rearrangement into the BamHI C region of viral sequences from the BamHI W and Y fragments.