The human cytomegalovirus UL36 gene controls caspase-dependent and -independent cell death programs activated by infection of monocytes differentiating to macrophages.

The cellular protease caspase-8 activates extrinsic apoptosis and also functions to promote monocyte-to-macrophage differentiation. Differentiation-induced alterations to antiviral caspase-8-dependent cell death pathways are unclear. Here, we show THP-1 monocyte-to-macrophage differentiation alters the specific cell death pathways activated in response to human cytomegalovirus (HCMV) infection. Employing viruses with mutations in UL36, the gene that encodes the viral inhibitor of caspase-8 activation (vICA), our data indicate that both caspase-dependent and -independent death pathways are activated in response to infection. Activation of caspase-dependent and -independent cell death responses restricted growth of vICA-deficient viruses, and vICA/pUL36 inhibited either response. Thus, these studies also reveal that the UL36 gene controls a caspase-independent cell death pathway. The impact of caspases on control of antiviral responses differed at early and late stages of macrophage differentiation. Early in differentiation, vICA-deficient virus-induced cell death was dependent on caspases and inhibited by the pan-caspase inhibitor z-VAD(OMe)-fluoromethyl ketone. In contrast, virus-induced death at late times of differentiation was caspase independent. Additional unlabeled and fluorescent inhibitors indicated that caspase-8 promoted death from within infected cells at early but not late stages of differentiation. These data highlight the multifunctional role of vICA/pUL36 as HCMV encounters various antiviral responses during macrophage differentiation.

Probing the spatial organization of measles virus fusion complexes.

The spatial organization of metastable paramyxovirus fusion (F) and attachment glycoprotein hetero-oligomers is largely unknown. To further elucidate the organization of functional fusion complexes of measles virus (MeV), an archetype of the paramyxovirus family, we subjected central predictions of alternative docking models to experimental testing using three distinct approaches. Carbohydrate shielding through engineered N-glycans indicates close proximity of a membrane-distal, but not membrane-proximal, section of the MeV attachment (H) protein stalk domain to F. Directed mutagenesis of this section identified residues 111, 114, and 118 as modulators of avidity of glycoprotein interactions and determinants of F triggering. Stalk-length variation through deletion or insertion of HR elements at positions flanking this section demonstrates that the location of the stalk segment containing these residues cannot be altered in functional fusion complexes. In contrast, increasing the distance between the H head domains harboring the receptor binding sites and this section through insertion of structurally rigid alpha-helical domains with a pitch of up to approximately 75 A downstream of stalk position 118 partially maintains functionality in transient expression assays and supports efficient growth of recombinant virions. In aggregate, these findings argue against specific protein-protein contacts between the H head and F head domains but instead support a docking model that is characterized by short-range contacts between the prefusion F head and the attachment protein stalk, possibly involving H residues 111, 114, and 118, and extension of the head domain of the attachment protein above prefusion F.