Establishment of B-Cell Lines Latently Infected with Reactivation-Competent Murine Gammaherpesvirus 68 Provides Evidence for Viral Alteration of a DNA Damage-Signaling Cascade

Abstract

Gammaherpesvirus 68 (␥HV68, or MHV68) is a naturally occurring rodent pathogen that replicates to high titer in cell culture and is amenable to in vivo experimental evaluation of viral and host determinants of gammaherpesvirus disease. However, the inability of MHV68 to transform primary murine B cells in culture, the absence of a robust cell culture latency system, and the paucity of MHV68-positive tumor cell lines have limited an understanding of the molecular mechanisms by which MHV68 modulates the host cell during latency and reactivation. To facilitate a more complete understanding of viral and host determinants that regulate MHV68 latency and reactivation in B cells, we generated a recombinant MHV68 virus that encodes a hygromycin resistance protein fused to enhanced green fluorescent protein as a means to select cells in culture that harbor latent virus. We utilized this virus to infect the A20 murine mature B-cell line and evaluate reactivation competence following treatment with diverse stimuli to reveal viral gene expression, DNA replication, and production of progeny virions. Comparative analyses of parental and infected A20 cells indicated a correlation between infection and alterations in DNA damage signaling following etoposide treatment. The data described in this study highlight the potential utility of this new cell culture-based system to dissect molecular mechanisms that regulate MHV68 latency and reactivation, as well as having the potential of illuminating biochemical alterations that contribute to gammaherpesvirus pathogenesis. In addition, such cell lines may be of value in evaluating targeted therapies to gammaherpesvirus-related tumors.Gammaherpesvirus 68 (␥HV68 or MHV68) is a rodent rhadinovirus found naturally in European bank voles and wood mice (6, 7). As a rodent pathogen, MHV68 infection of laboratory mice offers an experimental system to investigate aspects of gammaherpesvirus pathogenesis. Following experimental inoculation of laboratory mice, MHV68 undergoes productive replication at the site of inoculation, which facilitates dissemination to distal organs and is followed by quiescence or latency, a form of infection where viral gene expression is restricted and progeny virions are not produced (41,51,71). In the case of MHV68, latent infection is established in B lymphocytes, as well as epithelial cells, macrophages, and dendritic cells (19,56,58,74). Latency is maintained for the lifetime of the host, and latently infected cells retain the capacity to reinitiate the productive replication cycle, a process termed reactivation (45, 51, 52).The genetic malleability of MHV68 enables the identification of viral genes involved in specific aspects of viral pathogenesis by targeted mutagenesis (1). For example, such approaches have demonstrated the importance of the unique MHV68 M2 gene product in latency establishment, reactivation from latency, and promoting B-cell differentiation (24, 26), as well as defining the roles of conserved genes, such as orf73 (LANA homolog) in latency mai...

Is murine gammaherpesvirus-68 (MHV-68) a suitable immunotoxicological model for examining immunomodulatory drug-associated viral recrudescence?

“…Although the mechanisms are unclear, the DDR is utilized by gammaherpesviruses for viral replication (Barton et al, 2011;Sinclair et al, 2006;Weitzman et al, 2010). Several chemicals have been shown to induce the DDR from latently infected cells, including etoposide, camptothecin, cisplatin, and staurosporine (Feng et al, 2002;Forrest & Speck, 2008). These results suggest that the MHV-68 model will likely be sensitive to compounds with mechanisms of action that affect these pathways.…”

“…MHV-68 studies likewise discovered that histone acetylation was sufficient to re-activate the lytic cycle as a result of demethylation of the RTA promoter (Ben-Sasson & Klein, 1981;Chang & Liu, 2000;Chen et al, 2001;Countryman et al, 2008). In vitro, histone deacetylase inhibitors (such as butyric acid [NaB]) have also been shown to re-activate MHV-68 latently infected cell lines that might also have biological relevance in vivo (Forrest & Speck, 2008;Moser et al, 2005;Yang et al, 2009).…”

“…The MHV-68 model has also been studied extensively in vitro, and a few murine B-lymphocyte lines have been developed from MHV-68 latently infected mice (S11 and A20-HE) (Forrest & Speck, 2008;Usherwood et al, 1996a). These cell lines are both responsive to stimulation with the phorbol ester PMA, which results in virus re-activation from latency (Table 2).…”

“…These cell lines are both responsive to stimulation with the phorbol ester PMA, which results in virus re-activation from latency (Table 2). An investigation into some of the chemicals known to induce viral re-activation was done recently in A20-HE cells, a latently infected murine B-lymphocyte line (Forrest & Speck, 2008). Treatment of these cells with PMA resulted in the most robust viral re-activation, but these cells were also shown to be responsive to other known re-activation stimuli (see Table 2) including etoposide, camptothecin, and staurosporine (DNA damage inducers), BCR cross-linkers, and NaB (histone deacetylase inhibitor) (Forrest & Speck, 2008).…”

Murine gammaherpesvirus-68 (MHV-68) is not horizontally transmitted amongst laboratory mice by cage contact

“…A20-HE-2 cells are A20 cells latently infected with MHV-68-EGFP (enhanced green fluorescent protein) virus. These cells were obtained from Dr Craig Forrest and Dr Samuel Speck (Emory University, Atlanta, GA) who had previously characterized them (see Forrest & Speck, 2008). All cells used in these studies were either provided directly from Forrest and Speck or were re-derived from these cells.…”