IntroductionHuman cytomegalovirus (HCMV) infection generally follows a subclinical course, but may lead to severe disorders in immunocompromised individuals and is a main cause of infectious congenital diseases. HCMV remains latent in immunocompetent hosts, undergoing occasional reactivation. 1 Studies in murine models revealed that an effective defense against CMV requires the participation of natural killer (NK) and T cells. 2,3 Detection of antibodies and CD8 ϩ T lymphocytes specific for HCMV antigens allow an assessment of the adaptive immune response to the pathogen. 4,5 To escape from CD8 ϩ T cells, HCMV inhibits the expression of human leukocyte antigen (HLA) class I molecules and interferes with antigen presentation using a set of glycoproteins (US2, US3, US6, US10, and US11) whose genes are clustered within the unique short (US) region of the virus genome. [6][7][8] The loss of HLA class I molecules in HCMV-infected cells impairs the engagement of inhibitory receptors and prompts the activation of NK cell effector functions; reciprocally, the virus has developed several strategies to evade NK-mediated recognition. 9 The nature of receptor-ligand interactions involved in the NK cell response to CMV-infected cells is incompletely understood. In strains of mice expressing the Ly49H receptor, NK cell functions are triggered upon recognition of the m157 mouse CMV (MCMV) glycoprotein, becoming essential to control replication; 10,11 by contrast, human activating NK cell receptors (NKRs) specific for HCMV molecules have not been identified. The involvement of activating killer immunoglobulin (Ig)-like receptors (KIRs) and natural cytotoxicity receptors (NCRs; ie, NKp46, NKp30, and NKp44) in the response to HCMV is uncertain. The interaction of the pp65 HCMV tegument protein with NKp30 has been reported to inhibit rather than to activate NK cell functions. 12 The ability of the UL16 HCMV molecule to interfere with the surface expression of NKG2D ligands, [13][14][15] and the evidence that similar evasion mechanisms operate in MCMV infection, 16,17 support an important role for this killer lectinlike receptor (KLR) in the antiviral defense. 18 Recently, the UL141 HCMV molecule has been shown to inhibit the expression in infected cells of CD155, a ligand for the DNAM-1 stimulating receptor. 19 HCMV may also escape NK-mediated surveillance by keeping inhibitory receptors for HLA class I molecules engaged. The viral UL18 molecule binds with high affinity to the ILT2 (CD85j) inhibitory receptor, 20,21 though its role in immune evasion has not been precisely elucidated. 9 On the other hand, HLA-E appears constitutively resistant to the action of US2 and US11, 22 and it also becomes refractory to the action of US6 when bound to a peptide from the leader sequence of the HCMV UL40 protein. 23 For personal use only. on May 10, 2018. by guest www.bloodjournal.org From interfere with the NK cell response by engaging the inhibitory CD94/NKG2A KLR. 25 We recently reported 26 that healthy HCMV-seropositive individuals display...

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Members of the α- and β-subfamily of herpesviridae encode glycoproteins that specifically bind to the Fc part of immunoglobulin (Ig)G. Plasma membrane resident herpesviral Fc receptors seem to prevent virus-specific IgG from activating antibody-dependent effector functions. We show that the mouse cytomegalovirus (MCMV) molecule fcr-1 promotes a rapid down-regulation of NKG2D ligands murine UL16-binding protein like transcript (MULT)-1 and H60 from the cell surface. Deletion of the m138/fcr-1 gene from the MCMV genome attenuates viral replication to natural killer (NK) cell response in an NKG2D-dependent manner in vivo. A distinct N-terminal module within the fcr-1 ectodomain in conjunction with the fcr-1 transmembrane domain was required to dispose MULT-1 to degradation in lysosomes. In contrast, down-modulation of H60 required the complete fcr-1 ectodomain, implying independent modes of fcr-1 interaction with the NKG2D ligands. The results establish a novel viral strategy for down-modulating NK cell responses and highlight the impressive diversity of Fc receptor functions.

Cytomegaloviruses carry the US22 family of genes, which have common sequence motifs but diverse functions. Only two of the 12 US22 family genes of murine cytomegalovirus (MCMV) are essential for virus replication, but their functions have remained unknown. In the present study, we deleted the essential US22 family genes, m142 and m143, from the MCMV genome and propagated the mutant viruses on complementing cells. The m142 and the m143 deletion mutants were both unable to replicate in noncomplementing cells at low and high multiplicities of infection. In cells infected with the deletion mutants, viral immediate-early and early proteins were expressed, but viral DNA replication and synthesis of the late-gene product glycoprotein B were inhibited, even though mRNAs of late genes were present. Global protein synthesis was impaired in these cells, which correlated with phosphorylation of the double-stranded RNA-dependent protein kinase R (PKR) and its target protein, the eukaryotic translation initiation factor 2␣, suggesting that m142 and m143 are necessary to block the PKR-mediated shutdown of protein synthesis. Replication of the m142 and m143 knockout mutants was partially restored by expression of the human cytomegalovirus TRS1 gene, a known double-stranded-RNA-binding protein that inhibits PKR activation. These results indicate that m142 and m143 are both required for inhibition of the PKR-mediated host antiviral response.Cytomegaloviruses (CMVs) are prototypes of the ␤ subfamily of the Herpesviridae. Their genomes span 230 kb and are the largest among the herpesviruses. Of the approximately 170 genes on a CMV genome, about 46 are conserved among the herpesviruses (42). An additional 34 genes are characteristic of the ␤-herpesviruses (12,21,44,58), and the remaining genes are unique to a particular virus.A typical property of ␤-herpesvirus genomes is the presence of gene families, which probably arose by duplication from ancestral genes. One of the largest, the US22 gene family, was first identified in human cytomegalovirus (HCMV) and was later also found in other ␤-herpesviruses (12,21,44,51,58). It is characterized by four conserved sequence motifs consisting of hydrophobic residues interspersed with charged amino acids. HCMV and murine cytomegalovirus (MCMV) both include 12 members of the US22 gene family on their genomes (12, 51). The rat CMV and human herpesviruses 6 and 7, two other human ␤-herpesviruses, also possess up to 11 US22 family genes (20,21,44,58).Little is known about the functions of US22 gene products. However, 5 of the 12 US22 gene products of MCMV affect the virus' ability to replicate in macrophages: M36, M43, m139, m140, and m141 (28,29,32,41). The molecular mechanism of action for only one of these has been elucidated. The M36 gene encodes an antiapoptotic protein that binds to procaspase 8 and inhibits death receptor-mediated induction of apoptosis (41). The positional and sequence homolog of M36 in HCMV, UL36, was originally proposed to function as a transcriptional transactivator (16...

Double-stranded RNA (dsRNA) produced during viral infection activates several cellular antiviral responses.Among the best characterized is the shutoff of protein synthesis mediated by the dsRNA-dependent protein kinase (PKR) and the oligoadenylate synthetase (OAS)/RNase L system. As viral replication depends on protein synthesis, many viruses have evolved mechanisms for counteracting the PKR and OAS/RNase L pathways. The murine cytomegalovirus (MCMV) proteins m142 and m143 have been characterized as dsRNA binding proteins that inhibit PKR activation, phosphorylation of the translation initiation factor eIF2␣, and a subsequent protein synthesis shutoff. In the present study we analyzed the contribution of the PKR-and the OAS-dependent pathways to the control of MCMV replication in the absence or presence of m142 and m143. We show that the induction of eIF2␣ phosphorylation during infection with an m142-and m143-deficient MCMV is specifically mediated by PKR, not by the related eIF2␣ kinases PERK or GCN2. PKR antagonists of vaccinia virus (E3L) or herpes simplex virus (␥34.5) rescued the replication defect of an MCMV strain with deletions of both m142 and m143. Moreover, m142 and m143 bound to each other and interacted with PKR. By contrast, an activation of the OAS/RNase L pathway by MCMV was not detected in the presence or absence of m142 and m143, suggesting that these viral proteins have little or no influence on this pathway. Consistently, an m142-and m143-deficient MCMV strain replicated to high titers in fibroblasts lacking PKR but did not replicate in cells lacking RNase L. Hence, the PKR-mediated antiviral response is responsible for the essentiality of m142 and m143.

During viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved. To reduce ER stress, the UPR decreases protein synthesis, increases degradation of unfolded proteins, and upregulates chaperone expression to enhance protein folding. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage. However, the molecular mechanisms and the viral proteins responsible for UPR modulation remained to be identified. In this study, we investigated the modulation of IRE1 signaling by murine cytomegalovirus (MCMV) and found that IRE1-mediated mRNA splicing and expression of the X-box binding protein 1 (XBP1) is repressed in infected cells. By affinity purification, we identified the viral M50 protein as an IRE1-interacting protein. M50 expression in transfected or MCMV-infected cells induced a substantial downregulation of IRE1 protein levels. The N-terminal conserved region of M50 was found to be required for interaction with and downregulation of IRE1. Moreover, UL50, the human cytomegalovirus (HCMV) homolog of M50, affected IRE1 in the same way. Thus we concluded that IRE1 downregulation represents a previously undescribed viral strategy to curb the UPR.

A novel influenza A virus (IAV) of the H7N9 subtype has been isolated from severely diseased patients with pneumonia and acute respiratory distress syndrome and, apparently, from healthy poultry in March 2013 in Eastern China. We evaluated replication, tropism, and cytokine induction of the A/Anhui/1/2013 (H7N9) virus isolated from a fatal human infection and two low-pathogenic avian H7 subtype viruses in a human lung organ culture system mimicking infection of the lower respiratory tract. The A(H7N9) patient isolate replicated similarly well as a seasonal IAV in explanted human lung tissue, whereas avian H7 subtype viruses propagated poorly. Interestingly, the avian H7 strains provoked a strong antiviral type I interferon (IFN-I) response, whereas the A(H7N9) virus induced only low IFN levels. Nevertheless, all viruses analyzed were detected predominantly in type II pneumocytes, indicating that the A(H7N9) virus does not differ in its cellular tropism from other avian or human influenza viruses. Tissue culture-based studies suggested that the low induction of the IFN-β promoter correlated with an efficient suppression by the viral NS1 protein. These findings demonstrate that the zoonotic A(H7N9) virus is unusually well adapted to efficient propagation in human alveolar tissue, which most likely contributes to the severity of lower respiratory tract disease seen in many patients.

The RNA-dependent protein kinase (PKR) has broad antiviral activity inducing translational shutdown of viral and cellular genes and is therefore targeted by various viral proteins to facilitate pathogen propagation. The pleiotropic NS1 protein of influenza A virus acts as silencer of PKR activation and ensures high-level viral replication and virulence. However, the exact manner of this inhibition remains controversial. To elucidate the structural requirements within the NS1 protein for PKR inhibition, we generated a set of mutant viruses, identifying highly conserved arginine residues 35 and 46 within the NS1 N terminus as being most critical not only for binding to and blocking activation of PKR but also for efficient virus propagation. Biochemical and Förster resonance energy transfer (FRET)-based interaction studies showed that mutation of R35 or R46 allowed formation of NS1 dimers but eliminated any detectable binding to PKR as well as to double-stranded RNA (dsRNA). Using in vitro and in vivo approaches to phenotypic restoration, we demonstrated the essential role of the NS1 N terminus for blocking PKR. The strong attenuation conferred by NS1 mutation R35A or R46A was substantially alleviated by stable knockdown of PKR in human cells. Intriguingly, both NS1 mutant viruses did not trigger any signs of disease in PKR ϩ/ϩ mice, but replicated to high titers in lungs of PKR Ϫ/Ϫ mice and caused lethal infections. These data not only establish the NS1 N terminus as highly critical for neutralization of PKR's antiviral activity but also identify this blockade as an indispensable contribution of NS1 to the viral life cycle. IMPORTANCE Influenza A virus inhibits activation of the RNA-dependent protein kinase (PKR) by means of its nonstructural NS1 protein, but the underlying mode of inhibition is debated. Using mutational analysis, we identified arginine residues 35 and 46 within the N-terminal NS1 domain as highly critical for binding to and functional silencing of PKR. In addition, our data show that this is a main activity of amino acids 35 and 46, as the strong attenuation of corresponding mutant viruses in human cells was rescued to a large extent by lowering of PKR expression levels. Significantly, this corresponded with restoration of viral virulence for NS1 R35A and R46A mutant viruses in PKR Ϫ/Ϫ mice. Therefore, our data establish a model in which the NS1 N-terminal domain engages in a binding interaction to inhibit activation of PKR and ensure efficient viral propagation and virulence.KEYWORDS PKR, influenza, innate immunity, virulence regulation