Abstract
The inhibition of death-receptor apoptosis is a conserved viral function. The murine cytomegalovirus (MCMV) gene M36 is a sequence and functional homologue of the human cytomegalovirus gene UL36, and it encodes an inhibitor of apoptosis that binds to caspase-8, blocks downstream signaling and thus contributes to viral fitness in macrophages and in vivo. Here we show a direct link between the inability of mutants lacking the M36 gene (ΔM36) to inhibit apoptosis, poor viral growth in macrophage cell cultures and viral in vivo fitness and virulence. ΔM36 grew poorly in RAG1 knockout mice and in RAG/IL-2-receptor common gamma chain double knockout mice (RAGγC−/−), but the depletion of macrophages in either mouse strain rescued the growth of ΔM36 to almost wild-type levels. This was consistent with the observation that activated macrophages were sufficient to impair ΔM36 growth in vitro. Namely, spiking fibroblast cell cultures with activated macrophages had a suppressive effect on ΔM36 growth, which could be reverted by z-VAD-fmk, a chemical apoptosis inhibitor. TNFα from activated macrophages synergized with IFNγ in target cells to inhibit ΔM36 growth. Hence, our data show that poor ΔM36 growth in macrophages does not reflect a defect in tropism, but rather a defect in the suppression of antiviral mediators secreted by macrophages. To the best of our knowledge, this shows for the first time an immune evasion mechanism that protects MCMV selectively from the antiviral activity of macrophages, and thus critically contributes to viral pathogenicity in the immunocompromised host devoid of the adaptive immune system.
Highlights
The viral inhibition of programmed cell death is a conserved function in animal viruses [1,2,3,4]
murine CMV (MCMV) lacking the M36 gene (DM36) could not grow to high titers in macrophage cell lines or in primary macrophages, but showed no loss of fitness in fibroblasts [10], which is in line with the observations that UL36 is required for human CMV (HCMV) growth in macrophages derived from THP-1 monocytes [12], but not for its growth in fibroblasts [13]
The majority of adult people are infected with human cytomegalovirus (CMV), but in hosts with a healthy immune system it is kept in check and does not cause disease
Summary
The viral inhibition of programmed cell death is a conserved function in animal viruses [1,2,3,4]. The herpes simplex gene ICP34.5 and the cytomegalovirus (CMV) gene TRS1 both bind the host gene Beclin and block autophagy [5,6]. Both the human CMV (HCMV) and the murine CMV (MCMV) encode viral genes that block mitochondrial apoptosis [7,8] by blocking Bax, but not Bak signaling [7,9]. An even more remarkable conservation of gene function has been observed in the case of the viral Inhibitor of Caspase-8 Activation (vICA), encoded by the HCMV gene UL36, and its counterpart in the MCMV genome, a gene called M36 [10,11]. MCMV lacking the M36 gene (DM36) could not grow to high titers in macrophage cell lines or in primary macrophages, but showed no loss of fitness in fibroblasts [10], which is in line with the observations that UL36 is required for HCMV growth in macrophages derived from THP-1 monocytes [12], but not for its growth in fibroblasts [13]
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