Human cytomegalovirus (HCMV) is a clinically important herpesvirus that has co-evolved for millions of years with its human host, and establishes lifelong persistent infection. A substantial proportion of its 235kb genome is dedicated to manipulating host immunity through targeting antiviral host proteins for degradation or relocalisation. Quantitative proteomics of the infected cell has extensively characterised these processes, but the cell-free virion has been less well studied. We therefore carried out proteomic analysis of a clinical HCMV strain virion. This revealed multiple novel components, including the viral protein gpUL141, which is recognised as an NK immune-evasin that targets several host proteins (CD155, CD112, and TRAILR) when expressed in the cell. Co-Immunoprecipitation of gpUL141 from virions identified interactions with viral entry glycoproteins from the trimer (gH/gL/gO), pentamer (gH/gL/UL128/UL130/UL131A), and gH/gpUL116 complexes, as well as gB. These interactions occurred via direct interactions between gpUL141 and either gH or gB. Analysis supported a model in which gpUL141 homodimers independently interacted with separate gB/gH-containing complexes. gpUL141 encodes an ER retention domain that restricts trafficking through the ER/golgi, and limited the transport of glycoprotein complexes bound by gpUL141. As a result, gpUL141 reduced levels of multiple glycoprotein complexes on the infected cell surface as well as in the virion. This reduced syncytia formation, inhibited antibody-dependent cellular cytotoxicity (ADCC), and reduced susceptibility to neutralising antibodies. Thus, gpUL141 represents an immune-evasin that not only targets host proteins to limit NK-cell attack, but also alters the trafficking of multiple viral glycoprotein complexes in order to evade humoral immunity. ### Competing Interest Statement The authors have declared no competing interest.

Human cytomegalovirus (HCMV) UL141 induces protection against natural killer cell-mediated cytolysis by downregulating cell surface expression of CD155 (nectin-like molecule 5; poliovirus receptor), a ligand for the activating receptor DNAM-1 (CD226). However, DNAM-1 is also recognized to bind a second ligand, CD112 (nectin-2). We now show that HCMV targets CD112 for proteasome-mediated degradation by 48 h post-infection, thus removing both activating ligands for DNAM-1 from the cell surface during productive infection. Significantly, cell surface expression of both CD112 and CD155 was restored when UL141 was deleted from the HCMV genome. While gpUL141 alone is sufficient to mediate retention of CD155 in the endoplasmic reticulum, UL141 requires assistance from additional HCMV-encoded functions to suppress expression of CD112.

Human cytomegalovirus (HCMV) causes lifelong, persistent infections and its survival is under intense, continuous selective pressure from the immune system. A key aspect of HCMV's capacity for survival lies in immune avoidance. In this context, cells undergoing productive infection exhibit remarkable resistance to natural killer (NK) cell-mediated cytolysis in vitro. To date, six genes encoding proteins (UL16, UL18, UL40, UL83, UL141 and UL142) and one encoding a microRNA (miR-UL112) have been identified as capable of suppressing NK cell recognition. Even though HCMV infection efficiently activates expression of ligands for the NK cell activating receptor NKG2D, at least three functions (UL16, UL142 and miR-UL112) act in concert to suppress presentation of these ligands on the cell surface. Although HCMV downregulates expression of endogenous MHC-I, it encodes an MHC-I homologue (UL18) and also upregulates the expression of cellular HLA-E through the action of UL40. The disruption of normal intercellular connections exposes ligands for NK cell activating receptors on the cell surface, notably CD155. HCMV overcomes this vulnerability by encoding a function (UL141) that acts post-translationally to suppress cell surface expression of CD155. The mechanisms by which HCMV systematically evades (or, more properly, modulates) NK cell recognition constitutes an area of growing understanding that is enhancing our appreciation of the basic mechanisms of NK cell function in humans.