Bat-borne H9N2 influenza virus evades MxA restriction and exhibits efficient replication and transmission in ferrets.

Influenza A viruses (IAVs) of subtype H9N2 have reached an endemic stage in poultry farms in the Middle East and Asia. As a result, human infections with avian H9N2 viruses have been increasingly reported. In 2017, an H9N2 virus was isolated for the first time from Egyptian fruit bats (Rousettus aegyptiacus). Phylogenetic analyses revealed that bat H9N2 is descended from a common ancestor dating back centuries ago. However, the H9 and N2 sequences appear to be genetically similar to current avian IAVs, suggesting recent reassortment events. These observations raise the question of the zoonotic potential of the mammal-adapted bat H9N2. Here, we investigate the infection and transmission potential of bat H9N2 in vitro and in vivo, the ability to overcome the antiviral activity of the human MxA protein, and the presence of N2-specific cross-reactive antibodies in human sera. We show that bat H9N2 has high replication and transmission potential in ferrets, efficiently infects human lung explant cultures, and is able to evade antiviral inhibition by MxA in transgenic B6 mice. Together with its low antigenic similarity to the N2 of seasonal human strains, bat H9N2 fulfils key criteria for pre-pandemic IAVs.

The host RNA polymerase II C-terminal domain is the anchor for replication of the influenza virus genome.

The current model is that the influenza virus polymerase (FluPol) binds either to host RNA polymerase II (RNAP II) or to the acidic nuclear phosphoprotein 32 (ANP32), which drives its conformation and activity towards transcription or replication of the viral genome, respectively. Here, we provide evidence that the FluPol-RNAP II binding interface, beyond its well-acknowledged function in cap-snatching during transcription initiation, has also a pivotal role in replication of the viral genome. Using a combination of cell-based and in vitro approaches, we show that the RNAP II C-terminal-domain, jointly with ANP32, enhances FluPol replication activity. We observe successive conformational changes to switch from a transcriptase to a replicase conformation in the presence of the bound RNPAII C-terminal domain and propose a model in which the host RNAP II is the anchor for transcription and replication of the viral genome. Our data open new perspectives on the spatial coupling of viral transcription and replication and the coordinated balance between these two activities.

Advancing Precision Vaccinology by Molecular and Genomic Surveillance of Severe Acute Respiratory Syndrome Coronavirus 2 in Germany, 2021.

BACKGROUND: Comprehensive pathogen genomic surveillance represents a powerful tool to complement and advance precision vaccinology. The emergence of the Alpha variant in December 2020 and the resulting efforts to track the spread of this and other severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern led to an expansion of genomic sequencing activities in Germany. METHODS: At Robert Koch Institute (RKI), the German National Institute of Public Health, we established the Integrated Molecular Surveillance for SARS-CoV-2 (IMS-SC2) network to perform SARS-CoV-2 genomic surveillance at the national scale, SARS-CoV-2-positive samples from laboratories distributed across Germany regularly undergo whole-genome sequencing at RKI. RESULTS: We report analyses of 3623 SARS-CoV-2 genomes collected between December 2020 and December 2021, of which 3282 were randomly sampled. All variants of concern were identified in the sequenced sample set, at ratios equivalent to those in the 100-fold larger German GISAID sequence dataset from the same time period. Phylogenetic analysis confirmed variant assignments. Multiple mutations of concern emerged during the observation period. To model vaccine effectiveness in vitro, we employed authentic-virus neutralization assays, confirming that both the Beta and Zeta variants are capable of immune evasion. The IMS-SC2 sequence dataset facilitated an estimate of the SARS-CoV-2 incidence based on genetic evolution rates. Together with modeled vaccine efficacies, Delta-specific incidence estimation indicated that the German vaccination campaign contributed substantially to a deceleration of the nascent German Delta wave. CONCLUSIONS: SARS-CoV-2 molecular and genomic surveillance may inform public health policies including vaccination strategies and enable a proactive approach to controlling coronavirus disease 2019 spread as the virus evolves.

Archival influenza virus genomes from Europe reveal genomic variability during the 1918 pandemic.

The 1918 influenza pandemic was the deadliest respiratory pandemic of the 20th century and determined the genomic make-up of subsequent human influenza A viruses (IAV). Here, we analyze both the first 1918 IAV genomes from Europe and the first from samples prior to the autumn peak. 1918 IAV genomic diversity is consistent with a combination of local transmission and long-distance dispersal events. Comparison of genomes before and during the pandemic peak shows variation at two sites in the nucleoprotein gene associated with resistance to host antiviral response, pointing at a possible adaptation of 1918 IAV to humans. Finally, local molecular clock modeling suggests a pure pandemic descent of seasonal H1N1 IAV as an alternative to the hypothesis of origination through an intrasubtype reassortment.

Analysis of BNT162b2- and CVnCoV-elicited sera and of convalescent sera toward SARS-CoV-2 viruses.

BACKGROUND: The mRNA vaccine BNT162b2 (Comirnaty, BioNTech/Pfizer) and the vaccine candidate CVnCoV (Curevac) each encode a stabilized spike protein of SARS-CoV2 as antigen but differ with respect to the nature of the mRNA (modified versus unmodified nucleotides) and the mRNA amount (30 µg versus 12 µg RNA). This study characterizes antisera elicited by these two vaccines in comparison to convalescent sera. METHODS: Sera from BNT162b2 vaccinated healthcare workers, and sera from participants of a phase I trial vaccinated with 2, 4, 6, 8, or 12 µg CVnCoV and convalescent sera from hospitalized patients were analyzed by ELISA, neutralization tests, surface plasmon resonance (SPR), and peptide arrays. RESULTS: BNT162b2-elicited sera and convalescent sera have a higher titer of spike-RBD-specific antibodies and neutralizing antibodies as compared to the CVnCoV-elicited sera. For all analyzed sera a reduction in binding and neutralizing antibodies was found for the lineage B.1.351 variant of concern. SPR analyses revealed that the CVnCoV-elicited sera have a lower fraction of slow-dissociating antibodies. Accordingly, the CVnCoV sera almost fail to compete with the spike-ACE2 interaction. The significance of common VOC mutations K417N, E484K, or N501Y focused on linear epitopes was analyzed using a peptide array approach. The peptide arrays showed a strong difference between convalescent sera and vaccine-elicited sera. Specifically, the linear epitope at position N501 was affected by the mutation and elucidates the escape of viral variants to antibodies against this linear epitope. CONCLUSION: These data reveal differences in titer, neutralizing capacity, and affinity of the antibodies between BNT162b2- and CVnCoV-elicited sera, which could contribute to the apparent differences in vaccine efficacy.

Rise and Fall of SARS-CoV-2 Lineage A.27 in Germany.

Here, we report on the increasing frequency of the SARS-CoV-2 lineage A.27 in Germany during the first months of 2021. Genomic surveillance identified 710 A.27 genomes in Germany as of 2 May 2021, with a vast majority identified in laboratories from a single German state (Baden-Wuerttemberg, n = 572; 80.5%). Baden-Wuerttemberg is located near the border with France, from where most A.27 sequences were entered into public databases until May 2021. The first appearance of this lineage based on sequencing in a laboratory in Baden-Wuerttemberg can be dated to early January '21. From then on, the relative abundance of A.27 increased until the end of February but has since declined-meanwhile, the abundance of B.1.1.7 increased in the region. The A.27 lineage shows a mutational pattern typical of VOIs/VOCs, including an accumulation of amino acid substitutions in the Spike glycoprotein. Among those, L18F, L452R and N501Y are located in the epitope regions of the N-terminal- (NTD) or receptor binding domain (RBD) and have been suggested to result in immune escape and higher transmissibility. In addition, A.27 does not show the D614G mutation typical for all VOIs/VOCs from the B lineage. Overall, A.27 should continue to be monitored nationally and internationally, even though the observed trend in Germany was initially displaced by B.1.1.7 (Alpha), while now B.1.617.2 (Delta) is on the rise.

Evaluation of Multivalent Sialylated Polyglycerols for Resistance Induction in and Broad Antiviral Activity against Influenza A Viruses.

The development of multivalent sialic acid-based inhibitors active against a variety of influenza A virus (IAV) strains has been hampered by high genetic and structural variability of the targeted viral hemagglutinin (HA). Here, we addressed this challenge by employing sialylated polyglycerols (PGs). Efficacy of prototypic PGs was restricted to a narrow spectrum of IAV strains. To understand this restriction, we selected IAV mutants resistant to a prototypic multivalent sialylated PG by serial passaging. Resistance mutations mapped to the receptor binding site of HA, which was accompanied by altered receptor binding profiles of mutant viruses as detected by glycan array analysis. Specifying the inhibitor functionalization to 2,6-α-sialyllactose (SL) and adjusting the linker yielded a rationally designed inhibitor covering an extended spectrum of inhibited IAV strains. These results highlight the importance of integrating virological data with chemical synthesis and structural data for the development of sialylated PGs toward broad anti-influenza compounds.

The dynamic proteome of influenza A virus infection identifies M segment splicing as a host range determinant.

Pandemic influenza A virus (IAV) outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. The molecular basis of this species barrier is incompletely understood. Here we combine metabolic pulse labeling and quantitative proteomics to monitor protein synthesis upon infection of human cells with a human- and a bird-adapted IAV strain and observe striking differences in viral protein synthesis. Most importantly, the matrix protein M1 is inefficiently produced by the bird-adapted strain. We show that impaired production of M1 from bird-adapted strains is caused by increased splicing of the M segment RNA to alternative isoforms. Strain-specific M segment splicing is controlled by the 3' splice site and functionally important for permissive infection. In silico and biochemical evidence shows that avian-adapted M segments have evolved different conserved RNA structure features than human-adapted sequences. Thus, we identify M segment RNA splicing as a viral host range determinant.

The RNA Helicase DDX6 Associates with RIG-I to Augment Induction of Antiviral Signaling.

Virus infections induce sensitive antiviral responses within the host cell. The RNA helicase retinoic acid-inducible gene I (RIG-I) is a key sensor of influenza virus RNA that induces the expression of antiviral type I interferons. Recent evidence suggests a complex pattern of RIG-I regulation involving multiple interactions and cellular sites. In an approach employing affinity purification and quantitative mass spectrometry, we identified proteins with increased binding to RIG-I in response to influenza B virus infection. Among them was the RIG-I related RNA helicase DEAD box helicase 6 (DDX6), a known component of cytoplasmic mRNA-ribonucleoprotein (mRNP) granules like P-bodies and stress granules (SGs). RIG-I and DDX6 both localized to the cytosol and were detected in virus-induced SGs. Coimmunoprecipitation assays detected a basal level of complexes harboring RIG-I and DDX6 that increased after infection. Functionally, DDX6 augmented RIG-I mediated induction of interferon (IFN)-ß expression. Notably, DDX6 was found to bind viral RNA capable to stimulate RIG-I. These findings imply a novel function for DDX6 as an RNA co-sensor and signaling enhancer for RIG-I.

Linear polysialoside outperforms dendritic analogs for inhibition of influenza virus infection in vitro and in vivo.

Inhibition of influenza A virus infection by multivalent sialic acid inhibitors preventing viral hemagglutinin binding to host cells of the respiratory tract is a promising strategy. However, optimal geometry and optimal ligand presentation on multivalent scaffolds for efficient inhibition both in vitro and in vivo application are still unclear. Here, by comparing linear and dendritic polyglycerol sialosides (LPGSA and dPGSA) we identified architectural requirements and optimal ligand densities for an efficient multivalent inhibitor of influenza virus A/X31/1 (H3N2). Due to its large volume, the LPGSA at optimal ligand density sterically shielded the virus significantly better than the dendritic analog. A statistical mechanics model rationalizes the relevance of ligand density, morphology, and the size of multivalent scaffolds for the potential to inhibit virus-cell binding. Optimized LPGSA inhibited virus infection at IC50 in the low nanomolar nanoparticle concentration range and also showed potent antiviral activity against two avian influenza strains A/Mallard/439/2004 (H3N2) and A/turkey/Italy/472/1999 (H7N1) post infection. In vivo application of inhibitors clearly confirmed the higher inhibition potential of linear multivalent scaffolds to prevent infection. The optimized LPGSA did not show any acute toxicity, and was much more potent than the neuraminidase inhibitor oseltamivir carboxylate in vivo. Combined application of the LPGSA and oseltamivir carboxylate revealed a synergistic inhibitory effect and successfully prevented influenza virus infection in mice.

Quantitative Proteomic Approach Identifies Vpr Binding Protein as Novel Host Factor Supporting Influenza A Virus Infections in Human Cells.

Influenza A virus (IAV) infections are a major cause for respiratory disease in humans, which affects all age groups and contributes substantially to global morbidity and mortality. IAV have a large natural host reservoir in avian species. However, many avian IAV strains lack adaptation to other hosts and hardly propagate in humans. While seasonal or pandemic IAV strains replicate efficiently in permissive human cells, many avian IAV cause abortive nonproductive infections in these hosts despite successful cell entry. However, the precise reasons for these differential outcomes are poorly defined. We hypothesized that the distinct course of an IAV infection with a given virus strain is determined by the differential interplay between specific host and viral factors. By using Spike-in SILAC mass spectrometry-based quantitative proteomics we characterized sets of cellular factors whose abundance is specifically up- or downregulated in the course of permissive versus nonpermissive IAV infection, respectively. This approach allowed for the definition and quantitative comparison of about 3500 proteins in human lung epithelial cells in response to seasonal or low-pathogenic avian H3N2 IAV. Many identified proteins were similarly regulated by both virus strains, but also 16 candidates with distinct changes in permissive versus nonpermissive infection were found. RNAi-mediated knockdown of these differentially regulated host factors identified Vpr binding protein (VprBP) as proviral host factor because its downregulation inhibited efficient propagation of seasonal IAV whereas overexpression increased viral replication of both seasonal and avian IAV. These results not only show that there are similar differences in the overall changes during permissive and nonpermissive influenza virus infections, but also provide a basis to evaluate VprBP as novel anti-IAV drug target.

Influenza A Virus Virulence Depends on Two Amino Acids in the N-Terminal Domain of Its NS1 Protein To Facilitate Inhibition of the RNA-Dependent Protein Kinase PKR.

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+/+ mice, but replicated to high titers in lungs of PKR-/- 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-/- 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.

Elevation in type I interferons inhibits HCN1 and slows cortical neuronal oscillations.

Central nervous system (CNS) inflammation involves the generation of inducible cytokines such as interferons (IFNs) and alterations in brain activity, yet the interplay of both is not well understood. Here, we show that in vivo elevation of IFNs by viral brain infection reduced hyperpolarization-activated currents (Ih) in cortical pyramidal neurons. In rodent brain slices directly exposed to type I IFNs, the hyperpolarization-activated cyclic nucleotide (HCN)-gated channel subunit HCN1 was specifically affected. The effect required an intact type I receptor (IFNAR) signaling cascade. Consistent with Ih inhibition, IFNs hyperpolarized the resting membrane potential, shifted the resonance frequency, and increased the membrane impedance. In vivo application of IFN-ß to the rat and to the mouse cerebral cortex reduced the power of higher frequencies in the cortical electroencephalographic activity only in the presence of HCN1. In summary, these findings identify HCN1 channels as a novel neural target for type I IFNs providing the possibility to tune neural responses during the complex event of a CNS inflammation.

The novel human influenza A(H7N9) virus is naturally adapted to efficient growth in human lung tissue.

UNLABELLED: 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-ß 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. IMPORTANCE: Humans are usually not infected by avian influenza A viruses (IAV), but this large group of viruses contributes to the emergence of human pandemic strains. Transmission of virulent avian IAV to humans is therefore an alarming event that requires assessment of the biology as well as pathogenic and pandemic potentials of the viruses in clinically relevant models. Here, we demonstrate that an early virus isolate from the recent A(H7N9) outbreak in Eastern China replicated as efficiently as human-adapted IAV in explanted human lung tissue, whereas avian H7 subtype viruses were unable to propagate. Robust replication of the H7N9 strain correlated with a low induction of antiviral beta interferon (IFN-ß), and cell-based studies indicated that this is due to efficient suppression of the IFN response by the viral NS1 protein. Thus, explanted human lung tissue appears to be a useful experimental model to explore the determinants facilitating cross-species transmission of the H7N9 virus to humans.

Cytomegalovirus downregulates IRE1 to repress the unfolded protein response.

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.

Highly pathogenic H5N1 influenza A virus strains provoke heterogeneous IFN-α/ß responses that distinctively affect viral propagation in human cells.

The fatal transmissions of highly pathogenic avian influenza A viruses (IAV) of the H5N1 subtype to humans and high titer replication in the respiratory tract indicate that these pathogens can overcome the bird-to-human species barrier. While type I interferons (IFN-α/ß) are well described to contribute to the species barrier of many zoonotic viruses, current data to the role of these antiviral cytokines during human H5N1 IAV infections is limited and contradictory. We hypothesized an important role for the IFN system in limiting productive infection of avian H5N1 strains in human cells. Hence, we examined IFN-α/ß gene activation by different avian and human H5N1 isolates, if the IFN-α/ß response restricts H5N1 growth and whether the different strains were equally capable to regulate the IFN-α/ß system via their IFN-antagonistic NS1 proteins. Two human H5N1 isolates and a seasonal H3N2 strain propagated efficiently in human respiratory cells and induced little IFN-ß, whereas three purely avian H5N1 strains were attenuated for replication and provoked higher IFN secretion. Replication of avian viruses was significantly enhanced on interferon-deficient cells, and exogenous IFN potently limited the growth of all strains in human cells. Moreover, IFN-α/ß activation by all strains depended on retinoic acid-inducible gene I excluding principal differences in receptor activation between the different viruses. Interestingly, all H5N1 NS1 proteins suppressed IFN-α/ß induction comparably well to the NS1 of seasonal IAV. Thus, our study shows that H5N1 strains are heterogeneous in their capacity to activate human cells in an NS1-independent manner. Our findings also suggest that H5N1 viruses need to acquire adaptive changes to circumvent strong IFN-α/ß activation in human host cells. Since no single amino acid polymorphism could be associated with a respective high- or low induction phenotype we propose that the necessary adaptations to overcome the human IFN-α/ß barrier involve mutations in multiple H5N1 genes.

Construction of a lytically replicating Kaposi's sarcoma-associated herpesvirus.

Karposi's sarcoma-associated herpesvirus (KSHV) is found predominantly in a latent state in most cell types, impeding investigations of the lytic replication cycle. Here, we engineered the cloned KSHV genome, bacterial artificial chromosome 36 (BAC36), to enforce constitutive expression of the main lytic switch regulator, the replication and transcription activator (RTA) (open reading frame 50 [ORF50]). The resulting virus, KSHV-lyt, activated by default the lytic cycle and replicated to high titers in various cells. Using KSHV-lyt, we showed that ORF33 (encoding a tegument protein) is essential for lytic KSHV replication in cell culture, but ORF73 (encoding the latent nuclear antigen [LANA]) is not. Thus, KSHV-lyt should be highly useful to study viral gene function during lytic replication.

Specific inhibition of the PKR-mediated antiviral response by the murine cytomegalovirus proteins m142 and m143.

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 eIF2alpha, 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 eIF2alpha phosphorylation during infection with an m142- and m143-deficient MCMV is specifically mediated by PKR, not by the related eIF2alpha kinases PERK or GCN2. PKR antagonists of vaccinia virus (E3L) or herpes simplex virus (gamma34.5) 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.

Murine cytomegalovirus m142 and m143 are both required to block protein kinase R-mediated shutdown of protein synthesis.

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 2alpha, 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.

The herpesviral Fc receptor fcr-1 down-regulates the NKG2D ligands MULT-1 and H60.

Members of the alpha- and beta-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.