Selective thiazoline peptide cyclisation compatible with mRNA display and efficient synthesis.

Peptide display technologies are a powerful method for discovery of new bioactive sequences, but linear sequences are often very unstable in a biological setting. Macrocyclisation of such peptides is beneficial for target affinity, selectivity, stability, and cell permeability. However, macrocyclisation of a linear hit is unreliable and requires extensive structural knowledge. Genetically encoding macrocyclisation during the discovery process is a better approach, and so there is a need for diverse cyclisation options that can be deployed in the context of peptide display techniques such as mRNA display. In this work we show that meta-cyanopyridylalanine (mCNP) can be ribosomally incorporated into peptides, forming a macrocycle in a spontaneous and selective reaction with an N-terminal cysteine generated from bypassing the initiation codon in translation. This reactive amino acid can also be easily incorporated into peptides during standard Fmoc solid phase peptide synthesis, which can otherwise be a bottleneck in transferring from peptide discovery to peptide testing and application. We demonstrate the potential of this new method by discovery of macrocyclic peptides targeting influenza haemagglutinin, and molecular dynamics simulation indicates the mCNP cross-link stabilises a beta sheet structure in a representative of the most abundant cluster of active hits. Cyclisation by mCNP is also shown to be compatible with thioether macrocyclisation at a second cysteine to form bicycles of different architectures, provided that cysteine placement reinforces selectivity, with this bicyclisation happening spontaneously and in a controlled manner during peptide translation. Our new approach generates macrocycles with a more rigid cross-link and with better control of regiochemistry when additional cysteines are present, opening these up for further exploitation in chemical modification of in vitro translated peptides, and so is a valuable addition to the peptide discovery toolbox.

An Efficient, Site-Selective and Spontaneous Peptide Macrocyclisation During in†vitro Translation.

Macrocyclisation provides a means of stabilising the conformation of peptides, often resulting in improved stability, selectivity, affinity, and cell permeability. In this work, a new approach to peptide macrocyclisation is reported, using a cyanobenzothiazole-containing amino acid that can be incorporated into peptides by both in vitro translation and solid phase peptide synthesis, meaning it should be applicable to peptide discovery by mRNA display. This cyclisation proceeds rapidly, with minimal by-products, is selective over other amino acids including non N-terminal cysteines, and is compatible with further peptide elaboration exploiting such an additional cysteine in bicyclisation and derivatisation reactions. Molecular dynamics simulations show that the new cyclisation group is likely to influence the peptide conformation as compared to previous thioether-based approaches, through rigidity and intramolecular aromatic interactions, illustrating their complementarity.

Analysis of the Evolution of Pandemic Influenza A(H1N1) Virus Neuraminidase Reveals Entanglement of Different Phenotypic Characteristics.

The influenza A virus (IAV) neuraminidase (NA) is essential for virion release from cells and decoy receptors and an important target of antiviral drugs and antibodies. Adaptation to a new host sialome and escape from the host immune system are forces driving the selection of mutations in the NA gene. Phylogenetic analysis shows that until 2015, 16 amino acid substitutions in NA became fixed in the virus population after introduction in the human population of the pandemic IAV H1N1 (H1N1pdm09) in 2009. The accumulative effect of these substitutions, in the order in which they appeared, was analyzed using recombinant proteins and viruses in combination with different functional assays. The results indicate that NA activity did not evolve to a single optimum but rather fluctuated within a certain bandwidth. Furthermore, antigenic and enzymatic properties of NA were intertwined, with several residues affecting multiple properties. For example, the substitution K432E in the second sialic acid binding site, next to the catalytic site, was shown to affect catalytic activity, substrate specificity, and the pH optimum for maximum activity. This substitution also altered antigenicity of NA, which may explain its selection. We propose that the entanglement of NA phenotypes may be an important determining factor in the evolution of NA.IMPORTANCE Since its emergence in 2009, the pandemic H1N1 influenza A virus (IAV) has caused significant disease and mortality in humans. IAVs contain two envelope glycoproteins, the receptor-binding hemagglutinin (HA) and the receptor-destroying neuraminidase (NA). NA is essential for virion release from cells and decoy receptors, is an important target of antiviral drugs, and is increasingly being recognized as an important vaccine antigen. Not much is known, however, about the evolution of this protein upon the emergence of the novel pandemic H1N1 virus, with respect to its enzymatic activity and antigenicity. By reconstructing the evolutionary path of NA, we show that antigenic and enzymatic properties of NA are intertwined, with several residues affecting multiple properties. Understanding the entanglement of NA phenotypes will lead to better comprehension of IAV evolution and may help the development of NA-based vaccines.

Elephant Endotheliotropic Herpesvirus Is Omnipresent in Elephants in European Zoos and an Asian Elephant Range Country.

Elephant endotheliotropic herpesviruses (EEHVs) may cause acute, often lethal, hemorrhagic disease (EEHV-HD) in young elephants. Prevalence of EEHV in different elephant populations is still largely unknown. In order to improve diagnostic tools for the detection of EEHV infections and to obtain insight into its spread among elephants, we developed novel ELISAs based on EEHV1A gB and gH/gL. Performance of the ELISAs was assessed using sera from 41 European zoo elephants and 69 semi-captive elephants from Laos, one of the Asian elephant range countries. Sera from all (sub)adult animals tested (≥5 years of age) showed high reactivity with both gB and gH/gL, indicating that EEHV prevalence has been highly underestimated so far. Reactivity towards the antigens was generally lower for sera of juvenile animals (1 > 5 years). Only one (juvenile) animal, which was sampled directly after succumbing to EEHV-HD, was found to be seronegative for EEHV. The two other EEHV-HD cases tested showed low antibody levels, suggesting that all three cases died upon a primary EEHV infection. In conclusion, our study suggests that essentially all (semi-)captive (sub)adult elephants in European zoos and in Laos carry EEHV, and that young elephants with low antibody levels are at risk of dying from EEHV-HD.

Breast Milk Prefusion F Immunoglobulin G as a Correlate of Protection Against Respiratory Syncytial Virus Acute Respiratory Illness.

Background: Transplacental respiratory syncytial virus (RSV) antibody transfer has been characterized, but little is known about the protective effect of breast milk RSV-specific antibodies. Serum antibodies against the prefusion RSV fusion protein (pre-F) exhibit high neutralizing activity. We investigate protection of breast milk pre-F antibodies against RSV acute respiratory infection (ARI). Methods: Breast milk at 1, 3, and 6 months postpartum and midnasal swabs during infant illness episodes were collected in mother-infant pairs in Nepal. One hundred seventy-four infants with and without RSV ARI were matched 1:1 by risk factors for RSV ARI. Pre-F immunoglobulin A (IgA) and immunoglobulin G (IgG) antibody levels were measured in breast milk. Results: The median breast milk pre-F IgG antibody concentration before illness was lower in mothers of infants with RSV ARI (1.4 [interquartile range {IQR}, 1.1-1.6] log10 ng/mL) than without RSV ARI (1.5 [IQR, 1.3-1.8] log10 ng/mL) (P = .001). There was no difference in median maternal pre-F IgA antibody concentrations in cases vs controls (1.7 [IQR, 0.0-2.2] log10 ng/mL vs 1.7 [IQR, 1.2-2.2] log10 ng/mL, respectively; P = .58). Conclusions: Low breast milk pre-F IgG antibodies before RSV ARI support a potential role for pre-F IgG as a correlate of protection against RSV ARI. Induction of breast milk pre-F IgG may be a mechanism of protection for maternal RSV vaccines.

Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein.

Middle East respiratory syndrome coronavirus (MERS-CoV) targets the epithelial cells of the respiratory tract both in humans and in its natural host, the dromedary camel. Virion attachment to host cells is mediated by 20-nm-long homotrimers of spike envelope protein S. The N-terminal subunit of each S protomer, called S1, folds into four distinct domains designated S1A through S1D Binding of MERS-CoV to the cell surface entry receptor dipeptidyl peptidase 4 (DPP4) occurs via S1B We now demonstrate that in addition to DPP4, MERS-CoV binds to sialic acid (Sia). Initially demonstrated by hemagglutination assay with human erythrocytes and intact virus, MERS-CoV Sia-binding activity was assigned to S subdomain S1A When multivalently displayed on nanoparticles, S1 or S1A bound to human erythrocytes and to human mucin in a strictly Sia-dependent fashion. Glycan array analysis revealed a preference for α2,3-linked Sias over α2,6-linked Sias, which correlates with the differential distribution of α2,3-linked Sias and the predominant sites of MERS-CoV replication in the upper and lower respiratory tracts of camels and humans, respectively. Binding is hampered by Sia modifications such as 5-N-glycolylation and (7,)9-O-acetylation. Depletion of cell surface Sia by neuraminidase treatment inhibited MERS-CoV entry of Calu-3 human airway cells, thus providing direct evidence that virus-Sia interactions may aid in virion attachment. The combined observations lead us to propose that high-specificity, low-affinity attachment of MERS-CoV to sialoglycans during the preattachment or early attachment phase may form another determinant governing the host range and tissue tropism of this zoonotic pathogen.

In Vitro Enhancement of Respiratory Syncytial Virus Infection by Maternal Antibodies Does Not Explain Disease Severity in Infants.

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory illness in infants. At this young age, infants typically depend on maternally transferred antibodies (matAbs) and their innate immune system for protection against infections. RSV-specific matAbs are thought to protect from severe illness, yet severe RSV disease occurs mainly below 6 months of age, when neutralizing matAb levels are present. To investigate this discrepancy, we asked if disease severity is related to antibody properties other than neutralization. Some antibody effector functions are mediated via their Fc binding region. However, it has been shown that this binding may lead to antibody-dependent enhancement (ADE) of infection or reduction of neutralization, both possibly leading to more disease. In this study, we first showed that high levels of ADE of RSV infection occur in monocytic THP-1 cells in the presence of RSV antibodies and that neutralization by these antibodies was reduced in Vero cells when they were transduced with Fc gamma receptors. We then demonstrated that antibodies from cotton rats with formalin-inactivated (FI)-RSV-induced pulmonary pathology were capable of causing ADE. Human matAbs also caused ADE and were less neutralizing in vitro in cells that carry Fc receptors. However, these effects were unrelated to disease severity because they were seen both in uninfected controls and in infants hospitalized with different levels of RSV disease severity. We conclude that ADE and reduction of neutralization are unlikely to be involved in RSV disease in infants with neutralizing matAbs.IMPORTANCE It is unclear why severity of RSV disease peaks at the age when infants have neutralizing levels of maternal antibodies. Additionally, the exact reason for FI-RSV-induced enhanced disease, as seen in the 1960s vaccine trials, is still unclear. We hypothesized that antibodies present under either of these conditions could contribute to disease severity. Antibodies can have effects that may lead to more disease instead of protection. We investigated two of those effects: antibody-dependent enhancement of infection (ADE) and neutralization reduction. We show that ADE occurs in vitro with antibodies from FI-RSV-immunized RSV-infected cotton rats. Moreover, passively acquired maternal antibodies from infants had the capacity to induce ADE and reduction of neutralization. However, no clear association with disease severity was seen, ruling out that these properties explain disease in the presence of maternal antibodies. Our data contribute to a better understanding of the impact of antibodies on RSV disease in infants.

Characterization of Epitope-Specific Anti-Respiratory Syncytial Virus (Anti-RSV) Antibody Responses after Natural Infection and after Vaccination with Formalin-Inactivated RSV.

UNLABELLED: Antibodies against the fusion (F) protein of respiratory syncytial virus (RSV) play an important role in the protective immune response to this important respiratory virus. Little is known, however, about antibody levels against multiple F-specific epitopes induced by infection or after vaccination against RSV, while this is important to guide the evaluation of (novel) vaccines. In this study, we analyzed antibody levels against RSV proteins and F-specific epitopes in human sera and in sera of vaccinated and experimentally infected cotton rats and the correlation thereof with virus neutralization. Analysis of human sera revealed substantial diversity in antibody levels against F-, G (attachment)-, and F-specific epitopes between individuals. The highest correlation with virus neutralization was observed for antibodies recognizing prefusion-specific antigenic site Ø. Nevertheless, our results indicate that high levels of antibodies targeting other parts of the F protein can also mediate a potent antiviral antibody response. In agreement, sera of experimentally infected cotton rats contained high neutralizing activity despite lacking antigenic site Ø-specific antibodies. Strikingly, vaccination with formalin-inactivated RSV (FI-RSV) exclusively resulted in the induction of poorly neutralizing antibodies against postfusion-specific antigenic site I, although antigenic sites I, II, and IV were efficiently displayed in FI-RSV. The apparent immunodominance of antigenic site I in FI-RSV likely explains the low levels of neutralizing antibodies upon vaccination and challenge and may play a role in the vaccination-induced enhancement of disease observed with such preparations. IMPORTANCE: RSV is an importance cause of hospitalization of infants. The development of a vaccine against RSV has been hampered by the disastrous results obtained with FI-RSV vaccine preparations in the 1960s that resulted in vaccination-induced enhancement of disease. To get a better understanding of the antibody repertoire induced after infection or after vaccination against RSV, we investigated antibody levels against fusion (F) protein, attachment (G) protein, and F-specific epitopes in human and animal sera. The results indicate the importance of prefusion-specific antigenic site Ø antibodies as well as of antibodies targeting other epitopes in virus neutralization. However, vaccination of cotton rats with FI-RSV specifically resulted in the induction of weakly neutralizing, antigenic site I-specific antibodies, which may play a role in the enhancement of disease observed after vaccination with such preparations.

In silico structure-based design and synthesis of novel anti-RSV compounds.

Respiratory syncytial virus (RSV) is the major cause for respiratory tract disease in infants and young children. Currently, no licensed vaccine or a selective antiviral drug against RSV infections are available. Here, we describe a structure-based drug design approach that led to the synthesis of a novel series of zinc-ejecting compounds active against RSV replication. 30 compounds, sharing a common dithiocarbamate moiety, were designed and prepared to target the zinc finger motif of the M2-1 protein. A library of ∼ 12,000 small fragments was docked to explore the area surrounding the zinc ion. Among these, seven ligands were selected and used for the preparation of the new derivatives. The results reported here may help the development of a lead compound for the treatment of RSV infections.

Recombinant Soluble Respiratory Syncytial Virus F Protein That Lacks Heptad Repeat B, Contains a GCN4 Trimerization Motif and Is Not Cleaved Displays Prefusion-Like Characteristics.

The respiratory syncytial virus (RSV) fusion protein F is considered an attractive vaccine candidate especially in its prefusion conformation. We studied whether recombinant soluble RSV F proteins could be stabilized in a prefusion-like conformation by mutation of heptad repeat B (HRB). The results show that soluble, trimeric, non-cleaved RSV F protein, produced by expression of the furin cleavage site-mutated F ectodomain extended with a GCN4 trimerization sequence, is efficiently recognized by pre- as well as postfusion-specific antibodies. In contrast, a similar F protein completely lacking HRB displayed high reactivity with prefusion-specific antibodies recognizing antigenic site Ø, but did not expose postfusion-specific antigenic site I, in agreement with this protein maintaining a prefusion-like conformation. These features were dependent on the presence of the GCN4 trimerization domain. Absence of cleavage also contributed to binding of prefusion-specific antibodies. Similar antibody reactivity profiles were observed when the prefusion form of F was stabilized by the introduction of cysteine pairs in HRB. To study whether the inability to form the 6HB was responsible for the prefusion-like antibody reactivity profile, alanine mutations were introduced in HRB. Although introduction of alanine residues in HRB inhibited the formation of the 6HB, the exposure of postfusion-specific antigenic site I was not prevented. In conclusion, proteins that are not able to form the 6HB, due to mutation of HRB, may still display postfusion-specific antigenic site I. Replacement of HRB by the GCN4 trimerization domain in a non-cleaved soluble F protein resulted, however, in a protein with prefusion-like characteristics, suggesting that this HRB-lacking protein may represent a potential prefusion F protein subunit vaccine candidate.

ATP1A1-mediated Src signaling inhibits coronavirus entry into host cells.

UNLABELLED: In addition to transporting ions, the multisubunit Na(+),K(+)-ATPase also functions by relaying cardiotonic steroid (CTS)-binding-induced signals into cells. In this study, we analyzed the role of Na(+),K(+)-ATPase and, in particular, of its ATP1A1 α subunit during coronavirus (CoV) infection. As controls, the vesicular stomatitis virus (VSV) and influenza A virus (IAV) were included. Using gene silencing, the ATP1A1 protein was shown to be critical for infection of cells with murine hepatitis virus (MHV), feline infectious peritonitis virus (FIPV), and VSV but not with IAV. Lack of ATP1A1 did not affect virus binding to host cells but resulted in inhibited entry of MHV and VSV. Consistently, nanomolar concentrations of the cardiotonic steroids ouabain and bufalin, which are known not to affect the transport function of Na(+),K(+)-ATPase, inhibited infection of cells with MHV, FIPV, Middle East respiratory syndrome (MERS)-CoV, and VSV, but not IAV, when the compounds were present during virus inoculation. Cardiotonic steroids were shown to inhibit entry of MHV at an early stage, resulting in accumulation of virions close to the cell surface and, as a consequence, in reduced fusion. In agreement with an early block in infection, the inhibition of VSV by CTSs could be bypassed by low-pH shock. Viral RNA replication was not affected when these compounds were added after virus entry. The antiviral effect of ouabain could be relieved by the addition of different Src kinase inhibitors, indicating that Src signaling mediated via ATP1A1 plays a crucial role in the inhibition of CoV and VSV infections. IMPORTANCE: Coronaviruses (CoVs) are important pathogens of animals and humans, as demonstrated by the recent emergence of new human CoVs of zoonotic origin. Antiviral drugs targeting CoV infections are lacking. In the present study, we show that the ATP1A1 subunit of Na(+),K(+)-ATPase, an ion transporter and signaling transducer, supports CoV infection. Targeting ATP1A1 either by gene silencing or by low concentrations of the ATP1A1-binding cardiotonic steroids ouabain and bufalin resulted in inhibition of infection with murine, feline, and MERS-CoVs at an early entry stage. Infection with the control virus VSV was also inhibited. Src signaling mediated by ATP1A1 was shown to play a crucial role in the inhibition of virus entry by ouabain and bufalin. These results suggest that targeting the Na(+),K(+)-ATPase using cardiotonic steroids, several of which are FDA-approved compounds, may be an attractive therapeutic approach against CoV and VSV infections.

Dissecting virus entry: replication-independent analysis of virus binding, internalization, and penetration using minimal complementation of ß-galactosidase.

Studies of viral entry into host cells often rely on the detection of post-entry parameters, such as viral replication or the expression of a reporter gene, rather than on measuring entry per se. The lack of assays to easily detect the different steps of entry severely hampers the analysis of this key process in virus infection. Here we describe novel, highly adaptable viral entry assays making use of minimal complementation of the E. coli ß-galactosidase in mammalian cells. Enzyme activity is reconstituted when a small intravirion peptide (α-peptide) is complementing the inactive mutant form ΔM15 of ß-galactosidase. The method allows to dissect and to independently detect binding, internalization, and fusion of viruses during host cell entry. Here we use it to confirm and extend current knowledge on the entry process of two enveloped viruses: vesicular stomatitis virus (VSV) and murine hepatitis coronavirus (MHV).

A protective and safe intranasal RSV vaccine based on a recombinant prefusion-like form of the F protein bound to bacterium-like particles.

Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease in infants and the elderly. Currently, no licensed vaccine against RSV is available. Here we describe the development of a safe and effective intranasal subunit vaccine that is based on recombinant fusion (F) protein bound to the surface of immunostimulatory bacterium-like particles (BLPs) derived from the food-grade bacterium Lactococcus lactis. Different variants of F were analyzed with respect to their conformation and reactivity with neutralizing antibodies, assuming that F proteins mimicking the metastable prefusion form of RSV F expose a more extensive and relevant epitope repertoire than F proteins corresponding to the postfusion structure. Our results indicate that the recombinant soluble ectodomain of RSV F readily adopts a postfusion conformation, generation of which cannot be prevented by C-terminal addition of a trimerization motif, but whose formation is prevented by mutation of the two furin cleavage sites in F. While the putative postfusion form of F is recognized well by the monoclonal antibody Palivizumab, this is much less so for the more potently neutralizing, prefusion-specific antibodies D25 and AM22. Both addition of the trimerization motif and mutation of the furin cleavage sites increased the reactivity of F with D25 and AM22, with the highest reactivity being observed for F proteins in which both these features were combined. Intranasal vaccination of mice or cotton rats with BLPs loaded with this latter prefusion-like F protein (BLP-F), resulted in the potent induction of F-specific immunoglobulins and in significantly decreased virus titers in the lungs upon RSV challenge. Moreover, and in contrast to animals vaccinated with formalin-inactivated RSV, animals that received BLP-F exhibited high levels of F-specific secretory IgA in the nose and RSV-neutralizing antibodies in sera, but did not show symptoms of enhanced disease after challenge with RSV.

Coxsackievirus mutants that can bypass host factor PI4KIIIß and the need for high levels of PI4P lipids for replication.

RNA viruses can rapidly mutate and acquire resistance to drugs that directly target viral enzymes, which poses serious problems in a clinical context. Therefore, there is a growing interest in the development of antiviral drugs that target host factors critical for viral replication, since they are unlikely to mutate in response to therapy. We recently demonstrated that phosphatidylinositol-4-kinase IIIß (PI4KIIIß) and its product phosphatidylinositol-4-phosphate (PI4P) are essential for replication of enteroviruses, a group of medically important RNA viruses including poliovirus (PV), coxsackievirus, rhinovirus, and enterovirus 71. Here, we show that enviroxime and GW5074 decreased PI4P levels at the Golgi complex by directly inhibiting PI4KIIIß. Coxsackievirus mutants resistant to these inhibitors harbor single point mutations in the non-structural protein 3A. These 3A mutations did not confer compound-resistance by restoring the activity of PI4KIIIß in the presence of the compounds. Instead, replication of the mutant viruses no longer depended on PI4KIIIß, since their replication was insensitive to siRNA-mediated depletion of PI4KIIIß. The mutant viruses also did not rely on other isoforms of PI4K. Consistently, no high level of PI4P could be detected at the replication sites induced by the mutant viruses in the presence of the compounds. Collectively, these findings indicate that through specific single point mutations in 3A, CVB3 can bypass an essential host factor and lipid for its propagation, which is a new example of RNA viruses acquiring resistance against antiviral compounds, even when they directly target host factors.

Role of the SEL1L:LC3-I complex as an ERAD tuning receptor in the mammalian ER.

Several regulators of endoplasmic reticulum (ER)-associated degradation (ERAD) have a shorter half-life compared to conventional ER chaperones. At steady state, they are selectively removed from the ER by poorly defined events collectively referred to as ERAD tuning. Here we identify the complex comprising the type-I transmembrane protein SEL1L and the cytosolic protein LC3-I as an ERAD tuning receptor regulating the COPII-independent, vesicle-mediated removal of the lumenal ERAD regulators EDEM1 and OS-9 from the ER. Expression of folding-defective polypeptides enhances the lumenal content of EDEM1 and OS-9 by inhibiting their SEL1L:LC3-I-mediated segregation. This raises ERAD activity in the absence of UPR-induction. The mouse hepatitis virus (MHV) subverts ERAD tuning for replication. Consistently, SEL1L or LC3 silencing impair the MHV life cycle. Collectively, our data provide new molecular information about the ERAD tuning mechanisms that regulate ERAD in mammalian cells at the post translational level and how these mechanisms are hijacked by a pathogen.

Unconventional use of LC3 by coronaviruses through the alleged subversion of the ERAD tuning pathway.

Pathogens of bacterial and viral origin hijack pathways operating in eukaryotic cells in many ways in order to gain access into the host, to establish themselves and to eventually produce their progeny. The detailed molecular characterization of the subversion mechanisms devised by pathogens to infect host cells is crucial to generate targets for therapeutic intervention. Here we review recent data indicating that coronaviruses probably co-opt membranous carriers derived from the endoplasmic reticulum, which contain proteins that regulate disposal of misfolded polypeptides, for their replication. In addition, we also present models describing potential mechanisms that coronaviruses could employ for this hijacking.

Quantitative proteomic identification of host factors involved in the Salmonella typhimurium infection cycle.

To identify host factors involved in Salmonella replication, SILAC-based quantitative proteomics was used to investigate the interactions of Salmonella typhimurium with the secretory pathway in human epithelial cells. Protein profiles of Golgi-enriched fractions isolated from S. typhimurium-infected cells were compared with those of mock-infected cells, revealing significant depletion or enrichment of 105 proteins. Proteins annotated to play a role in membrane traffic were overrepresented among the depleted proteins whereas proteins annotated to the cytoskeleton showed a diverse behavior with some proteins being enriched, others being depleted from the Golgi fraction upon Salmonella infection. To study the functional relevance of identified proteins in the Salmonella infection cycle, small interfering RNA (siRNA) experiments were performed. siRNA-mediated depletion of a selection of affected proteins identified five host factors involved in Salmonella infection. Depletion of peroxiredoxin-6 (PRDX6), isoform ß-4c of integrin ß-4 (ITGB4), isoform 1 of protein lap2 (erbin interacting protein; ERBB2IP), stomatin (STOM) or TBC domain containing protein 10b (TBC1D10B) resulted in increased Salmonella replication. Surprisingly, in addition to the effect on Salmonella replication, depletion of STOM or ITGB4 resulted in a dispersal of intracellular Salmonella microcolonies. It can be concluded that by using SILAC-based quantitative proteomics we were able to identify novel host cell proteins involved in the complex interplay between Salmonella and epithelial cells.

Dissection of the influenza A virus endocytic routes reveals macropinocytosis as an alternative entry pathway.

Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the amiloride derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis.

Identification of host factors involved in coronavirus replication by quantitative proteomics analysis.

In this study, we applied a quantitative proteomic approach, based on SILAC, to investigate the interactions of coronaviruses with the secretory pathway of the host cell, with the aim to identify host factors involved in coronavirus replication. Comparison of the protein profiles of Golgi-enriched fractions of cells that were either mock infected or infected with mouse hepatitis virus revealed the significant depletion or enrichment of 116 proteins. Although ribosomal/nucleic acid binding proteins were enriched in the Golgi-fractions of mouse hepatitis virus-infected cells, proteins annotated to localize to several organelles of the secretory pathway were overrepresented among the proteins that were depleted from these fractions upon infection. We hypothesized that proteins, of which the abundance or distribution is affected by infection, are likely to be involved in the virus life cycle. Indeed, depletion of a small subset of the affected proteins by using small interfering RNAs identified several host factors involved in coronavirus infection. Transfection of small interfering RNAs targeting either C11orf59 or Golgi apparatus glycoprotein 1 resulted in increased virus replication, whereas depletion of vesicle-trafficking protein vesicle-trafficking protein sec22b enhanced the release of infectious progeny virus. Overexpression of these proteins, on the other hand, had a negative effect on virus replication. Overall, our study shows that the SILAC approach is a suitable tool to study host-pathogen interactions and to identify host proteins involved in virus replication.

The proteasome inhibitor Velcade enhances rather than reduces disease in mouse hepatitis coronavirus-infected mice.

Many viruses, including coronaviruses (CoVs), depend on a functional cellular proteasome for efficient infection in vitro. Hence, the proteasome inhibitor Velcade (bortezomib), a clinically approved anticancer drug, shown in an accompanying study (M. Raaben et al., J. Virol. 84:7869-7879, 2010) to strongly inhibit mouse hepatitis CoV (MHV) infection in cultured cells, seemed an attractive candidate for testing its antiviral properties in vivo. Surprisingly, however, the drug did not reduce replication of the virus in mice. Rather, inhibition of the proteasome caused enhanced infection with lethal outcome, calling for caution when using this type of drug during infection.