ADAM17 targeting by human cytomegalovirus remodels the cell surface proteome to simultaneously regulate multiple immune pathways.

Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defenses. In exploring the finding that HCMV infection up-regulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory antiviral cytokine TNFα, we found that the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype 'sheddase', a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its UL/b' region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with an HCMV double-deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins (P < 0.05) in an ADAM17-dependent fashion. These included reported substrates of ADAM17 with established immunological functions such as TNFR2 and jagged1, but also numerous unreported host and viral targets, such as nectin1, UL8, and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation.

Suppression of costimulation by human cytomegalovirus promotes evasion of cellular immune defenses.

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.

The pentameric complex drives immunologically covert cell-cell transmission of wild-type human cytomegalovirus.

Human cytomegalovirus (HCMV) strains that have been passaged in vitro rapidly acquire mutations that impact viral growth. These laboratory-adapted strains of HCMV generally exhibit restricted tropism, produce high levels of cell-free virus, and develop susceptibility to natural killer cells. To permit experimentation with a virus that retained a clinically relevant phenotype, we reconstructed a wild-type (WT) HCMV genome using bacterial artificial chromosome technology. Like clinical virus, this genome proved to be unstable in cell culture; however, propagation of intact virus was achieved by placing the RL13 and UL128 genes under conditional expression. In this study, we show that WT-HCMV produces extremely low titers of cell-free virus but can efficiently infect fibroblasts, epithelial, monocyte-derived dendritic, and Langerhans cells via direct cell-cell transmission. This process of cell-cell transfer required the UL128 locus, but not the RL13 gene, and was significantly less vulnerable to the disruptive effects of IFN, cellular restriction factors, and neutralizing antibodies compared with cell-free entry. Resistance to neutralizing antibodies was dependent on high-level expression of the pentameric gH/gL/gpUL128-131A complex, a feature of WT but not passaged strains of HCMV.

Genetic Stability of Bacterial Artificial Chromosome-Derived Human Cytomegalovirus during Culture In Vitro.

UNLABELLED: Clinical human cytomegalovirus (HCMV) strains invariably mutate when propagatedin vitro Mutations in gene RL13 are selected in all cell types, whereas in fibroblasts mutants in the UL128 locus (UL128L; genes UL128, UL130, and UL131A) are also selected. In addition, sporadic mutations are selected elsewhere in the genome in all cell types. We sought to investigate conditions under which HCMV can be propagated without incurring genetic defects. Bacterial artificial chromosomes (BACs) provide a stable, genetically defined source of viral genome. Viruses were generated from BACs containing the genomes of strains TR, TB40, FIX, and Merlin, as well as from Merlin-BAC recombinants containing variant nucleotides in UL128L from TB40-BAC4 or FIX-BAC. Propagation of viruses derived from TR-BAC, TB40-BAC4, and FIX-BAC in either fibroblast or epithelial cells was associated with the generation of defects around the prokaryotic vector, which is retained in the unique short (US) region of viruses. This was not observed for Merlin-BAC, from which the vector is excised in derived viruses; however, propagation in epithelial cells was consistently associated with mutations in the unique longb' (UL/b') region, all impacting on gene UL141. Viruses derived from Merlin-BAC in fibroblasts had mutations in UL128L, but mutations occurred less frequently with recombinants containing UL128L nucleotides from TB40-BAC4 or FIX-BAC. Viruses derived from a Merlin-BAC derivative in which RL13 and UL128L were either mutated or repressed were remarkably stable in fibroblasts. Thus, HCMV containing a wild-type gene complement can be generatedin vitroby deriving virus from a self-excising BAC in fibroblasts and repressing RL13 and UL128L. IMPORTANCE: Researchers should aim to study viruses that accurately represent the causative agents of disease. This is problematic for HCMV because clinical strains mutate rapidly when propagatedin vitro, becoming less cell associated, altered in tropism, more susceptible to natural killer cells, and less pathogenic. Following isolation from clinical material, HCMV genomes can be stabilized by cloning into bacterial artificial chromosomes (BACs), and then virus is regenerated by DNA transfection. However, mutations can occur not only during isolation prior to BAC cloning but also when virus is regenerated. We have identified conditions under which BAC-derived viruses containing an intact, wild-type genome can be propagatedin vitrowith minimal risk of mutants being selected, enabling studies of viruses expressing the gene complement of a clinical strain. However, even under these optimized conditions, sporadic mutations can occur, highlighting the advisability of sequencing the HCMV stocks used in experiments.

Plasma membrane profiling defines an expanded class of cell surface proteins selectively targeted for degradation by HCMV US2 in cooperation with UL141.

Human cytomegalovirus (HCMV) US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I). Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin ß1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2's impact on HCMV pathogenesis.

Two novel human cytomegalovirus NK cell evasion functions target MICA for lysosomal degradation.

NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, αß and γδ T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)1-6) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12-US21; a genetic arrangement, which is suggestive of an 'accordion' expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on MICA may have contributed to sustaining the US12 gene family.

Inhibition of post-transcriptional RNA processing by CDK inhibitors and its implication in anti-viral therapy.

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and RNA polymerase II mediated transcription. Several pharmacological CDK inhibitors are currently in clinical trials as potential cancer therapeutics and some of them also exhibit antiviral effects. Olomoucine II and roscovitine, purine-based inhibitors of CDKs, were described as effective antiviral agents that inhibit replication of a broad range of wild type human viruses. Olomoucine II and roscovitine show high selectivity for CDK7 and CDK9, with important functions in the regulation of RNA polymerase II transcription. RNA polymerase II is necessary for viral transcription and following replication in cells. We analyzed the effect of inhibition of CDKs by olomoucine II on gene expression from viral promoters and compared its effect to widely-used roscovitine. We found that both roscovitine and olomoucine II blocked the phosphorylation of RNA polymerase II C-terminal domain. However the repression of genes regulated by viral promoters was strongly dependent on gene localization. Both roscovitine and olomoucine II inhibited expression only when the viral promoter was not integrated into chromosomal DNA. In contrast, treatment of cells with genome-integrated viral promoters increased their expression even though there was decreased phosphorylation of the C-terminal domain of RNA polymerase II. To define the mechanism responsible for decreased gene expression after pharmacological CDK inhibitor treatment, the level of mRNA transcription from extrachromosomal DNA was determined. Interestingly, our results showed that inhibition of RNA polymerase II C-terminal domain phosphorylation increased the number of transcribed mRNAs. However, some of these mRNAs were truncated and lacked polyadenylation, which resulted in decreased translation. These results suggest that phosphorylation of RNA polymerase II C-terminal domain is critical for linking transcription and posttrancriptional processing of mRNA expressed from extrachromosomal DNA.

Human cytomegalovirus suppresses Fas expression and function.

Human cytomegalovirus (HCMV) is known to evade extrinsic pro-apoptotic pathways not only by downregulating cell surface expression of the death receptors TNFR1, TRAIL receptor 1 (TNFRSF10A) and TRAIL receptor 2 (TNFRSF10B), but also by impeding downstream signalling events. Fas (CD95/APO-1/TNFRSF6) also plays a prominent role in apoptotic clearance of virus-infected cells, so its fate in HCMV-infected cells needs to be addressed. Here, we show that cell surface expression of Fas was suppressed in HCMV-infected fibroblasts from 24 h onwards through the late phase of productive infection, and was dependent on de novo virus-encoded gene expression but not virus DNA replication. Significant levels of the fully glycosylated (endoglycosidase-H-resistant) Fas were retained within HCMV-infected cells throughout the infection within intracellular membranous structures. HCMV infection provided cells with a high level of protection against Fas-mediated apoptosis. Downregulation of Fas was observed with HCMV strains AD169, FIX, Merlin and TB40.

Impact of sequence variation in the UL128 locus on production of human cytomegalovirus in fibroblast and epithelial cells.

The human cytomegalovirus (HCMV) virion envelope contains a complex consisting of glycoproteins gH and gL plus proteins encoded by the UL128 locus (UL128L): pUL128, pUL130, and pUL131A. UL128L is necessary for efficient infection of myeloid, epithelial, and endothelial cells but limits replication in fibroblasts. Consequently, disrupting mutations in UL128L are rapidly selected when clinical isolates are cultured in fibroblasts. In contrast, bacterial artificial chromosome (BAC)-cloned strains TB40-BAC4, FIX, and TR do not contain overt disruptions in UL128L, yet no virus reconstituted from them has been reported to acquire mutations in UL128L in vitro. We performed BAC mutagenesis and reconstitution experiments to test the hypothesis that these strains contain subtle mutations in UL128L that were acquired during passage prior to BAC cloning. Compared to strain Merlin containing wild-type UL128L, all three strains produced higher yields of cell-free virus. Moreover, TB40-BAC4 and FIX spread cell to cell more rapidly than wild-type Merlin in fibroblasts but more slowly in epithelial cells. The differential growth properties of TB40-BAC4 and FIX (but not TR) were mapped to single-nucleotide substitutions in UL128L. The substitution in TB40-BAC4 reduced the splicing efficiency of UL128, and that in FIX resulted in an amino acid substitution in UL130. Introduction of these substitutions into Merlin dramatically increased yields of cell-free virus and increased cell-to-cell spread in fibroblasts but reduced the abundance of pUL128 in the virion and the efficiency of epithelial cell infection. These substitutions appear to represent mutations in UL128L that permit virus to be propagated in fibroblasts while retaining epithelial cell tropism.

Human cytomegalovirus glycoprotein UL141 targets the TRAIL death receptors to thwart host innate antiviral defenses.

Death receptors (DRs) of the TNFR superfamily contribute to antiviral immunity by promoting apoptosis and regulating immune homeostasis during infection, and viral inhibition of DR signaling can alter immune defenses. Here we identify the human cytomegalovirus (HCMV) UL141 glycoprotein as necessary and sufficient to restrict TRAIL DR function. Despite showing no primary sequence homology to TNF family cytokines, UL141 binds the ectodomains of both human TRAIL DRs with affinities comparable to the natural ligand TRAIL. UL141 binding promotes intracellular retention of the DRs, thus protecting virus infected cells from TRAIL and TRAIL-dependent NK cell-mediated killing. The identification of UL141 as a herpesvirus modulator of the TRAIL DRs strongly implicates this pathway as a regulator of host defense to HCMV and highlights UL141 as a pleiotropic inhibitor of NK cell effector function.

Sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture.

Mutations that occurred during adaptation of human cytomegalovirus to cell culture were monitored by isolating four strains from clinical samples, passaging them in various cell types and sequencing ten complete virus genomes from the final passages. Mutational dynamics were assessed by targeted sequencing of intermediate passages and the original clinical samples. Gene RL13 and the UL128 locus (UL128L, consisting of genes UL128, UL130 and UL131A) mutated in all strains. Mutations in RL13 occurred in fibroblast, epithelial and endothelial cells, whereas those in UL128L were limited to fibroblasts and detected later than those in RL13. In addition, a region containing genes UL145, UL144, UL142, UL141 and UL140 mutated in three strains. All strains exhibited numerous mutations in other regions of the genome, with a preponderance in parts of the inverted repeats. An investigation was carried out on the kinetic growth yields of viruses derived from selected passages that were predominantly non-mutated in RL13 and UL128L (RL13+UL128L+), or that were largely mutated in RL13 (RL13-UL128L+) or both RL13 and UL128L (RL13-UL128L-). RL13-UL128L- viruses produced greater yields of infectious progeny than RL13-UL128L+ viruses, and RL13-UL128L+ viruses produced greater yields than RL13+UL128L+ viruses. These results suggest strongly that RL13 and UL128L exert at least partially independent suppressive effects on growth in fibroblasts. As all isolates proved genetically unstable in all cell types tested, caution is advised in choosing and monitoring strains for experimental studies of vulnerable functions, particularly those involved in cell tropism, immune evasion or growth temperance.

The inhibitor of cyclin-dependent kinases, olomoucine II, exhibits potent antiviral properties.

BACKGROUND: Olomoucine II, the most recent derivative of roscovitine, is an exceptionally potent pharmacological inhibitor of cyclin-dependent kinase activities. Here, we report that olomoucine II is also an effective antiviral agent. METHODS: Antiviral activities of olomoucine II were tested on a range of human viruses in in vitro assays that evaluated viral growth and replication. RESULTS: Olomoucine II inhibited replication of a broad range of wild-type human viruses, including herpes simplex virus, human adenovirus type-4 and human cytomegalovirus. Olomoucine II also inhibited replication of vaccinia virus and herpes simplex virus mutants resistant to conventional acyclovir treatment. This report is the first demonstration of a poxvirus being sensitive to a cyclin-dependent kinase inhibitor. The antiviral effects of olomoucine II could be observed at lower concentrations than with roscovitine, although both were short-term. A remarkable observation was that olomoucine II, when used in combination with the DNA polymerase inhibitor cidofovir, was able to almost completely eliminate the spread of infectious adenovirus type-4 progeny from infected cells. CONCLUSIONS: Our results show that when targeting two complementary antiviral mechanisms, strongly additive effects could be observed.

Adenovirus E3/19K promotes evasion of NK cell recognition by intracellular sequestration of the NKG2D ligands major histocompatibility complex class I chain-related proteins A and B.

The adenovirus (Ad) early transcription unit 3 (E3) encodes multiple immunosubversive functions that are presumed to facilitate the establishment and persistence of infection. Indeed, the capacity of E3/19K to inhibit transport of HLA class I (HLA-I) to the cell surface, thereby preventing peptide presentation to CD8(+) T cells, has long been recognized as a paradigm for viral immune evasion. However, HLA-I downregulation has the potential to render Ad-infected cells vulnerable to natural killer (NK) cell recognition. Furthermore, expression of the immediate-early Ad gene E1A is associated with efficient induction of ligands for the key NK cell-activating receptor NKG2D. Here we show that while infection with wild-type Ad enhances synthesis of the NKG2D ligands, major histocompatibility complex class I chain-related proteins A and B (MICA and MICB), their expression on the cell surface is actively suppressed. Both MICA and MICB are retained within the endoplasmic reticulum as immature endoglycosidase H-sensitive forms. By analyzing a range of cell lines and viruses carrying mutated versions of the E3 gene region, E3/19K was identified as the gene responsible for this activity. The structural requirements within E3/19K necessary to sequester MICA/B and HLA-I are similar. In functional assays, deletion of E3/19K rendered Ad-infected cells more sensitive to NK cell recognition. We report the first NK evasion function in the Adenoviridae and describe a novel function for E3/19K. Thus, E3/19K has a dual function: inhibition of T-cell recognition and NK cell activation.

Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function.

With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.

Adenovirus vector delivery stimulates natural killer cell recognition.

We report that delivery of first-generation replication-deficient adenovirus (RDAd) vectors into primary human fibroblasts is associated with the induction of natural killer (NK) cell-mediated cytolysis in vitro. RDAd vector delivery induced cytolysis by a range of NK cell populations including the NK cell clone NKL, primary polyclonal NK lines and a proportion of NK clones (36 %) in autologous HLA-matched assays. Adenovirus-induced cytolysis was inhibited by antibody blocking of the NK-activating receptor NKG2D, implicating this receptor in this function. NKG2D is ubiquitously expressed on NK cells and CD8(+) T cells. Significantly, gamma-irradiation of the vector eliminated the effect, suggesting that breakthrough expression from the vector induces at least some of the pro-inflammatory responses of unknown aetiology following the application of RDAd vectors during in vivo gene delivery.

The human cytomegalovirus MHC class I homolog UL18 inhibits LIR-1+ but activates LIR-1- NK cells.

The inhibitory leukocyte Ig-like receptor 1 (LIR-1, also known as ILT2, CD85j, or LILRB1) was identified by its high affinity for the human CMV (HCMV) MHC class I homolog gpUL18. The role of this LIR-1-gpUL18 interaction in modulating NK recognition during HCMV infection has previously not been clearly defined. In this study, LIR-1(+) NKL cell-mediated cytotoxicity was shown to be inhibited by transduction of targets with a replication-deficient adenovirus vector encoding UL18 (RAd-UL18). Fibroblasts infected with an HCMV UL18 mutant (DeltaUL18) also exhibited enhanced susceptibility to NKL killing relative to cells infected with the parental virus. In additional cytolysis assays, UL18-mediated protection was also evident in the context of adenovirus vector transduction and HCMV infection of autologous fibroblast targets using IFN-alpha-activated NK bulk cultures derived from a donor with a high frequency of LIR-1(+) NK cells. A single LIR-1(high) NK clone derived from this donor was inhibited by UL18, while 3 of 24 clones were activated. CD107 mobilization assays revealed that LIR-1(+) NK cells were consistently inhibited by UL18 in all tested donors, but this effect was often masked in the global response by UL18-mediated activation of a subset of LIR-1(-) NK cells. Although Ab-blocking experiments support UL18 inhibition being induced by a direct interaction with LIR-1, the UL18-mediated activation is LIR-1 independent.

Human cytomegalovirus encodes an MHC class I-like molecule (UL142) that functions to inhibit NK cell lysis.

Clinical and low passage strains of human CMV (HCMV) encode an additional MHC class I-related molecule UL142, in addition to the previously described UL18. The UL142 open reading frame is encoded within the ULb' region which is missing from a number of common high passage laboratory strains. Cells expressing UL142 following transfection, and fibroblasts infected with a recombinant adenovirus-expressing UL142, were used to screen both polyclonal NK cells and NK cell clones, in a completely autologous system. Analysis of 100 NK cell clones derived from five donors, revealed 23 clones that were inhibited by fibroblasts expressing UL142 alone. Small-interfering RNA-mediated knockdown of UL142 mRNA expression in HCMV-infected cells resulted in increased sensitivity to lysis. From these data we conclude that UL142 is a novel HCMV-encoded MHC class I-related molecule which inhibits NK cell killing in a clonally dependent manner.

Characterization of a highly glycosylated form of the human cytomegalovirus HLA class I homologue gpUL18.

Human cytomegalovirus (HCMV) gpUL18 is a HLA class I (HLA-I) homologue with high affinity for the inhibitory receptor LIR-1/ILT2. The previously described 67 kDa form of gpUL18 is shown here to be sensitive to endoglycosidase-H (EndoH). A novel form of gpUL18 with a molecular mass of approximately 160 kDa and resistance to EndoH was identified in cells infected with HCMV strain AD169 or the low passage HCMV isolates Merlin and Toledo. The 67 kDa EndoH-sensitive gpUL18 glycoform was detected earlier in a productive infection (from 24 h post-infection) than the slower-migrating EndoH-resistant glycoform (from 72 h post-infection). Deletion of the US2-US11 region from the HCMV genome was associated with a substantial up-regulation of endogenous HLA-I in infected cells, but had no obvious effect on the gpUL18 expression pattern. Vaccinia virus and adenovirus vectors were used to further analyse gpUL18 expression. Depending on the delivery vector system, differences in the electrophoretic motility of the EndoH-resistant >105 kDa form of gpUL18, but not the EndoH-sensitive 67 kDa form, were observed; post-translational modification of the higher molecular mass glycoform appears to be influenced by active virus infection and vector delivery. The EndoH-sensitive 67 kDa gpUL18 had a rapid turnover, while the maturation to the EndoH-resistant >105 kDa form was relatively slow and inefficient. However, synthesis of the EndoH-resistant >105 kDa form was enhanced with elevated levels of beta2-microglobulin. When expressed by using an adenovirus vector, both the EndoH-sensitive 67 kDa and the EndoH-resistant >105 kDa gpUL18 forms could be detected on the cell surface.

UL40-mediated NK evasion during productive infection with human cytomegalovirus.

Human cytomegalovirus (HCMV) exploits a range of strategies to evade and modulate the immune response. Its capacity to down-regulate MHC I expression was anticipated to render infected cells vulnerable to natural killer (NK) attack. Kinetic analysis revealed that during productive infection, HCMV strain AD169 first enhanced and then inhibited lysis of primary skin fibroblasts by a CD94/NKG2A(+)NKG2D(+)ILT2(+) NK line. The inhibition of cytotoxicity against strain AD169-infected fibroblasts was abolished by prior treatment of targets or effectors with anti-MHC I and anti-CD94 monoclonal antibodies, respectively, implying a CD94/HLA-E-dependent mechanism. An HCMV strain AD169, UL40 deletion mutant could not inhibit CD94/NKG2A(+) NK killing against skin fibroblasts. The contribution of UL40 to evasion of primary NK cells then was tested in a system where targets and effectors were MHC-matched. Primary NK cells activated with IFNalpha as well as cultured primary NK cell lines showed increased killing against DeltaUL40-infected fibroblasts compared with AD169-infected targets. This effect was abrogated by depletion of CD94(+) cells. These findings demonstrate that HCMV encodes a mechanism of evasion specifically targeted against a proportion of CD94(+) NK cells and show that this system functions during a productive infection.