Mary Hummel

A model for reactivation of CMV from latency

BACKGROUND Reactivation of CMV from latency results in serious morbidity and mortality in immunocompromised transplant recipients. The mechanism by which CMV reactivates from latency has not been well understood. OBJECTIVE In this review we discuss three models for reactivation from latency and present evidence in favor of the model that reactivation is a multi-step process which is initiated by the allogeneic response to the transplanted organ. Study design (J. Virol. 75 (2001) 4814). Mice latently infected with murine cytomegalovirus (MCMV) were used as donors for allogeneic or syngeneic kidney transplants into immunocompetent recipients. The contralateral donor kidneys were used as controls. Transplanted kidneys were removed at various times after transplant and analyzed for expression of viral genes associated with productive infection and for expression of inflammatory cytokines. Electrophoretic mobility shift assay was performed on nuclear extracts of control and transplanted kidneys to examine activation of AP-1 and NFkappaB. Latently infected mice were also injected with tumor necrosis factor (TNF) to examine the effect of TNF alone on induction of MCMV immediate-early (IE) gene expression. Transgenic major immediate early promoter-lacZ mice carrying a beta-galactosidase reporter gene under the control of the human cytomegalovirus (HCMV) IE promoter/enhancer were used as donors for allogeneic kidney transplants to study the effect of allogeneic transplantation on induction of HCMV IE gene expression. RESULTS Allogeneic, but not syngeneic transplantation induces MCMV IE-1 expression and expression of inflammatory cytokines, including TNF. Allogeneic transplantation activates transcription factors, including NFkappaB and AP-1. TNF alone can induce MCMV IE-1 gene expression and activation of NFkappaB and AP-1 in some tissues. CONCLUSIONS We propose that induction of IE-1 gene expression is the first step in reactivation of the virus in an immunocompromised transplant recipient, and that it occurs as a result of the allogeneic response, which induces expression of TNF and subsequent activation of NFkappaB, and ischemia/reperfusion injury, which induces activation of AP-1. We speculate that the natural stimulus for reactivation in an immunocompetent host is an inflammatory immune response to infection and that allogeneic transplantation mimics this process.

Allogeneic Transplantation Induces Expression of Cytomegalovirus Immediate-Early Genes In Vivo: a Model for Reactivation from Latency

ABSTRACT Reactivation of cytomegalovirus (CMV) from latency is a frequent complication of organ transplantation, and the molecular mechanism by which this occurs is unknown. Previous studies have shown that allogeneic stimulation induces reactivation of human CMV (HCMV) in vitro (64). We find that transplantation of vascularized allogeneic kidneys induces murine CMV (MCMV) and HCMV immediate-early (ie) gene expression. This induction is accompanied by increased expression of transcripts encoding inflammatory cytokines, including tumor necrosis factor (TNF), interleukin-2, and gamma interferon, and by activation of NF-κB. TNF alone can substitute for allogeneic transplantation in inducing HCMV and MCMV iegene expression in some tissues. Our studies suggest that reactivation is a multistep process which is initiated by factors that induceie gene expression, including TNF and NF-κB. Allogeneic transplantation combined with immunosuppression may be required to achieve complete reactivation in vivo.

The E8E2C protein, a negative regulator of viral transcription and replication, is required for extrachromosomal maintenance of human papillomavirus type 31 in keratinocytes.

ABSTRACT The viral E2 protein is a major regulator of papillomavirus DNA replication. An important way to influence viral replication is through modulation of the activity of the E2 protein. This could occur through the action of truncated E2 proteins, called E2 repressors, whose role in the replication cycle of human papillomaviruses (HPVs) has not been determined. In this study, using cell lines that contain episomal copies of the “high-risk” HPV type 31 (HPV31), we have identified viral transcripts with a splice from nucleotide (nt) 1296 to 3295. These transcripts are similar to RNAs from other animal and human papillomaviruses and have the potential to fuse a small open reading frame (E8) to the C terminus of E2, resulting in an E8 ̂E2C fusion protein. E8 ̂E2C transcripts were present throughout the complete replication cycle of HPV31. A genetic analysis of E8 ̂E2C in the context of the HPV31 genome revealed that mutation of the single ATG of the E8 gene, introduction of a stop codon downstream of the ATG, or disruption of the splice donor site at nt 1296 led to a dramatic 30- to 40-fold increase in the transient DNA replication levels in both normal and immortalized human keratinocytes. High-level expression of E8 ̂E2C from heterologous vectors was found to inhibit E1-E2-dependent DNA replication of an HPV31 origin of replication construct as well as to interfere with E2s ability to transactivate reporter gene constructs. In addition, HPV31 E8 ̂E2C strongly repressed the basal activity of the major viral early promoter P97 independent of E2. E8 ̂E2C may therefore exert its negative effect on viral DNA replication through modulating E2s ability to enhance E1-dependent DNA replication as well as by regulating viral gene expression. Surprisingly, HPV31 genomes that were unable to express E8 ̂E2C could not be maintained extrachromosomally in human keratinocytes in long-term assays despite high transient DNA replication levels. This suggests that the E8 ̂E2C protein may play a role in copy number control as well as in the stable maintenance of HPV episomes.

Renal Ischemia/Reperfusion Injury Activates the Enhancer Domain of the Human Cytomegalovirus Major Immediate Early Promoter

Reactivation of latent human cytomegalovirus is of significant concern in immunocompromised transplant patients and is likely to occur through transcriptional activation of immediate early (ie) gene expression through mechanisms that are not well understood. TNF‐mediated activation of NF‐κB has been proposed to be one pathway leading to transcriptional activation of CMV ie gene expression. Using transgenic mice carrying a lacZ reporter gene under the control of the HCMV major ie promoter/enhancer (MIEP‐lacZ mice) and MIEP‐lacZ mice deficient in TNF receptor 1 and TNF receptor 2 (MIEP‐lac Z TNFR DKO mice), we demonstrate that renal ischemia/reperfusion (I/R) injury activates the HCMV enhancer independently of TNF. Induction of MIEP‐lacZ expression was preceded by TNFR‐independent formation of reactive oxygen species (ROS), weak and transient activation of 
 NF‐κB and strong and sustained activation of AP‐1. Our studies show that, in addition to TNF‐mediated signaling, TNF‐independent signaling induced by I/R injury can contribute to the activation of the HCMV enhancer. This likely occurs through ROS‐mediated activation of AP‐1. Targeting MAP kinase signaling pathways as well as NF‐κB may be of therapeutic value in patients with CMV infection.

Establishment of Murine Cytomegalovirus Latency In Vivo Is Associated with Changes in Histone Modifications and Recruitment of Transcriptional Repressors to the Major Immediate-Early Promoter

ABSTRACT Human cytomegalovirus (CMV) is a ubiquitous herpesvirus with the ability to establish a lifelong latent infection. The mechanism by which this occurs is not well understood. Regulation of, for example, immediate-early (IE) gene expression is thought to be a critical control point in transcriptional control of the switch between latency and reactivation. Here, we present evidence that supports previous studies showing that the majority of genomes are quiescent with respect to gene expression. To study the possible role of epigenetic factors that may be involved in repression of ie gene expression in latency, we have analyzed changes in the patterns of modifications of histones bound to the major IE promoter (MIEP) in the kidneys of acutely and latently infected mice. Our studies show that, like herpes simplex virus, murine CMV genomes become relatively enriched in histones in latent infection. There are dramatic changes in modifications of histones associated with the MIEP when latency is established: H3 and H4 become hypoacetylated and H3 is hypomethylated at lysine 4, while H3 lysine 9 is hypermethylated in latently infected mice. These changes are accompanied by a relative loss of RNA polymerase and gain of heterochromatin protein 1γ and Yin-Yang 1 bound to the MIEP. Our studies suggest that, in the majority of cells, CMV establishes a true latent infection, defined as the lack of expression of genes associated with productive infection, and that this occurs through changes in histone modifications and recruitment of transcriptional silencing factors to the MIEP.

Epigenetic Control of Cytomegalovirus Latency and Reactivation

Cytomegalovirus (CMV) gene expression is repressed in latency due to heterochromatinization of viral genomes. In murine CMV (MCMV) latently infected mice, viral genomes are bound to histones with heterochromatic modifications, to enzymes that mediate these modifications, and to adaptor proteins that may recruit co-repressor complexes. Kinetic analyses of repressor binding show that these repressors are recruited at the earliest time of infection, suggesting that latency may be the default state. Kidney transplantation leads to epigenetic reprogramming of latent viral chromatin and reactivation of immediate early gene expression. Inflammatory signaling pathways, which activate transcription factors that regulate the major immediate early promoter (MIEP), likely mediate the switch in viral chromatin.

Biphasic recruitment of transcriptional repressors to the murine cytomegalovirus major immediate-early promoter during the course of infection in vivo.

ABSTRACT Our previous studies showed that establishment of murine cytomegalovirus (MCMV) latency in vivo is associated with repression of immediate-early gene expression, deacetylation of histones bound to the major immediate-early promoter (MIEP), changes in patterns of methylation of histones, and recruitment of cellular repressors of transcription to the MIEP. Here, we have quantitatively analyzed the kinetics of changes in viral RNA expression, DNA copy number, and recruitment of repressors and activators of transcription to viral promoters during the course of infection. Our results show that changes in viral gene expression correlate with changes in recruitment of RNA polymerase and acetylated histones to viral promoters. Binding of the transcriptional repressors histone deacetylase type 2 (HDAC2), HDAC3, YY1, CBF-1/RBP-Jk, Daxx, and CIR to the MIEP and HDACs to other promoters showed a biphasic pattern: some binding was detectable prior to activation of viral gene expression, then decreased with the onset of transcription and increased again as repression of viral gene expression occurred. Potential binding sites for CBF-1/RBP-Jk and YY1 in the MIEP and for YY1 in the M100 promoter (M100P) were identified by in silico analysis. While recruitment of HDACs was not promoter specific, binding of CBF-1/RBP-Jk and YY1 was restricted to promoters with their cognate sites. Our results suggest that sequences within viral promoters may contribute to establishment of latency through recruitment of transcriptional repressors to these genes. The observation that repressors are bound to the MIEP and other promoters immediately upon infection suggests that latency may be established in some cells very early in infection.

Intragraft TNF receptor signaling contributes to activation of innate and adaptive immunity in a renal allograft model.

Background. Increased levels of tumor necrosis factor (TNF) are a risk factor for allograft rejection. In vitro studies have shown that binding of TNF to its receptor activates signaling cascades that induce expression of many genes involved in inflammation. The role of intragraft TNF receptor (TNFR) signaling in activation of gene expression in allografts has not been studied. Methods. Gene expression profiling and quantitative real-time polymerase chain reaction analysis were used to investigate the role of TNFR signaling in the early intragraft activation of cellular gene expression in renal allografts at 2 days posttransplant. Results. The TNFRs play a critical role in activating intragraft expression of transcription factors controlling innate and adaptive immunity and stress responses (interferon regulatory factor {lsqb;IRF{rsqb;1, IRF 8, Isgf3g, and ATF3) of cytokines and receptors mediating inflammation (TNF, interleukin [IL]-6, interferon-γ, oncostatin M receptor {lsqb;OMCR{rsqb;, toll-like receptor {lsqb;TLR{rsqb;2, and IL-2Rγ), of chemokines and adhesion molecules that recruit inflammatory cells (Cxcl9, Cxcl11, E-selectin, and intracellular adhesion molecule {lsqb;ICAM{rsqb;-1), of genes involved in costimulation of T cells and processing and presentation of antigens (H2-DMb, Psmb8, and CD40), and genes that mediate the response to interferons. In addition to its proinflammatory role, TNFR signaling induces expression of SOCS3, a negative regulator of IL-6 and OSMR signaling and Nfkbie, and a negative regulator of TNFR signal transduction. Conclusions. These studies illustrate the pleiotropic effect of TNF in both activation and down-modulation of the immune response and the complex interactions between the TNFRs and other cytokine signaling pathways in the early allograft response.

Immunosuppression is not required for reactivation of latent murine cytomegalovirus.

We have shown, for the first time, that TNF induces expression of MCMV IE RNA in the lungs of latently infected mice in the absence of immunosuppression. These initial data suggest that TNF may play an important role in the reactivation of latent MCMV, in the absence of immunosuppression, and provide a provocative insight into the mechanisms of CMV reactivation. Studies are in progress to determine whether genes associated with later stages of the viral life cycle are induced by TNF and whether infectious virus is produced.

Biochemistry of Epstein—Barr Virus

The objective of this introduction is to develop concisely the context in which biochemical studies of Epstein—Barr virus (EBV) have been undertaken. Elements of history and biology are relevant to an understanding of the somewhat unusual course of biochemical research with EBV.