A Myeloid Progenitor Cell Line Capable of Supporting Human Cytomegalovirus Latency and Reactivation, Resulting in Infectious Progeny

Abstract

<jats:p>Human cytomegalovirus (HCMV) is a herpesvirus that establishes a lifelong, latent infection within a host. At times when the immune system is compromised, the virus undergoes a lytic reactivation producing infectious progeny. The identification and understanding of the biological mechanisms underlying HCMV latency and reactivation are not completely defined. To this end, we have developed a tractable<jats:italic>in vitro</jats:italic>model system to investigate these phases of viral infection using a clonal population of myeloid progenitor cells (Kasumi-3 cells). Infection of these cells results in maintenance of the viral genome with restricted viral RNA expression that is reversed with the addition of the phorbol ester 12-<jats:italic>O</jats:italic>-tetradecanoylphorbol-13-acetate (TPA, also known as PMA). Additionally, a latent viral transcript (LUNA) is expressed at times where viral lytic transcription is suppressed. Infected Kasumi-3 cells initiate production of infectious virus following TPA treatment, which requires cell-to-cell contact for efficient transfer of virus to other cell types. Importantly, lytically infected fibroblast, endothelial, or epithelial cells can transfer virus to Kasumi-3 cells, which fail to initiate lytic replication until stimulated with TPA. Finally, inflammatory cytokines, in addition to the pharmacological agent TPA, are sufficient for transcription of immediate-early (IE) genes following latent infection. Taken together, our findings argue that the Kasumi-3 cell line is a tractable<jats:italic>in vitro</jats:italic>model system with which to study HCMV latency and reactivation.</jats:p>

Transplant-induced reactivation of murine cytomegalovirus immediate early gene expression is associated with recruitment of NF-κB and AP-1 to the major immediate early promoter

“…The goal of the present study is to identify additional candidates that may contribute to reactivation of IE gene expression in allogeneic transplants, with the long-term goal of developing novel therapeutic approaches to preventing reactivation of the virus. In addition to the TNF/NF-k B and IL-6/mitogen-activated protein kinase (MAPK) signalling pathways, which have been previously implicated in reactivation of HCMV (Döcke et al, 1994;Fietze et al, 1994;Hargett & Shenk, 2010;Huang et al, 2012;Kew et al, 2014;O'Connor & Murphy, 2012;Prösch et al, 1995;Reeves & Compton, 2011;Stein et al, 1993), we have identified novel signalling pathways that have not to our knowledge been previously associated with reactivation of HCMV. Our results suggest that it may be necessary to target multiple signalling pathways to prevent transcriptional reactivation of CMV.…”

“…Allograft rejection, sepsis, acute illness, IL-6 and TNF have long been implicated in reactivation of HCMV in patients (Cook et al, 1998;Döcke et al, 1994;Fietze et al, 1994;Grattan et al, 1989;Heininger et al, 2001;Hibberd et al, 1992;Kalil & Florescu, 2009;Kutza et al, 1998;Lao et al, 1997;Limaye et al, 2008;Mutimer et al, 1997;Portela et al, 1995;Razonable et al, 2001;Reinke et al, 1994a) and allogeneic stimulation, TNF, IL-6 and lipopolysaccharide have been shown to induce reactivation of HCMV in experimental models (Hargett & Shenk, 2010;Huang et al, 2012;Kew et al, 2014;O'Connor & Murphy, 2012;Reeves & Compton, 2011;Söderberg-Nauclér et al, 1997). Allogeneic transplantation and TNF are sufficient to activate both an HCMV MIEP-lacZ transgene and MCMV IE gene expression (Hummel et al, 2001).…”

“…Murine cytomegalovirus (MCMV) is very similar to HCMV in many important ways, including: (i) ability to establish latency and to reactivate; (ii) hierarchical regulation of viral gene expression; (iii) structure, function and organization of the major immediate early (IE) genes (Keil et al, 1987a, b;Meier & Stinski, 2013;Stenberg et al, 1984); (iv) repression of IE gene expression during latency by heterochromatinization of viral genomes (Grzimek et al, 2001;Hummel & Abecassis, 2002;Hummel et al, 2001;Kurz et al, 1999;Liu et al, 2008;Reeves & Sinclair, 2013;Reeves et al, 2005;Seckert et al, 2013); (v) the presence of similar regulatory elements (e.g. NF-k B, AP-1, Sp1 and CREB/ATF binding sites) in the viral IE enhancers Meier & Stinski, 2013); and (vi) (re)activation of the MIEP and/or IE transcription in response to inflammatory mediators or allogeneic stimulation (Döcke et al, 1994;Fietze et al, 1994;Huang et al, 2012;Hummel & Abecassis, 2002;Hummel et al, 2001;Kew et al, 2014;Lee et al, 2004;Liu et al, 2013;O'Connor & Murphy, 2012;Prösch et al, 1995;Reeves & Compton, 2011;Simon et al, 2005;Stein et al, 1993).…”

Estrogen-related receptor α is required for efficient human cytomegalovirus replication

“…To determine the importance of ERRα during infection of a different cell type with a clinical isolate of HCMV, TB40/E-GFP (28,33), was used to infect retinal pigmented epithelial cells (ARPE-19) (34). TB40/E spreads in a cell-to-cell manner without release of significant quantities of extracellular infectious particles in these cells (35).…”

Inhibition of the FACT Complex Reduces Transcription from the Human Cytomegalovirus Major Immediate Early Promoter in Models of Lytic and Latent Replication

“…Using our Kasumi-3 (K3) HPC latency model system, we reported that phorbol ester (e.g., tetradecanoyl phorbol acetate [TPA]) treatment following latent infection stimulates MIEP-driven transcription and the production of infectious virus (10). Also, tumor necrosis factor alpha (TNF-␣) or interleukin-1␤ (IL-1␤) treatment of latently infected K3 cells (K3s) (10) or primary HPCs induced MIEP transcription (8,11). These inflammatory cytokines, as well as TPA, activate NF-B (12,13).…”