Use of a Virus-Encoded Enzymatic Marker Reveals that a Stable Fraction of Memory B Cells Expresses Latency-Associated Nuclear Antigen throughout Chronic Gammaherpesvirus Infection

Abstract

An integral feature of gammaherpesvirus infections is the ability to establish lifelong latency in B cells. During latency, the viral genome is maintained as an extrachomosomal episome, with stable maintenance in dividing cells mediated by the viral proteins Epstein-Barr nuclear antigen 1 (EBNA-1) for Epstein-Barr virus and latencyassociated nuclear antigen (LANA) for Kaposi's sarcoma-associated herpesvirus. It is believed that the expression of episome maintenance proteins is turned off in the predominant long-term latency reservoir of resting memory B cells, suggesting that chronic gammaherpesvirus infection is primarily dormant. However, the kinetics of LANA/ EBNA-1 expression in individual B-cell subsets throughout a course of infection has not been examined. The infection of mice with murine gammaherpesvirus 68 (MHV68, ␥HV68) provides a model to determine the specific cellular and molecular events that occur in vivo during lifelong gammaherpesvirus latency. In work described here, we make use of a heterologously expressed enzymatic marker to define the types of B cells that express the LANA homolog (mLANA) during chronic MHV68 infection. Our data demonstrate that mLANA is expressed in a stable fraction of B cells throughout chronic infection, with a prominent peak at 28 days. The expression of mLANA was detected in naïve follicular B cells, germinal-center B cells, and memory B cells throughout infection, with germinalcenter and memory B cells accounting for more than 80% of the mLANA-expressing cells during the maintenance phase of latency. These findings suggest that the maintenance phase of latency is an active process that involves the ongoing proliferation or reseeding of latently infected memory B cells.Gammaherpesviruses such as Epstein-Barr virus (EBV), Kaposi's sarcoma-associated virus (KSHV, HHV-8), and murine gammaherpesvirus 68 (MHV68, ␥HV68) are associated with lymphoproliferative diseases and a variety of malignancies of both epithelial and lymphoid origin. The strict species specificity exhibited by gammaherpesviruses has limited research on the human viruses primarily to in vitro studies. MHV68 is genetically colinear to the human gammaherpesviruses and exhibits many similar pathogenic features (54, 62). MHV68 is a natural pathogen of rodents (6, 9, 44), making the inoculation of mice with MHV68 a useful small-animal model to study gammaherpesvirus infection in vivo.A hallmark of gammaherpesvirus infections is the establishment of lifelong latency in B cells. During latency, the viral genome is maintained as an extrachromosomal episome, viral gene expression is highly restricted, and no new progeny virions are generated. The stable maintenance of the episome in dividing cells requires regulated plasmid DNA replication and the efficient partitioning of replicated genomes to daughter cells. These processes are mediated by critical episome maintenance protein Epstein-Barr nuclear antigen 1 (EBNA-1) for EBV and latency-associated nuclear antigen (LANA) for KSHV. EBNA-1 and LANA facilitate the re...

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

“…One powerful tool used in the MHV68 model of gammaherpesvirus pathogenesis is the ability to generate recombinant viruses with the insertion of transgenes, including cre, to mediate recombinatorial deletion of loxP-flanked host genes (63), fluorescent proteins (13,27), luciferase reporters (37,61), or a ␤-lactamase tag for marking infected cells (64) or with the expression of a transdominant negative mutant factor that targets a specific signaling pathway (44). However, care must be taken to avoid disruption of neighboring genes.…”

“…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).…”

Critical roles of chemokine receptor CCR10 in regulating memory IgA responses in intestines

“…(Fig. 7A) (22)(23)(24). Although frequencies of total CCR10 + and CCR10 − IgA + memory-like B cells in intestines were not significantly different in CCR10 EGFP/EGFP vs. CCR10 +/EGFP mice (Fig.…”

Immature and Transitional B Cells Are Latency Reservoirs for a Gammaherpesvirus

“…mLANA is expressed in a substantial fraction of latently infected cells, and as such the fusion marker is detectable in mature B cells throughout long-term infection (25). For experiments here, B6 mice were inoculated i.n.…”