UL36 Rescues Apoptosis Inhibition and In vivo Replication of a Chimeric MCMV Lacking the M36 Gene

Abstract

Apoptosis is an important defense mechanism mounted by the immune system to control virus replication. Hence, cytomegaloviruses (CMV) evolved and acquired numerous anti-apoptotic genes. The product of the human CMV (HCMV) UL36 gene, pUL36 (also known as vICA), binds to pro-caspase-8, thus inhibiting death-receptor apoptosis and enabling viral replication in differentiated THP-1 cells. In vivo studies of the function of HCMV genes are severely limited due to the strict host specificity of cytomegaloviruses, but CMV orthologues that co-evolved with other species allow the experimental study of CMV biology in vivo. The mouse CMV (MCMV) homolog of the UL36 gene is called M36, and its protein product (pM36) is a functional homolog of vICA that binds to murine caspase-8 and inhibits its activation. M36-deficient MCMV is severely growth impaired in macrophages and in vivo. Here we show that pUL36 binds to the murine pro-caspase-8, and that UL36 expression inhibits death-receptor apoptosis in murine cells and can replace M36 to allow MCMV growth in vitro and in vivo. We generated a chimeric MCMV expressing the UL36 ORF sequence instead of the M36 one. The newly generated MCMVUL36 inhibited apoptosis in macrophage lines RAW 264.7, J774A.1, and IC-21 and its growth was rescued to wild type levels. Similarly, growth was rescued in vivo in the liver and spleen, but only partially in the salivary glands of BALB/c and C57BL/6 mice. In conclusion, we determined that an immune-evasive HCMV gene is conserved enough to functionally replace its MCMV counterpart and thus allow its study in an in vivo setting. As UL36 and M36 proteins engage the same molecular host target, our newly developed model can facilitate studies of anti-viral compounds targeting pUL36 in vivo.

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

“…The vICA protein from HCMV is a determinant of virus fitness in cultured macrophages (38). The MCMV homolog M36 confers virus growth fitness in vivo (21)(22)(23), where the macrophages have been shown to be the major cell type that limits the virus spread and growth (24). Here, we show that UL36 and M36 proteins also protect the virus-infected cells from CD8 T cell control by inhibiting cell-extrinsic apoptosis at the Casp8 level.…”

“…Casp8induced apoptosis is inhibited by the HCMV protein vICA (viral inhibitor of caspase-8 activation), a product of the viral UL36 gene (19), or by the MCMV homolog, M36 (20). Both of these gene products inhibit Casp8 activation and are interchangeable across species (21). MCMV lacking the M36 is attenuated in vivo (22,23) because of a failure to inhibit FADD signaling to Casp8 (22).…”

Die Another Day: Inhibition of Cell Death Pathways by Cytomegalovirus

“…It is dispensable for viral replication in fibroblasts, but required for optimal replication in macrophages in vitro [ 109 ]. The replication defect of an MCMV M36 deletion mutant in macrophages was fully rescued by expression of HCMV UL36 or overexpression of a dominant-negative FADD [ 110 , 111 ] and partially rescued by expression of MC159, a viral FLIP of the molluscum contagiosum virus [ 112 ]. Remarkably, the in vivo growth defect of MCMV mutants lacking M36 was rescued by depletion of macrophages [ 113 ].…”

Tuning the Orchestra: HCMV vs. Innate Immunity

“…The fact that homologs of HCMV vICA have been identified in the vast majority of mammalian betaherpesviruses implies that the function of vICA is important and conserved. This is exemplified by M36, the vICA counterpart of MCMV, which also displays an anti-apoptotic activity by interacting with procaspase-8, and that has been shown to be rescued by vICA in order to allow viral replication, confirming the reliability of the murine model (Chaudhry et al, 2017).…”