An optimised protocol for the expression and purification of adenovirus core protein VII.

Adenovirus protein VII (pVII) is a highly basic core protein, bearing resemblance to mammalian histones. Despite its diverse functions, a comprehensive understanding of its structural intricacies and the mechanisms underlying its functions remain elusive, primarily due to the complexity of producing a good amount of soluble pVII. This study aimed to optimise the expression and purification of recombinant pVII from four different adenoviruses with a simple vector construct. This study successfully determined the optimal conditions for efficiently purifying pVII across four adenovirus species, revealing the differential preference for bacterial expression systems. The One Shot BL21 Star (DE3) proved favourable over Rosetta 2 (DE3) pLysS with consistent levels of expression between IPTG-induced and auto-induction. We demonstrated that combining chemical and mechanical cell lysis is possible and highly effective. Other noteworthy benefits were observed in using RNase during sample processing. The addition of RNase has significantly improved the quality and quantity of the purified protein as confirmed by chromatographic and western blot analyses. These findings established a solid groundwork for pVII purification methodologies and carry the significant potential to assist in unveiling the core structure of pVII, its arrangement within the core, DNA condensation intricacies, and potential pathways for nuclear transport.

Structural and functional characterization of siadenovirus core protein VII nuclear localization demonstrates the existence of multiple nuclear transport pathways.

Adenovirus protein VII (pVII) plays a crucial role in the nuclear localization of genomic DNA following viral infection and contains nuclear localization signal (NLS) sequences for the importin (IMP)-mediated nuclear import pathway. However, functional analysis of pVII in adenoviruses to date has failed to fully determine the underlying mechanisms responsible for nuclear import of pVII. Therefore, in the present study, we extended our analysis by examining the nuclear trafficking of adenovirus pVII from a non-human species, psittacine siadenovirus F (PsSiAdV). We identified a putative classical (c)NLS at pVII residues 120-128 (120PGGFKRRRL128). Fluorescence polarization and electrophoretic mobility shift assays demonstrated direct, high-affinity interaction with both IMPα2 and IMPα3 but not IMPß. Structural analysis of the pVII-NLS/IMPα2 complex confirmed a classical interaction, with the major binding site of IMPα occupied by K124 of pVII-NLS. Quantitative confocal laser scanning microscopy showed that PsSiAdV pVII-NLS can confer IMPα/ß-dependent nuclear localization to GFP. PsSiAdV pVII also localized in the nucleus when expressed in the absence of other viral proteins. Importantly, in contrast to what has been reported for HAdV pVII, PsSiAdV pVII does not localize to the nucleolus. In addition, our study demonstrated that inhibition of the IMPα/ß nuclear import pathway did not prevent PsSiAdV pVII nuclear targeting, indicating the existence of alternative pathways for nuclear localization, similar to what has been previously shown for human adenovirus pVII. Further examination of other potential NLS signals, characterization of alternative nuclear import pathways, and investigation of pVII nuclear targeting across different adenovirus species is recommended to fully elucidate the role of varying nuclear import pathways in the nuclear localization of pVII.

Suppression of MR1 by human cytomegalovirus inhibits MAIT cell activation.

Introduction: The antigen presentation molecule MHC class I related protein-1 (MR1) is best characterized by its ability to present bacterially derived metabolites of vitamin B2 biosynthesis to mucosal-associated invariant T-cells (MAIT cells). Methods: Through in vitro human cytomegalovirus (HCMV) infection in the presence of MR1 ligand we investigate the modulation of MR1 expression. Using coimmunoprecipitation, mass spectrometry, expression by recombinant adenovirus and HCMV deletion mutants we investigate HCMV gpUS9 and its family members as potential regulators of MR1 expression. The functional consequences of MR1 modulation by HCMV infection are explored in coculture activation assays with either Jurkat cells engineered to express the MAIT cell TCR or primary MAIT cells. MR1 dependence in these activation assays is established by addition of MR1 neutralizing antibody and CRISPR/Cas-9 mediated MR1 knockout. Results: Here we demonstrate that HCMV infection efficiently suppresses MR1 surface expression and reduces total MR1 protein levels. Expression of the viral glycoprotein gpUS9 in isolation could reduce both cell surface and total MR1 levels, with analysis of a specific US9 HCMV deletion mutant suggesting that the virus can target MR1 using multiple mechanisms. Functional assays with primary MAIT cells demonstrated the ability of HCMV infection to inhibit bacterially driven, MR1-dependent activation using both neutralizing antibodies and engineered MR1 knockout cells. Discussion: This study identifies a strategy encoded by HCMV to disrupt the MR1:MAIT cell axis. This immune axis is less well characterized in the context of viral infection. HCMV encodes hundreds of proteins, some of which regulate the expression of antigen presentation molecules. However the ability of this virus to regulate the MR1:MAIT TCR axis has not been studied in detail.

Adenoviruses in Avian Hosts: Recent Discoveries Shed New Light on Adenovirus Diversity and Evolution.

While adenoviruses cause infections in a wide range of vertebrates, members of the genus Atadenovirus, Siadenovirus, and Aviadenovirus predominantly infect avian hosts. Several recent studies on avian adenoviruses have encouraged us to re-visit previously proposed adenovirus evolutionary concepts. Complete genomes and partial DNA polymerase sequences of avian adenoviruses were extracted from NCBI and analysed using various software. Genomic analyses and constructed phylogenetic trees identified the atadenovirus origin from an Australian native passerine bird in contrast to the previously established reptilian origin. In addition, we demonstrated that the theories on higher AT content in atadenoviruses are no longer accurate and cannot be considered as a species demarcation criterion for the genus Atadenovirus. Phylogenetic reconstruction further emphasised the need to reconsider siadenovirus origin, and we recommend extended studies on avian adenoviruses in wild birds to provide finer evolutionary resolution.

Restriction of Human Cytomegalovirus Infection by Galectin-9.

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus. While HCMV infection is generally asymptomatic in the immunocompetent, it can have devastating consequences in those with compromised or underdeveloped immune systems, including transplant recipients and neonates. Galectins are a widely expressed protein family that have been demonstrated to modulate both antiviral immunity and regulate direct host-virus interactions. The potential for galectins to directly modulate HCMV infection has not previously been studied, and our results reveal that galectin-9 (Gal-9) can potently inhibit HCMV infection. Gal-9-mediated inhibition of HCMV was dependent upon its carbohydrate recognition domains and thus dependent on glycan interactions. Temperature shift studies revealed that Gal-9 specific inhibition was mediated primarily at the level of virus-cell fusion and not binding. Additionally, we found that during reactivation of HCMV in hematopoietic stem cell transplant (HSCT) patients soluble Gal-9 is upregulated. This study provides the first evidence for Gal-9 functioning as a potent antiviral defense effector molecule against HCMV infection and identifies it as a potential clinical candidate to restrict HCMV infections.IMPORTANCE Human cytomegalovirus (HCMV) continues to cause serious and often life-threatening disease in those with impaired or underdeveloped immune systems. This virus is able to infect and replicate in a wide range of human cell types, which enables the virus to spread to other individuals in a number of settings. Current antiviral drugs are associated with a significant toxicity profile, and there is no vaccine; these factors highlight a need to identify additional targets for the development of anti-HCMV therapies. We demonstrate for the first time that secretion of a member of the galectin family of proteins, galectin-9 (Gal-9), is upregulated during natural HCMV-reactivated infection and that this soluble cellular protein possesses a potent capacity to block HCMV infection by inhibiting virus entry into the host cell. Our findings support the possibility of harnessing the antiviral properties of Gal-9 to prevent HCMV infection and disease.

Varicella-Zoster Virus ORF63 Protects Human Neuronal and Keratinocyte Cell Lines from Apoptosis and Changes Its Localization upon Apoptosis Induction.

There are many facets of varicella-zoster virus (VZV) pathogenesis that are not fully understood, such as the mechanisms involved in the establishment of lifelong latency, reactivation, and development of serious conditions like postherpetic neuralgia (PHN). Virus-encoded modulation of apoptosis has been suggested to play an important role in these processes. VZV open reading frame 63 (ORF63) has been shown to modulate apoptosis in a cell-type-specific manner, but the impact of ORF63 on cell death pathways has not been examined in isolation in the context of human cells. We sought to elucidate the effect of VZV ORF63 on apoptosis induction in human neuron and keratinocyte cell lines. VZV ORF63 was shown to protect differentiated SH-SY5Y neuronal cells against staurosporine-induced apoptosis. In addition, VZV infection did not induce high levels of apoptosis in the HaCaT human keratinocyte line, highlighting a delay in apoptosis induction. VZV ORF63 was shown to protect HaCaT cells against both staurosporine- and Fas ligand-induced apoptosis. Confocal microscopy was utilized to examine VZV ORF63 localization during apoptosis induction. In VZV infection and ORF63 expression alone, VZV ORF63 became more cytoplasmic, with aggregate formation during apoptosis induction. Taken together, this suggests that VZV ORF63 protects both differentiated SH-SY5Y cells and HaCaT cells from apoptosis induction and may mediate this effect through its localization change during apoptosis. VZV ORF63 is a prominent VZV gene product in both productive and latent infection and thus may play a critical role in VZV pathogenesis by aiding neuron and keratinocyte survival.IMPORTANCE VZV, a human-specific alphaherpesvirus, causes chicken pox during primary infection and establishes lifelong latency in the dorsal root ganglia (DRG). Reactivation of VZV causes shingles, which is often followed by a prolonged pain syndrome called postherpetic neuralgia. It has been suggested that the ability of the virus to modulate cell death pathways is linked to its ability to establish latency and reactivate. The significance of our research lies in investigating the ability of ORF63, a VZV gene product, to inhibit apoptosis in novel cell types crucial for VZV pathogenesis. This will allow an increased understanding of critical enigmatic components of VZV pathogenesis.

Nuclear domain 10 components upregulated via interferon during human cytomegalovirus infection potently regulate viral infection.

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that causes life-threatening disease in immunocompromised and immunonaïve individuals. Type I interferons (IFNs) are crucial molecules in the innate immune response to HCMV and are also known to upregulate several components of the interchromosomal multiprotein aggregates collectively referred to as nuclear domain 10 (ND10). In the context of herpesvirus infection, ND10 components are known to restrict gene expression. This raises the question as to whether key ND10 components (PML, Sp100 and hDaxx) act as anti-viral IFN-stimulated genes (ISGs) during HCMV infection. In this study, analysis of ND10 component transcription during HCMV infection demonstrated that PML and Sp100 were significantly upregulated whilst hDaxx expression remained unchanged. In cells engineered to block the production of, or response to, type I IFNs, upregulation of PML and Sp100 was not detected during HCMV infection. Furthermore, pre-treatment with an IFN-ß neutralizing antibody inhibited upregulation of PML and Sp100 during both infection and treatment with HCMV-infected cell supernatant. The significance of ND10 components functioning as anti-viral ISGs during HCMV infection was determined through knockdown of PML, Sp100 and hDaxx. ND10 knockdown cells were significantly more permissive to HCMV infection, as previously described but, in contrast to control cells, could support HCMV plaque formation following IFN-ß pre-treatment. This ability of HCMV to overcome the potently anti-viral effects of IFN-ß in ND10 expression deficient cells provides evidence that ND10 component upregulation is a key mediator of the anti-viral activity of IFN-ß.

Human Natural Killer cell expression of ULBP2 is associated with a mature functional phenotype.

NKG2D is an important activating receptor expressed on NK cells. Ligands (termed NKG2DL) for this receptor include ULBP1-6, MICA and MICB in humans; they are upregulated in stressed, cancerous or infected cells where they engage NKG2D to induce NK cell cytotoxicity and cytokine production. Expression of NKG2DL on effector cells has been described in mice and more recently in human cells. We confirm that NK cell lines and IL-2 stimulated primary human NK cells also express the NKG2DL, ULBP2. However, expression of ULBP2 was not a result of transfer from a non-NK cell to an NK cell and in contrast to recent reports we saw no evidence that ULBP2 expression targeted these NK cells for fratricide or for cytotoxicity by NKG2D-expressing, non-NK effector cells. ULBP2 expression was however linked to expression of mature CD57(+) NK cells. In particular, expression of ULBP2 was strongest on those NK cells that had evidence of recent activation and proliferation. We suggest that ULBP2 could be used to identify recently activated "mature" NK cells. Defining this phenotype would be useful for understanding the ontogeny on human NK cells.

Human Cytomegalovirus-Encoded Human Interleukin-10 (IL-10) Homolog Amplifies Its Immunomodulatory Potential by Upregulating Human IL-10 in Monocytes.

UNLABELLED: The human cytomegalovirus (HCMV) gene UL111A encodes cytomegalovirus-encoded human interleukin-10 (cmvIL-10), a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). This viral homolog exhibits a range of immunomodulatory functions, including suppression of proinflammatory cytokine production and dendritic cell (DC) maturation, as well as inhibition of major histocompatibility complex (MHC) class I and class II. Here, we present data showing that cmvIL-10 upregulates hIL-10, and we identify CD14(+)monocytes and monocyte-derived macrophages and DCs as major sources of hIL-10 secretion in response to cmvIL-10. Monocyte activation was not a prerequisite for cmvIL-10-mediated upregulation of hIL-10, which was dose dependent and controlled at the transcriptional level. Furthermore, cmvIL-10 upregulated expression of tumor progression locus 2 (TPL2), which is a regulator of the positive hIL-10 feedback loop, whereas expression of a negative regulator of the hIL-10 feedback loop, dual-specificity phosphatase 1 (DUSP1), remained unchanged. Engagement of the hIL-10 receptor (hIL-10R) by cmvIL-10 led to upregulation of heme oxygenase 1 (HO-1), an enzyme linked with suppression of inflammatory responses, and this upregulation was required for cmvIL-10-mediated upregulation of hIL-10. We also demonstrate an important role for both phosphatidylinositol 3-kinase (PI3K) and STAT3 in the upregulation of HO-1 and hIL-10 by cmvIL-10. In addition to upregulating hIL-10, cmvIL-10 could exert a direct immunomodulatory function, as demonstrated by its capacity to upregulate expression of cell surface CD163 when hIL-10 was neutralized. This study identifies a mechanistic basis for cmvIL-10 function, including the capacity of this viral cytokine to potentially amplify its immunosuppressive impact by upregulating hIL-10 expression. IMPORTANCE: Human cytomegalovirus (HCMV) is a large, double-stranded DNA virus that causes significant human disease, particularly in the congenital setting and in solid-organ and hematopoietic stem cell transplant patients. A prominent feature of HCMV is the wide range of viral gene products that it encodes which function to modulate host defenses. One of these is cmvIL-10, which is a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). In this study, we report that, in addition to exerting a direct biological impact, cmvIL-10 upregulates the expression of hIL-10 by primary blood-derived monocytes and that it does so by modulating existing cellular pathways. This capacity of cmvIL-10 to upregulate hIL-10 represents a mechanism by which HCMV may amplify its immunomodulatory impact during infection.

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.

Human cytomegalovirus interleukin-10 polarizes monocytes toward a deactivated M2c phenotype to repress host immune responses.

Several human cytomegalovirus (HCMV) genes encode products that modulate cellular functions in a manner likely to enhance viral pathogenesis. This includes UL111A, which encodes homologs of human interleukin-10 (hIL-10). Depending upon signals received, monocytes and macrophages become polarized to either classically activated (M1 proinflammatory) or alternatively activated (M2 anti-inflammatory) subsets. Skewing of polarization toward an M2 subset may benefit the virus by limiting the proinflammatory responses to infection, and so we determined whether HCMV-encoded viral IL-10 influenced monocyte polarization. Recombinant viral IL-10 protein polarized CD14(+) monocytes toward an anti-inflammatory M2 subset with an M2c phenotype, as demonstrated by high expression of CD163 and CD14 and suppression of major histocompatibility complex (MHC) class II. Significantly, in the context of productive HCMV infection, viral IL-10 produced by infected cells polarized uninfected monocytes toward an M2c phenotype. We also assessed the impact of viral IL-10 on heme oxygenase 1 (HO-1), which is an enzyme linked with suppression of inflammatory responses. Polarization of monocytes by viral IL-10 resulted in upregulation of HO-1, and inhibition of HO-1 function resulted in a loss of capacity of viral IL-10 to suppress tumor necrosis factor alpha (TNF-α) and IL-1ß, implicating HO-1 in viral IL-10-induced suppression of proinflammatory cytokines by M2c monocytes. In addition, a functional consequence of monocytes polarized with viral IL-10 was a decreased capacity to activate CD4(+) T cells. This study identifies a novel role for viral IL-10 in driving M2c polarization, which may limit virus clearance by restricting proinflammatory and CD4(+) T cell responses at sites of infection.

Differential relocation and stability of PML-body components during productive human cytomegalovirus infection: detailed characterization by live-cell imaging.

In controlling the switch from latency to lytic infection, the immediate early (IE) genes lie at the core of herpesvirus pathogenesis. To image the 72kDa human cytomegalovirus (HCMV) major IE protein (IE1-72K), a recombinant virus encoding IE1 fused with EGFP was constructed. Using this construct, the IE1-EGFP fusion was detected at ND10 (PML-bodies) within 2h post infection (p.i.) and the complete disruption of ND10 imaged through to 6h p.i. HCMV genomes and IE2-86K protein could be detected adjacent to the slowly degrading IE1-72K/ND10 foci. IE1-72K associates with metaphase chromatin, recruiting both PML and STAT2. hDaxx, STAT1 and IE2-86K did not re-locate to metaphase chromatin; the fate of hDaxx is particularly important as this protein contributes to an intrinsic barrier to HCMV infection. While IE1-72K participates in a complex with chromatin, PML, STAT2 and Sp100, IE1-72K releases hDaxx from ND10 yet does not appear to remain associated with it.

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 TNF-like protein 1A-death receptor 3 pathway promotes macrophage foam cell formation in vitro.

TNF-like protein 1A (TL1A), a TNF superfamily cytokine that binds to death receptor 3 (DR3), is highly expressed in macrophage foam cell-rich regions of atherosclerotic plaques, although its role in foam cell formation has yet to be elucidated. We investigated whether TL1A can directly stimulate macrophage foam cell formation in both THP-1 and primary human monocyte-derived macrophages with the underlying mechanisms involved. We demonstrated that TL1A promotes foam cell formation in human macrophages in vitro by increasing both acetylated and oxidized low-density lipoprotein uptake, by enhancing intracellular total and esterified cholesterol levels and reducing cholesterol efflux. This imbalance in cholesterol homeostasis is orchestrated by TL1A-mediated changes in the mRNA and protein expression of several genes implicated in the uptake and efflux of cholesterol, such as scavenger receptor A and ATP-binding cassette transporter A1. Furthermore, through the use of virally delivered DR3 short-hairpin RNA and bone marrow-derived macrophages from DR3 knockout mice, we demonstrate that DR3 can regulate foam cell formation and contributes significantly to the action of TL1A in this process in vitro. We show, for the first time, a novel proatherogenic role for both TL1A and DR3 that implicates this pathway as a target for the therapeutic intervention of atherosclerosis.

Differential requirements of the C terminus of Nbs1 in suppressing adenovirus DNA replication and promoting concatemer formation.

Adenoviruses (Ad) with the early region E4 deleted (E4-deleted virus) are defective for DNA replication and late protein synthesis. Infection with E4-deleted viruses results in activation of a DNA damage response, accumulation of cellular repair factors in foci at viral replication centers, and joining together of viral genomes into concatemers. The cellular DNA repair complex composed of Mre11, Rad50, and Nbs1 (MRN) is required for concatemer formation and full activation of damage signaling through the protein kinases Ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR). The E4orf3 and E4orf6 proteins expressed from the E4 region of Ad type 5 (Ad5) inactivate the MRN complex by degradation and mislocalization, and prevent the DNA damage response. Here we investigated individual contributions of the MRN complex, concatemer formation, and damage signaling to viral DNA replication during infection with E4-deleted virus. Using virus mutants, short hairpin RNA knockdown and hypomorphic cell lines, we show that inactivation of MRN results in increased viral replication. We demonstrate that defective replication in the absence of E4 is not due to concatemer formation or DNA damage signaling. The C terminus of Nbs1 is required for the inhibition of Ad DNA replication and recruitment of MRN to viral replication centers. We identified regions of Nbs1 that are differentially required for concatemer formation and inhibition of Ad DNA replication. These results demonstrate that targeting of the MRN complex explains the redundant functions of E4orf3 and E4orf6 in promoting Ad DNA replication. Understanding how MRN impacts the adenoviral life cycle will provide insights into the functions of this DNA damage sensor.

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.

Complex I binding by a virally encoded RNA regulates mitochondria-induced cell death.

Human cytomegalovirus infection perturbs multiple cellular processes that could promote the release of proapoptotic stimuli. Consequently, it encodes mechanisms to prevent cell death during infection. Using rotenone, a potent inhibitor of the mitochondrial enzyme complex I (reduced nicotinamide adenine dinucleotide-ubiquinone oxido-reductase), we found that human cytomegalovirus infection protected cells from rotenone-induced apoptosis, a protection mediated by a 2.7-kilobase virally encoded RNA (beta2.7). During infection, beta2.7 RNA interacted with complex I and prevented the relocalization of the essential subunit genes associated with retinoid/interferon-induced mortality-19, in response to apoptotic stimuli. This interaction, which is important for stabilizing the mitochondrial membrane potential, resulted in continued adenosine triphosphate production, which is critical for the successful completion of the virus' life cycle. Complex I targeting by a viral RNA represents a refined strategy to modulate the metabolic viability of the infected host cell.

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.