Immature and Transitional B Cells Are Latency Reservoirs for a Gammaherpesvirus

Gammaherpesviruses are ubiquitous pathogens that are associated with the development of B cell lymphomas. Gammaherpesviruses employ multiple mechanisms to transiently stimulate a broad, polyclonal germinal center reaction, an inherently mutagenic stage of B cell differentiation that is thought to be the primary target of malignant transformation in virus-driven lymphomagenesis. We found that this gammaherpesvirus-driven germinal center expansion was exaggerated and lost its transient nature in the absence of interferon-regulatory factor 1 (IRF-1), a transcription factor with antiviral and tumor suppressor functions. Uncontrolled and persistent expansion of germinal center B cells led to pathological changes in the spleens of chronically infected IRF-1-deficient animals. Additionally, we found decreased IRF-1 expression in cases of human posttransplant lymphoproliferative disorder, a malignant condition associated with gammaherpesvirus infection. The results of our study define an unappreciated role for IRF-1 in B cell biology and provide insight into the potential mechanism of gammaherpesvirus-driven lymphomagenesis. IMPORTANCE Gammaherpesviruses establish lifelong infection in most adults and are associated with B cell lymphomas. While the infection is asymptomatic in many hosts, it is critical to identify individuals who may be at an increased risk of virus-induced cancer. Such identification is currently impossible Interferon-regulatory factor 1 (IRF-1) is a conserved transcription factor that restricts the replication of diverse RNA and DNA viruses in vitro via a poorly understood mechanism (1, 2). Additionally, IRF-1 suppresses replication and restricts the tropism of West Nile virus (WNV) (3), vesicular stomatitis virus (VSV) (4), and murine norovirus (5) during the acute phase of infection in vivo. However, there is a paucity of studies defining the role of IRF-1 during chronic virus infection. Such a role undoubtedly exists, as distinct IRF-1 polymorphisms are associated with either susceptibility to chronic hepatitis B and C virus infection (6) or resistance to HIV-1 (7). Additionally, IRF-1 is a tumor suppressor, as evidenced by the synergy between IRF-1 insufficiency and oncogene deregulation in animal models (8) and decreased IRF-1 expression in several human cancers (9). Importantly, this tumor suppressor nature of IRF-1 may be of particular relevance in the context of chronic infection with cancer-associated viruses.Our study focuses on the interaction between IRF-1 and gammaherpesviruses, ubiquitous pathogens that establish lifelong infection in a majority of the human population and are associated with the development of B cell lymphomas (10). In spite of the widespread nature of gammaherpesvirus infection, the host risk factors that predispose individuals toward gammaherpesvirus-driven lymphomagenesis are still poorly understood. Gammaherpesviruses establish long-term latency in memory B cells. Accordingly, these viruses employ both viral and host mechanisms to stimulate a germinal cent...

Section: Irf-1 Suppresses the Establishment Of Latent Gammaherpesvirumentioning

confidence: 99%

Tumor Suppressor Interferon-Regulatory Factor 1 Counteracts the Germinal Center Reaction Driven by a Cancer-Associated Gammaherpesvirus

Mboko

Olteanu

Ray

et al. 2016

“…While the frequency of viral genome-positive splenocytes in the G50DblKo virus-infected animals was around 8.5-fold lower than that in the wild-type virus-infected animals, these analyses demonstrate that this mutant virus is able to get to the spleen and establish latency. Thus, the lack of detectable reactivation of the G50DblKo mutant reflects a significant defect in virus reactivation from B cells, the dominant latently infected cell population in the spleen (19,35).…”

Section: Characterization Of the Orf50 Distal Promoter Activity In VImentioning

confidence: 99%

Identification of Alternative Transcripts Encoding the Essential Murine Gammaherpesvirus Lytic Transactivator RTA

Wakeman¹,

Johnson²,

Paden³

et al. 2014

The essential immediate early transcriptional activator RTA, encoded by gene 50, is conserved among all characterized gammaherpesviruses. Analyses of a recombinant murine gammaherpesvirus 68 (MHV68) lacking both of the known gene 50 promoters (G50DblKo) revealed that this mutant retained the ability to replicate in the simian kidney epithelial cell line Vero but not in permissive murine fibroblasts following low-multiplicity infection. However, G50DblKo replication in permissive fibroblasts was partially rescued by high-multiplicity infection. In addition, replication of the G50DblKo virus was rescued by growth on mouse embryonic fibroblasts (MEFs) isolated from IFN-␣/␤R ؊/؊ mice, while growth on Vero cells was suppressed by the addition of alpha interferon (IFN-␣). 5= rapid amplification of cDNA ends (RACE) analyses of RNAs prepared from G50DblKo and wild-type MHV68-infected murine macrophages identified three novel gene 50 transcripts initiating from 2 transcription initiation sites located upstream of the currently defined proximal and distal gene 50 promoters. In transient promoter assays, neither of the newly identified gene 50 promoters exhibited sensitivity to IFN-␣ treatment. Furthermore, in a single-step growth analysis RTA levels were higher at early times postinfection with the G50DblKo mutant than with wild-type virus but ultimately fell below the levels of RTA expressed by wild-type virus at later times in infection. Infection of mice with the MHV68 G50DblKo virus demonstrated that this mutant virus was able to establish latency in the spleen and peritoneal exudate cells (PECs) of C57BL/6 mice with about 1/10 the efficiency of wild-type virus or marker rescue virus. However, despite the ability to establish latency, the G50DblKo virus mutant was severely impaired in its ability to reactivate from either latently infected splenocytes or PECs. Consistent with the ability to rescue replication of the G50DblKo mutant by growth on type I interferon receptor null MEFs, infection of IFN-␣/␤R

“…Dysfunction of immune control mechanisms during MHV68 infection leads to increased reactivation (6), recrudescence (37,43,88), and numerous pathologies, including arteritis, pneumonia, fibrosis, lymphoid hyperplasia, and increased mortality (5, 20). MHV68 is detected in multiple cell types during chronic infection, including fibroblasts, epithelial and endothelial cells, macrophages, and multiple B cell types (5,10,42,58,64,89). B cells are the predominant latency reservoir; B cell activation and terminal differentiation to plasma cells are in vivo mechanisms for driving MHV68 reactivation from latency (51, 75).…”

mentioning

confidence: 99%

Tiled Microarray Identification of Novel Viral Transcript Structures and Distinct Transcriptional Profiles during Two Modes of Productive Murine Gammaherpesvirus 68 Infection

Cheng

Zhi

Santana

et al. 2012

cWe applied a custom tiled microarray to examine murine gammaherpesvirus 68 (MHV68) polyadenylated transcript expression in a time course of de novo infection of fibroblast cells and following phorbol ester-mediated reactivation from a latently infected B cell line. During de novo infection, all open reading frames (ORFs) were transcribed and clustered into four major temporal groups that were overlapping yet distinct from clusters based on the phorbol ester-stimulated B cell reactivation time course. High-density transcript analysis at 2-h intervals during de novo infection mapped gene boundaries with a 20-nucleotide resolution, including a previously undefined ORF73 transcript and the MHV68 ORF63 homolog of Kaposi's sarcoma-associated herpesvirus vNLRP1. ORF6 transcript initiation was mapped by tiled array and confirmed by 5= rapid amplification of cDNA ends. The ϳ1.3-kb region upstream of ORF6 was responsive to lytic infection and MHV68 RTA, identifying a novel RTA-responsive promoter. Transcription in intergenic regions consistent with the previously defined expressed genomic regions was detected during both types of productive infection. We conclude that the MHV68 transcriptome is dynamic and distinct during de novo fibroblast infection and upon phorbol ester-stimulated B cell reactivation, highlighting the need to evaluate further transcript structure and the context-dependent molecular events that govern viral gene expression during chronic infection. Murine gammaherpesvirus 68 (MHV68; also known as ␥HV68 or murid herpesvirus 4) infection of mice is a model pathogenesis system for gammaherpesviruses such as Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/ HHV-8) and Epstein-Barr virus (EBV) (5, 20). The life cycles of these lymphotropic and transforming viruses in the host involve the initial transit across a mucosal barrier to gain access to and establish latency in a leukocyte reservoir, followed by intermittent reactivation and dissemination to the mucosal surfaces for spread to new hosts (32,74,77,82).Replication at the site of primary infection impacts MHV68 dissemination and latency establishment in secondary lymphoid tissues of mice. The absence of proteins essential for lytic replication, such as the viral transactivator ORF50/mRTA (61) and the ORF6/single-stranded DNA-binding protein (ssDBP) (49), or the inhibition of viral DNA replication by the administration of cidofovir (62) impairs the establishment of latency in the splenic B cell compartment. In addition, virus replication at the mucosa triggers the innate and adaptive immune responses critical for host control (6,45,72,73). These responses might play a critical role in the recruitment and activation of target cells such as dendritic cells that precede dissemination to distal reservoirs, such as splenic B cells and peritoneal macrophages (5, 25). Thus, the lytic gene expression program in newly infected cells that are permissive for productive infection is an important aspect of pathogenesis.Reactivation from latency is a...