CD58 is an adhesion molecule that is known to play a critical role in costimulation of effector cells and is intrinsic to immune synapse structure. Herein, we describe a virally encoded gene that inhibits CD58 surface expression. Human cytomegalovirus (HCMV) UL148 was necessary and sufficient to promote intracellular retention of CD58 during HCMV infection. Blocking studies with antagonistic anti-CD58 mAb and an HCMV UL148 deletion mutant (HCMV∆UL148) with restored CD58 expression demonstrated that the CD2/CD58 axis was essential for the recognition of HCMV-infected targets by CD8 HCMV-specific cytotoxic T lymphocytes (CTLs). Further, challenge of peripheral blood mononuclear cells ex vivo with HCMV∆UL148 increased both CTL and natural killer (NK) cell degranulation against HCMV-infected cells, including NK-driven antibody-dependent cellular cytotoxicity, showing that UL148 is a modulator of the function of multiple effector cell subsets. Our data stress the effect of HCMV immune evasion functions on shaping the immune response, highlighting the capacity for their potential use in modulating immunity during the development of anti-HCMV vaccines and HCMV-based vaccine vectors.

Human cytomegalovirus (HCMV) is under constant selective pressure from the immune system in vivo. Study of HCMV genes that have been lost in the absence of, or genetically altered by, such selection can focus research toward findings of in vivo significance. We have been particularly interested in the most pronounced change in the highly passaged laboratory strains AD169 and Towne—the deletion of 13–15 kb of sequence (designated the UL/b′ region) that encodes up to 22 canonical genes, UL133-UL150. At least 5 genes have been identified in UL/b′ that inhibit NK cell function. UL135 suppresses formation of the immunological synapse (IS) by remodeling the actin cytoskeleton, thereby illustrating target cell cooperation in IS formation. UL141 inhibits expression of two activating ligands (CD155, CD112) for the activating receptor CD226 (DNAM-1), and two receptors (TRAIL-R1, R2) for the apoptosis-inducing TRAIL. UL142, ectopically expressed in isolation, and UL148A, target specific MICA allotypes that are ligands for NKG2D. UL148 impairs expression of CD58 (LFA-3), the co-stimulatory cell adhesion molecule for CD2 found on T and NK cells. Outside UL/b′, studies on natural variants have shown UL18 mutants change affinity for their inhibitory ligand LIR-1, while mutations in UL40's HLA-E binding peptide differentially drive NKG2C+ NK expansions. Research into HCMV genomic stability and its effect on NK function has provided important insights into virus:host interactions, but future studies will require consideration of genetic variability and the effect of genes expressed in the context of infection to fully understand their in vivo impact.

Regulation of the lectin galectin 9 (Gal-9) was investigated for the first time during human cytomegalovirus (HCMV) infection. Gal-9 transcription was significantly upregulated in transplant recipients with reactivated HCMV in vivo. In vitro, Gal-9 was potently upregulated by HCMV independently of viral gene expression, with interferon beta (IFN-␤) identified as the mediator of this effect. This study defines an immunoregulatory protein potently increased by HCMV infection and a novel mechanism to control Gal-9 through IFN-␤ induction. P rimary human cytomegalovirus (HCMV) infection is followed by lifelong latency (1). Reactivation from latency is associated with severe morbidity and mortality in the immunocompromised, especially in the allogeneic hematopoietic stem cell transplant (HSCT) setting, where donor or recipient HCMV seropositivity is associated with adverse outcomes. In addition, HCMV is the leading infectious cause of birth defects in the developed world (2).Galectins are a family of lectins that preferentially bind ␤-galactosides. Galectin 9 (Gal-9) can modulate diverse biological activities, including cell adhesion, proliferation, apoptosis, and cell cycle progression (3). Despite such varied functions, regulation of Gal-9 is very poorly understood. Functionally, Gal-9 is best characterized as an immunoregulatory molecule controlling T-cell activity via interaction with its receptor, Tim-3 (4), although Tim-3-independent functions have also been described (5). Gal-9 can play an important role in virus life cycles. It modulates human immunodeficiency virus type 1 (HIV-1) entry (6, 7), while Gal-9-knockout mice exhibit more potent antiviral T-cell responses than wild-type mice (8, 9), and infection with Epstein-Barr virus (EBV) modulates Gal-9 expression to evade immune clearance, inducing apoptosis of EBV-specific CD4 ϩ T cells (10, 11). We investigated the expression of Gal-9 in the context of HCMV infection both in vivo and in vitro, identifying a novel, virally induced mechanism to promote Gal-9 expression.Galectin 9 is upregulated in hematopoietic stem cell transplant recipients with reactivated HCMV infection. We hypothesized that Gal-9 is upregulated in patients with active HCMV replication. Blood samples were drawn from HSCT recipients before peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation from patients with or without HCMV reactivation at a range of time points posttransplant (detailed in Tables 1 and 2). Patients were monitored for HCMV reactivation by quantitative PCR (qPCR; Roche Cobas Amplicor CMV Monitor test), with a sustained rise in serum HCMV genome copies above assay detection limits over at least two time points used to define HCMV reactivation.RNA was then isolated from PBMCs using an RNAqueous kit (Ambion). RNA was converted to cDNA using random primers and SuperScript III reverse transcriptase (Life Technologies). cDNA levels were assayed by qPCR (StepOnePlus real-time PCR system; Life Technologies) using 2ϫ Brilliant II SYBR g...

dThe effect of abrogating the interferon (IFN) response on human cytomegalovirus (HCMV) replication was investigated using primary human cells engineered to block either the production of or the response to type I IFNs. In IFN-deficient cells, HCMV produced larger plaques and spread and replicated more rapidly than in parental cells. These cells demonstrate the vital role of IFNs in controlling HCMV replication and provide useful tools to investigate the IFN response to HCMV. T ype I interferons (IFNs) play a crucial role in the control of viral infection through inducing expression of a suite of interferon-stimulated genes (ISGs), with many of these ISGs exhibiting direct antiviral activity that serves to control viral replication (1, 2). IFNs can also act indirectly in the antiviral response by promoting the activation and proliferation of innate and adaptive immune effectors, including natural killer cells, dendritic cells, and T and B cells (3, 4). Human cytomegalovirus (HCMV) is a large double-stranded DNA (dsDNA) virus that is an important pathogen associated with severe morbidity and mortality in the immunosuppressed, especially in the allogeneic hematopoietic stem cell transplant (HSCT) setting (5). HCMV is also the leading infectious cause of birth defects in the developed world (6).The importance of the IFN response in controlling cytomegalovirus infection is exemplified by the hypersensitivity of engineered mice with defects in the IFN response to murine cytomegalovirus (MCMV) replication and disease (7,8). Although CMV encodes a number of gene functions that modulate the IFN response by inhibiting both the production of and response to IFNs (9-17), HCMV infection is still capable of inducing IFN-␤, via an interferon regulatory factor 3 (IRF3)-dependent pathway, in human fibroblasts (HF) (9,15,(18)(19)(20)(21)(22)(23)(24)(25). Furthermore, treatment with exogenous type I and type II IFNs is known to restrict HCMV infection/replication in vitro and in vivo (9,12,(26)(27)(28), confirming the sensitivity of HCMV to IFN-mediated control. Despite a number of reports investigating the IFN response to HCMV, the effect of abrogating the IFN response on HCMV infection and replication, to our knowledge, has not previously been investigated and was studied here using engineered cell lines.Generation of IFN-deficient cell lines. To investigate the effect of abrogating the IFN response on HCMV replication, the known abilities of the nPro protein of bovine viral diarrhea virus (BVDV) to target IRF3 (blocking IFN-␤ production) (29) and of the V protein of parainfluenza virus type 5 (PIV-5) to target STAT1 (blocking IFN responsiveness) (30, 31) were utilized. Lentivirus vectors expressing the nPro and V genes, respectively, were generated as described previously (29). Primary HF from the ATCC (HFF-1) were transduced with the lentiviruses, and cells were selected using 1 g/ml puromycin. To test whether nPro/HF could produce IFN-␤ in response to HCMV infection, parental HF and nPro/HF were infected with HCMV strain Mer...