Novel insights into the host cell glycan binding profile of human metapneumovirus.

Numerous viruses have been found to exploit glycoconjugates expressed on human cells as their initial attachment factor for viral entry and infection. The virus-cell glycointeractome, when characterized, may serve as a template for antiviral drug design. Heparan sulfate proteoglycans extensively decorate the human cell surface and were previously described as a primary receptor for human metapneumovirus (HMPV). After respiratory syncytial virus, HMPV is the second most prevalent respiratory pathogen causing respiratory tract infection in young children. To date, there is neither vaccine nor drug available to prevent or treat HMPV infection. Using a multidisciplinary approach, we report for the first time the glycointeractome of the HMPV fusion (F) protein, a viral surface glycoprotein that is essential for target-cell recognition, attachment, and entry. Our glycan microarray and surface plasmon resonance results suggest that Galß1-3/4GlcNAc moieties that may be sialylated or fucosylated are readily recognized by HMPV F. The bound motifs are highly similar to the N-linked and O-linked glycans primarily expressed on the human lung epithelium. We demonstrate that the identified glycans have the potential to compete with the cellular receptors used for HMPV entry and consequently block HMPV infection. We found that lacto-N-neotetraose demonstrated the strongest HMPV binding inhibition in a cell infection assay. Our current findings offer an encouraging and novel avenue for the design of anti-HMPV drug candidates using oligosaccharide templates.IMPORTANCEAll cells are decorated with a dense coat of sugars that makes a sugar code. Many respiratory viruses exploit this sugar code by binding to these sugars to cause infection. Human metapneumovirus is a leading cause for acute respiratory tract infections. Despite its medical importance, there is no vaccine or antiviral drug available to prevent or treat human metapneumovirus infection. This study investigates how human metapneumovirus binds to sugars in order to more efficiently infect the human host. We found that human metapneumovirus binds to a diverse range of sugars and demonstrated that these sugars can ultimately block viral infection. Understanding how viruses can take advantage of the sugar code on our cells could identify new intervention and treatment strategies to combat viral disease.

A neutralizing single-domain antibody that targets the trimer interface of the human metapneumovirus fusion protein.

IMPORTANCE: Human metapneumovirus (hMPV) is an important respiratory pathogen for which no licensed antivirals or vaccines exist. Single-domain antibodies represent promising antiviral biologics that can be easily produced and formatted. We describe the isolation and detailed characterization of two hMPV-neutralizing single-domain antibodies that are directed against the fusion protein F. One of these single-domain antibodies broadly neutralizes hMPV A and B strains, can prevent proteolytic maturation of F, and binds to an epitope in the F trimer interface. This suggests that hMPV pre-F undergoes trimer opening or "breathing" on infectious virions, exposing a vulnerable site for neutralizing antibodies. Finally, we show that this single-domain antibody, fused to a human IgG1 Fc, can protect cotton rats against hMPV replication, an important finding for potential future clinical applications.

Structural basis for respiratory syncytial virus and human metapneumovirus neutralization.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) continue to be a global burden to infants, the elderly, and immunocompromised individuals. In the past ten years, there has been substantial progress in the development of new vaccine candidates and therapies against these viruses. These advancements were guided by the structural elucidation of the major surface glycoproteins for these viruses, the fusion (F) protein and attachment (G) protein. The identification of immunodominant epitopes on the RSV F and hMPV F proteins has expanded current knowledge on antibody-mediated immune responses, which has led to new approaches for vaccine and therapeutic development through the stabilization of pre-fusion constructs of the F protein and pre-fusion-specific monoclonal antibodies with high potency and efficacy. In this review, we describe structural characteristics of known antigenic sites on the RSV and hMPV proteins, their influence on the immune response, and current progress in vaccine and therapeutic development.

Potent cross-neutralization of respiratory syncytial virus and human metapneumovirus through a structurally conserved antibody recognition mode.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) infections pose a significant health burden. Using pre-fusion conformation fusion (F) proteins, we isolated a panel of anti-F antibodies from a human donor. One antibody (RSV-199) potently cross-neutralized 8 RSV and hMPV strains by recognizing antigenic site III, which is partially conserved in RSV and hMPV F. Next, we determined the cryoelectron microscopy (cryo-EM) structures of RSV-199 bound to RSV F trimers, hMPV F monomers, and an unexpected dimeric form of hMPV F. These structures revealed how RSV-199 engages both RSV and hMPV F proteins through conserved interactions of the antibody heavy-chain variable region and how variability within heavy-chain complementarity-determining region 3 (HCDR3) can be accommodated at the F protein interface in site-III-directed antibodies. Furthermore, RSV-199 offered enhanced protection against RSV A and B strains and hMPV in cotton rats. These findings highlight the mechanisms of broad neutralization and therapeutic potential of RSV-199.

Avian Metapneumovirus Subgroup C Phosphoprotein Suppresses Type I Interferon Production by Blocking Interferon Regulatory Factor 3 Nuclear Translocation.

Avian metapneumovirus subgroup C (aMPV/C) is an important pathogen that causes upper respiratory symptoms and egg production decline in turkeys and chickens. aMPV/C infection leads to inhibition of the host antiviral immune response. However, our understanding of the molecular mechanisms underlying host immune response antagonized by aMPV/C infection is limited. In this study, we demonstrated that the aMPV/C phosphoprotein (P) inhibits the IFN antiviral signaling pathway triggered by melanoma differentiation gene 5 (MDA5) and reduces interferon ß (IFN-ß) production and IFN-stimulated genes (ISGs) by targeting IFN regulatory factor 7 (IRF7) but not nuclear factor κB (NF-κB) in DF-1 cells. Moreover, we found that aMPV/C P protein only blocks the nuclear translocation of IRF3 by interacting with IRF3 in HEK-293T cells, instead of affecting IRF3 phosphorylation and inducing IRF3 degradation, which suppresses IRF3 signaling activation and results in a decrease in IFN-ß production. Collectively, these results reveal a novel mechanism by which aMPV/C infection disrupts IFN-ß production in the host. IMPORTANCE The innate immune response is the first defense line of host cells and organisms against viral infections. When RNA viruses infect cells, viral RNA induces activation of retinoic acid-induced gene I and melanoma differentiation gene 5, which initiates downstream molecules and finally produces type I interferon (IFN-I) to regulate antiviral immune responses. The mechanism for avian metapneumovirus (aMPV) modulating IFN-I production to benefit its replication remains unknown. Here, we demonstrate that phosphoprotein of aMPV subgroup C (aMPV/C) selectively inhibits the nuclear translocation of interferon regulatory 3 (IRF3), instead of affecting the expression and phosphorylation of IRF3, which finally downregulates IFN-I production. This study showed a novel mechanism for aMPV/C infection antagonizing the host IFN response.

Evidence against the Human Metapneumovirus G, SH, and M2-2 Proteins as Bona Fide Interferon Antagonists.

The production of type I interferon (IFN) is the hallmark of the innate immune response. Most, if not all, mammalian viruses have a way to circumvent this response. Fundamental knowledge on viral evasion of innate immune responses may facilitate the design of novel antiviral therapies. To investigate how human metapneumovirus (HMPV) interacts with the innate immune response, recombinant viruses lacking G, short hydrophobic (SH), or M2-2 protein expression were assessed for IFN induction in A549 cells. HMPV lacking G or SH protein expression induced similarly low levels of IFN, compared to the wild-type virus, whereas HMPV lacking M2-2 expression induced significantly more IFN than the wild-type virus. However, sequence analysis of the genomes of M2-2 mutant viruses revealed large numbers of mutations throughout the genome. Over 70% of these nucleotide substitutions were A-to-G and T-to-C mutations, consistent with the properties of the adenosine deaminase acting on RNA (ADAR) protein family. Knockdown of ADAR1 by CRISPR interference confirmed the role of ADAR1 in the editing of M2-2 deletion mutant virus genomes. More importantly, Northern blot analyses revealed the presence of defective interfering RNAs (DIs) in M2-2 mutant viruses and not in the wild-type virus or G and SH deletion mutant viruses. DIs are known to be potent inducers of the IFN response. The presence of DIs in M2-2 mutant virus stocks and hypermutated virus genomes interfere with studies on HMPV and the innate immune response and should be addressed in future studies. IMPORTANCE Understanding the interaction between viruses and the innate immune response is one of the barriers to the design of antiviral therapies. Here, we investigated the role of the G, SH, and M2-2 proteins of HMPV as type I IFN antagonists. In contrast to other studies, no IFN-antagonistic functions could be observed for the G and SH proteins. HMPV with a deletion of the M2-2 protein did induce type I IFN production upon infection of airway epithelial cells. However, during generation of virus stocks, these viruses rapidly accumulated DIs, which are strong activators of the type I IFN response. Additionally, the genomes of these viruses were hypermutated, which was prevented by generating stocks in ADAR knockdown cells, confirming a role for ADAR in hypermutation of HMPV genomes or DIs. These data indicate that a role of the HMPV M2-2 protein as a bona fide IFN antagonist remains elusive.

SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses.

Viruses possessing class I fusion proteins require proteolytic activation by host cell proteases to mediate fusion with the host cell membrane. The mammalian SPINT2 gene encodes a protease inhibitor that targets trypsin-like serine proteases. Here we show the protease inhibitor, SPINT2, restricts cleavage-activation efficiently for a range of influenza viruses and for human metapneumovirus (HMPV). SPINT2 treatment resulted in the cleavage and fusion inhibition of full-length influenza A/CA/04/09 (H1N1) HA, A/Aichi/68 (H3N2) HA, A/Shanghai/2/2013 (H7N9) HA and HMPV F when activated by trypsin, recombinant matriptase or KLK5. We also demonstrate that SPINT2 was able to reduce viral growth of influenza A/CA/04/09 H1N1 and A/X31 H3N2 in cell culture by inhibiting matriptase or TMPRSS2. Moreover, inhibition efficacy did not differ whether SPINT2 was added at the time of infection or 24 h post-infection. Our data suggest that the SPINT2 inhibitor has a strong potential to serve as a novel broad-spectrum antiviral.

Human metapneumovirus fusion protein triggering: Increasing complexities by analysis of new HMPV fusion proteins.

The human metapneumovirus (HMPV) fusion protein (F) mediates fusion of the viral envelope and cellular membranes to establish infection. HMPV F from some, but not all, viral strains promotes fusion only after exposure to low pH. Previous studies have identified several key residues involved in low pH triggering, including H435 and a proposed requirement for glycine at position 294. We analyzed the different levels of fusion activity, protein expression and cleavage of three HMPV F proteins not previously examined. Interestingly, low pH-triggered fusion in the absence of G294 was identified in one F protein, while a novel histidine residue (H434) was identified that enhanced low pH promoted fusion in another. The third F protein failed to promote cell-to-cell fusion, suggesting other requirements for F protein triggering. Our results demonstrate HMPV F triggering is more complex than previously described and suggest a more intricate mechanism for fusion protein function and activation.

Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.

Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.

Human Metapneumovirus Attachment Protein Contributes to Neutrophil Recruitment into the Airways of Infected Mice.

Human Metapneumovirus (HMPV) is a leading respiratory pathogen that causes lower respiratory tract infections worldwide. Acute HMPV infection induces an exacerbated inflammatory neutrophilic response leading to bronchiolitis and pneumonia. However, the mechanism by which the virus regulates neutrophil infiltration into the airways still remains unexplored. In this work, we used an experimental mouse model of HMPV infection to demonstrate that the attachment (G) protein of HMPV contributes to the recruitment of neutrophils into the airways and modulate the production of neutrophil chemoattractants and Type I IFN responses, specifically IFN-α. These findings provide the first evidence that the HMPV G protein contributes to the in vivo neutrophilic response to HMPV infection and furthers our understanding on virus induced inflammatory responses in the airways.

X-ray structure and activities of an essential Mononegavirales L-protein domain.

The L protein of mononegaviruses harbours all catalytic activities for genome replication and transcription. It contains six conserved domains (CR-I to -VI; Fig. 1a). CR-III has been linked to polymerase and polyadenylation activity, CR-V to mRNA capping and CR-VI to cap methylation. However, how these activities are choreographed is poorly understood. Here we present the 2.2-Å X-ray structure and activities of CR-VI+, a portion of human Metapneumovirus L consisting of CR-VI and the poorly conserved region at its C terminus, the +domain. The CR-VI domain has a methyltransferase fold, which besides the typical S-adenosylmethionine-binding site ((SAM)P) also contains a novel pocket ((NS)P) that can accommodate a nucleoside. CR-VI lacks an obvious cap-binding site, and the (SAM)P-adjoining site holding the nucleotides undergoing methylation ((SUB)P) is unusually narrow because of the overhanging +domain. CR-VI+ sequentially methylates caps at their 2'O and N7 positions, and also displays nucleotide triphosphatase activity.

Trypsin- and low pH-mediated fusogenicity of avian metapneumovirus fusion proteins is determined by residues at positions 100, 101 and 294.

Avian metapneumovirus (aMPV) and human metapneumovirus (hMPV) are members of the genus Metapneumovirus in the subfamily Pneumovirinae. Metapneumovirus fusion (F) protein mediates the fusion of host cells with the virus membrane for infection. Trypsin- and/or low pH-induced membrane fusion is a strain-dependent phenomenon for hMPV. Here, we demonstrated that three subtypes of aMPV (aMPV/A, aMPV/B, and aMPV/C) F proteins promoted cell-cell fusion in the absence of trypsin. Indeed, in the presence of trypsin, only aMPV/C F protein fusogenicity was enhanced. Mutagenesis of the amino acids at position 100 and/or 101, located at a putative cleavage region in aMPV F proteins, revealed that the trypsin-mediated fusogenicity of aMPV F proteins is regulated by the residues at positions 100 and 101. Moreover, we demonstrated that aMPV/A and aMPV/B F proteins mediated cell-cell fusion independent of low pH, whereas the aMPV/C F protein did not. Mutagenesis of the residue at position 294 in the aMPV/A, aMPV/B, and aMPV/C F proteins showed that 294G played a critical role in F protein-mediated fusion under low pH conditions. These findings on aMPV F protein-induced cell-cell fusion provide new insights into the molecular mechanisms underlying membrane fusion and pathogenesis of aMPV.

Effect of amino acid sequence variations at position 149 on the fusogenic activity of the subtype B avian metapneumovirus fusion protein.

The entry of enveloped viruses into host cells requires the fusion of viral and cell membranes. These membrane fusion reactions are mediated by virus-encoded glycoproteins. In the case of avian metapneumovirus (aMPV), the fusion (F) protein alone can mediate virus entry and induce syncytium formation in vitro. To investigate the fusogenic activity of the aMPV F protein, we compared the fusogenic activities of three subtypes of aMPV F proteins using a TCSD50 assay developed in this study. Interestingly, we found that the F protein of aMPV subtype B (aMPV/B) strain VCO3/60616 (aMPV/vB) was hyperfusogenic when compared with F proteins of aMPV/B strain aMPV/f (aMPV/fB), aMPV subtype A (aMPV/A), and aMPV subtype C (aMPV/C). We then further demonstrated that the amino acid (aa) residue 149F contributed to the hyperfusogenic activity of the aMPV/vB F protein. Moreover, we revealed that residue 149F had no effect on the fusogenic activities of aMPV/A, aMPV/C, and human metapneumovirus (hMPV) F proteins. Collectively, we provide the first evidence that the amino acid at position 149 affects the fusogenic activity of the aMPV/B F protein, and our findings will provide new insights into the fusogenic mechanism of this protein.

The human metapneumovirus fusion protein mediates entry via an interaction with RGD-binding integrins.

Paramyxoviruses use a specialized fusion protein to merge the viral envelope with cell membranes and initiate infection. Most paramyxoviruses require the interaction of two viral proteins to enter cells; an attachment protein binds cell surface receptors, leading to the activation of a fusion (F) protein that fuses the viral envelope and host cell plasma membrane. In contrast, human metapneumovirus (HMPV) expressing only the F protein is replication competent, suggesting a primary role for HMPV F in attachment and fusion. We previously identified an invariant arginine-glycine-aspartate (RGD) motif in the HMPV F protein and showed that the RGD-binding integrin αVß1-promoted HMPV infection. Here we show that both HMPV F-mediated binding and virus entry depend upon multiple RGD-binding integrins and that HMPV F can mediate binding and fusion in the absence of the viral attachment (G) protein. The invariant F-RGD motif is critical for infection, as an F-RAE virus was profoundly impaired. Further, F-integrin binding is required for productive viral RNA transcription, indicating that RGD-binding integrins serve as receptors for the HMPV fusion protein. Thus, HMPV F is triggered to induce virus-cell fusion by interactions with cellular receptors in a manner that is independent of the viral G protein. These results suggest a stepwise mechanism of HMPV entry mediated by the F protein through its interactions with cellular receptors, including RGD-binding integrins.

Human metapneumovirus (HMPV) binding and infection are mediated by interactions between the HMPV fusion protein and heparan sulfate.

Human metapneumovirus (HMPV) is a major worldwide respiratory pathogen that causes acute upper and lower respiratory tract disease. The mechanism by which this virus recognizes and gains access to its target cell is still largely unknown. In this study, we addressed the initial steps in virus binding and infection and found that the first binding partner for HMPV is heparan sulfate (HS). While wild-type CHO-K1 cells are permissive to HMPV infection, mutant cell lines lacking the ability to synthesize glycosaminoglycans (GAGs), specifically, heparan sulfate proteoglycans (HSPGs), were resistant to binding and infection by HMPV. The permissiveness to HMPV infection was also abolished when CHO-K1 cells were treated with heparinases. Importantly, using recombinant HMPV lacking both the G and small hydrophobic (SH) proteins, we report that this first virus-cell binding interaction is driven primarily by the fusion protein (HMPV F) and that this interaction is needed to establish a productive infection. Finally, HMPV binding to cells did not require ß1 integrin expression, and RGD-mediated interactions were not essential in promoting HMPV F-mediated cell-to-cell membrane fusion. Cells lacking ß1 integrin, however, were less permissive to HMPV infection, indicating that while ß1 integrins play an important role in promoting HMPV infection, the interaction between integrins and HMPV occurs after the initial binding of HMPV F to heparan sulfate proteoglycans.

Topology and cellular localization of the small hydrophobic protein of avian metapneumovirus.

The small hydrophobic protein (SH) is a type II integral membrane protein that is packaged into virions and is only present in certain paramyxoviruses including metapneumovirus. In addition to a highly divergent primary sequence, SH proteins vary significantly in size amongst the different viruses. Human respiratory syncytial virus (HRSV) encodes the smallest SH protein consisting of only 64 amino acids, while metapneumoviruses have the longest SH protein ranging from 174 to 179 amino acids in length. Little is currently known about the cellular localization and topology of the metapneumovirus SH protein. Here we characterize for the first time metapneumovirus SH protein with respect to topology, subcellular localization, and transport using avian metapneumovirus subgroup C (AMPV-C) as a model system. We show that AMPV-C SH is an integral membrane protein with N(in)C(out) orientation located in both the plasma membrane as well as within intracellular compartments, which is similar to what has been described previously for SH proteins of other paramyxoviruses. Furthermore, we demonstrate that AMPV-C SH protein localizes in the endoplasmic reticulum (ER), Golgi, and cell surface, and is transported through ER-Golgi secretory pathway.

The synthetic bacterial lipopeptide Pam3CSK4 modulates respiratory syncytial virus infection independent of TLR activation.

Respiratory syncytial virus (RSV) is an important cause of acute respiratory disease in infants, immunocompromised subjects and the elderly. However, it is unclear why most primary RSV infections are associated with relatively mild symptoms, whereas some result in severe lower respiratory tract infections and bronchiolitis. Since RSV hospitalization has been associated with respiratory bacterial co-infections, we have tested if bacterial Toll-like receptor (TLR) agonists influence RSV-A2-GFP infection in human primary cells or cell lines. The synthetic bacterial lipopeptide Pam3-Cys-Ser-Lys4 (Pam3CSK4), the prototype ligand for the heterodimeric TLR1/TLR2 complex, enhanced RSV infection in primary epithelial, myeloid and lymphoid cells. Surprisingly, enhancement was optimal when lipopeptides and virus were added simultaneously, whereas addition of Pam3CSK4 immediately after infection had no effect. We have identified two structurally related lipopeptides without TLR-signaling capacity that also modulate RSV infection, whereas Pam3CSK4-reminiscent TLR1/2 agonists did not, and conclude that modulation of infection is independent of TLR activation. A similar TLR-independent enhancement of infection could also be demonstrated for wild-type RSV strains, and for HIV-1, measles virus and human metapneumovirus. We show that the effect of Pam3CSK4 is primarily mediated by enhanced binding of RSV to its target cells. The N-palmitoylated cysteine and the cationic lysines were identified as pivotal for enhanced virus binding. Surprisingly, we observed inhibition of RSV infection in immortalized epithelial cell lines, which was shown to be related to interactions between Pam3CSK4 and negatively charged glycosaminoglycans on these cells, which are known targets for binding of laboratory-adapted but not wild-type RSV. These data suggest a potential role for bacterial lipopeptides in enhanced binding of RSV and other viruses to their target cells, thus affecting viral entry or spread independent of TLR signaling. Moreover, our results also suggest a potential application for these synthetic lipopeptides as adjuvants for live-attenuated viral vaccines.

Detection of respiratory viruses with a multiplex polymerase chain reaction assay (MultiCode-PLx Respiratory Virus Panel) in patients with hematologic malignancies.

Respiratory viral pathogens are a common cause of morbidity in patients with hematologic malignancies. Sensitive molecular assays have increased the detection of common respiratory viruses and expanded the panel of detectable viruses. Both a rapid viral culture with direct fluorescence antibody (DFA) staining and a PCR-based assay (MultiCode-PLx Respiratory Virus Panel) were performed on patients with hematologic malignancies, who underwent collection of a nasopharyngeal swab or bronchoalveolar lavage from October 2006 to April 2007. Eighty-two samples from 70 patients were obtained; all patients had upper respiratory tract symptoms. Respiratory viruses were detected in 10 samples (12%) by conventional virological methods and in 31 samples (38%) by the MultiCode-PLx assay. This increased diagnostic yield resulted from better sensitivity for those viruses detectable by both methods and detection of viruses not covered by the antigen detection/rapid culture method (human metapneumovirus, coronaviruses and rhinoviruses). The MultiCode-PLx assay frequently identified respiratory viral infections which are not detected by rapid viral culture/DFA; 40% of these patients had pneumonia in addition to the upper respiratory tract symptoms. Improved diagnostics for respiratory viruses may lead to more effective management and better outcomes in this patient population.

Human metapneumovirus small hydrophobic protein inhibits NF-kappaB transcriptional activity.

Human metapneumovirus, a leading cause of respiratory tract infections in infants, encodes a small hydrophobic (SH) protein of unknown function. In this study, we showed that infection of airway epithelial cells or mice with recombinant human metapneumovirus lacking SH expression (rhMPV-DeltaSH) enhanced secretion of proinflammatory mediators, including interleukin 6 (IL-6) and IL-8, encoded by two NF-kB-dependent genes, compared to infection with wild-type rhMPV. RhMPV-DeltaSH infection resulted in enhanced NF-kB-dependent gene transcription and in increased levels of phosphorylated and acetylated NF-kB without affecting its nuclear translocation, identifying a possible novel mechanism by which paramyxovirus SH proteins modulate NF-kB activation.

Intracellular processing, glycosylation, and cell surface expression of human metapneumovirus attachment glycoprotein.

The biosynthesis and posttranslational processing of human metapneumovirus attachment G glycoprotein were investigated. After pulse-labeling, the G protein accumulated as three species with molecular weights of 45,000, 50,000, and 53,000 (45K, 50K, and 53K, respectively). N-Glycosidase digestion indicated that these forms represent the unglycosylated precursor and N-glycosylated intermediate products, respectively. After an appropriate chase, these three naive forms were further processed to a mature 97K form. The presence of O-linked sugars in mature G protein was confirmed by O-glycanase digestion and lectin-binding assay using Arachis hypogaea (peanut agglutinin), an O-glycan-specific lectin. In addition, in the O-glycosylation-deficient cell line (CHO ldlD cell), the G protein could not be processed to the mature form unless the exogenous Gal and GalNAc were supplemented, which provided added evidence supporting the O-linked glycosylation of G protein. The maturation of G was completely blocked by monensin but was partially sensitive to brefeldin A (BFA), suggesting the O-linked glycosylation of G initiated in the trans-Golgi compartment and terminated in the trans-Golgi network. Enzymatic deglycosylation analysis confirmed that the BFA-G was a partial mature form containing N-linked oligosaccharides and various amounts of O-linked carbohydrate side chains. The expression of G protein at the cell surface could be detected by indirect immunofluorescence staining assay. Furthermore, cell surface immunoprecipitation displayed an efficient intracellular transport of G protein.