Myeloid Dendritic Cells Repress Human Cytomegalovirus Gene Expression and Spread by Releasing Interferon-Unrelated Soluble Antiviral Factors

Abstract

Cytomegalovirus (CMV) is a betaherpesvirus that latently infects most adult humans worldwide and is a major cause of morbidity and mortality in immunocompromised hosts. Latent human CMV (HCMV) is believed to reside in precursors of myeloid-lineage leukocytes and monocytes, which give rise to macrophages and dendritic cells (DC). We report here that human monocyte-derived DC (mo-DC) suppress HCMV infection in coculture with infected fibroblast target cells in a manner dependent on the effector-to-target ratio. Intriguingly, optimal activation of mo-DC was achieved under coculture conditions and not by direct infection with HCMV, implying that mo-DC may recognize unique molecular patterns on, or within, infected fibroblasts. We show that HCMV is controlled by secreted factors that act by priming defenses in target cells rather than by direct viral neutralization, but we excluded a role for interferons (IFNs) in this control. The expression of lytic viral genes in infected cells and the progression of infection were significantly slowed, but this effect was reversible, indicating that the control of infection depended on the transient induction of antiviral effector molecules in target cells. Using immediate early or late-phase reporter HCMVs, we show that soluble factors secreted in the cocultures suppress HCMV replication at both stages of the infection and that their antiviral effects are robust and comparable in numerous batches of mo-DC as well as in primary fibroblasts and stromal cells. Human cytomegalovirus is a widespread opportunistic pathogen that can cause severe disease and complications in vulnerable individuals. This includes newborn children, HIV AIDS patients, and transplant recipients. Although the majority of healthy humans carry this virus throughout their lives without symptoms, it is not exactly clear which tissues in the body are the main reservoirs of latent virus infection or how the delicate balance between the virus and the immune system is maintained over an individual's lifetime. Here, for the first time, we provide evidence for a novel mechanism of direct virus control by a subset of human innate immune cells called dendritic cells, which are regarded as a major site of virus latency and reactivation. Our findings may have important implications in HCMV disease prevention as well as in development of novel therapeutic approaches.

Cytomegalovirus inhibition of extrinsic apoptosis determines fitness and resistance to cytotoxic CD8 T cells

“…Similarly, UL36 gene ORF was disrupted from HCMV genome by replacing the methionine in first exon at position 49,087 with stop codon. Generation of the reporter TB40E IEr has been described in detail previously (51). Briefly, mNeonGreen gene linked to the P2A peptide was inserted before the start codon of UL122/123 exon 2.…”

Triple RNA-Seq Reveals Synergy in a Human Virus-Fungus Co-infection Model

“…Reporter strain human CMV TB40/E-mNeonGreen and CMV TB40/SE (non-fluorescent) were generated according to published protocols (Kasmapour et al, 2017;Paijo et al, 2016;Sampaio et al, 2017). A. fumigatus (ATCC 46645) germ tubes were prepared overnight in RPMI (Invitrogen) and used for dual or triple RNA-seq.…”

Chromatin-Remodeling Factor SPOC1 Acts as a Cellular Restriction Factor against Human Cytomegalovirus by Repressing the Major Immediate Early Promoter

“…UV-inactivated AD169 was generated by the exposure of wild-type AD169 to UV light (0.12 J/cm 2 ). The recombinant reporter HCMV strain used, TB40/E-IE-mNeonGreen, was generated as described previously (32). One day prior to infection, HFF cells were seeded into six-well dishes (3 ϫ 10 5 cells/well).…”

“…In order to monitor the infection in real time, a Leica TCS SP5 confocal microscope with a complete environmental incubation system was used. Thereby, images of 5 fields under each condition were acquired every 15 min for up to 30 h. The acquired stacks of time series for all fields were automatically analyzed by using an ImageJ macro created by B. Kasmapour, Braunschweig, Germany, reporting the mean IE signal associated with the cell nuclei in each frame (32).…”

“…HFF/SPOC1 (Tet-On) cells were induced with doxycycline 24 h prior to infection, while untreated cells were used in parallel as a control. For real-time monitoring, we made use of the recombinant reporter virus TB40/E-IE-mNeonGreen, which was used previously by Kasmapour and colleagues to monitor IE protein expression kinetics (32). The dynamics of early viral gene expression (up to 30 hpi) were investigated after infection at an MOI of 0.05, followed by automated whole-frame evaluation and quantification with ImageJ-based software (Fig.…”