Experimental human cytomegalovirus latency in CD14⁺monocytes

Abstract

CD14 + monocytes are a reservoir for latent human cytomegalovirus, and virus replication is reactivated during their differentiation to macrophages or dendritic cells. It has not been clear whether the virus can establish latency upon direct infection of monocytes or whether it must first become quiescent in a progenitor cell that subsequently differentiates to generate a monocyte. We report that infection of primary human monocytes with a clinical strain of human cytomegalovirus exhibits the hallmarks of latency. We established conditions for culturing monocytes that prevent differentiation for at least 25 d, as evidenced by cell surface marker expression. Infection of these monocytes with the FIX clinical strain resulted in transient accumulation of many viral lytic RNAs and sustained expression of four previously described latency-associated transcripts. The amount of viral DNA remained constant after infection, and cell surface and total HLA-DR proteins were substantially reduced on a continuing basis after infection. When treated with cytokine mixtures that stimulate differentiation to a macrophage or dendritic cell phenotype, infected monocytes reactivated virus replication and produced infectious progeny. Treatment of infected monocytes with IL-6 alone also was sufficient for reactivation, and the particles produced after exposure to this cytokine were about fivefold more infectious than virions produced by other treatments. We propose that in vivo microenvironments influence not only the efficiency of reactivation but also the infectivity of the virions produced from latently infected monocytes.herpesvirus | myeloid biology | cell culture | antigen presentation | immunology H uman cytomegalovirus (HCMV) is a dangerous opportunistic pathogen (1) that replicates in many cell types but enters latency in others, allowing persistence of the viral genome without production of progeny. Viral DNA and a small subset of viral RNAs have been found in naturally infected CD34 + hematopoietic stem cells (HSCs) and CD33 + progenitor cells (2). Experimental infections of CD34 + and CD33 + cells also display the hallmarks of latency. HCMV DNA and a subset of viral transcripts are present in these cells after infection in culture, and the virus can be reactivated to produce progeny if the cells differentiate (2). The choice of HCMV strain can influence the outcome of infections (3). Two clinical isolates entered and exited latency, whereas the AD169 laboratory strain failed to become latent in CD34 + cells. AD169 lacks the UL138 gene, which is important for entry into latency (3, 4).Like HSCs, naturally infected CD14 + monocytes harbor HCMV DNA (5-7), and viral replication is activated by differentiation (8, 9). Monocytes from peripheral blood are nonpermissive for HCMV replication (10), but when differentiated to a macrophage phenotype, they support the production of infectious progeny (11, 12). Thus, natural and experimental infections argue that differentiation from monocyte to macrophage or dendritic cell marks a divi...

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).…”

The immunology of human cytomegalovirus latency: could latent infection be cleared by novel immunotherapeutic strategies?

“…CD34 1 progenitors, monocytes and granulocyte-macrophage progenitors, as well as some established myeloid cell lines, can be infected in culture allowing the maintenance of latent viral genomes which can then be reactivated by differentiation signals. 21,30,[35][36][37][45][46][47] However, it is clear that some experimental models using established cell lines do not appear to fully recapitulate all aspects of control of latency and reactivation observed in primary myeloid cells. 48 A totally quiescent viral genome during latent infection would clearly be the ideal way to avoid immune surveillance-if viral proteins are not expressed at all there would be no processing and presentation of viral antigens to specific T cells and, thus, latently infected cells would be ignored by the host immune response.…”

“…28 Consistent with cells of the myeloid lineage being sites of latent infection, analyses of the viral transcription programme in these cells generally shows a suppression of viral lytic gene expression 2,29-32 but concomitant expression of known latency-associated viral genes. 31,[33][34][35][36][37] Importantly, these cells do not produce infectious virions; an essential characteristic of latent infection. In latent myeloid cells in vivo, this suppression of the lytic transcription programme appears to involve repression of the viral major immediate early promoter (MIEP), which would normally drive lytic cycle, through post-translational modification of histones around the MIEP resulting in the presence of well characterized repressive chromatin marks (reviewed in Ref.…”

“…Also, importantly, this latent transcription programme can be reactivated to lytic cycle by differentiation of latent CD34 1 cells or monocytes to macrophages or dendritic cells resulting in expression of the established lytic temporal cascade of viral gene expression, leading to viral DNA replication and de novo virus production. 36,[38][39][40] Crucially, this reactivation of the lytic cascade of gene expression is initiated by the expression of the major immediate early proteins (IE72 and IE86); IE gene expression, thus, acts as a master regulator to initiate lytic cycle [41][42][43][44] (Figure 4). It is worth noting that many of these analyses of naturally latently infected cells can also be, in general, recapitulated in experimental models of latency in vitro.…”