TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells.

The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2' site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.

Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike.

Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits.

Surface glycoprotein of Borna disease virus mediates virus spread from cell to cell.

Borna disease virus (BDV) is a non-segmented negative-stranded RNA virus that maintains a strictly neurotropic and persistent infection in affected end hosts. The primary target cells for BDV infection are brain cells, e.g. neurons and astrocytes. The exact mechanism of how infection is propagated between these cells and especially the role of the viral glycoprotein (GP) for cell-cell transmission, however, are still incompletely understood. Here, we use different cell culture systems, including rat primary astrocytes and mixed cultures of rat brain cells, to show that BDV primarily spreads through cell-cell contacts. We employ a highly stable and efficient peptidomimetic inhibitor to inhibit the furin-mediated processing of GP and demonstrate that cleaved and fusion-active GP is strictly necessary for the cell-to-cell spread of BDV. Together, our quantitative observations clarify the role of Borna disease virus-glycoprotein for viral dissemination and highlight the regulation of GP expression as a potential mechanism to limit viral spread and maintain persistence. These findings furthermore indicate that targeting host cell proteases might be a promising approach to inhibit viral GP activation and spread of infection.

Influenza virus activating host proteases: Identification, localization and inhibitors as potential therapeutics.

Cellular proteases are reponsible for activation of influenza virus hemagglutinin (HA) in epithelial tissues of the respiratory tract. The trans-Golgi network (TGN) is the main subcellular compartment where HA cleavage occurs during its biosynthesis. The proteolytic HA cleavage is an indispensable prerequisite for the fusion of viral with endosomal membrane and the delivery of the virus genome into the cell. Both, the structure and accessibility of the HA cleavage site determine the responsible host protease(s) for cutting. Most influenza virus strains contain a HA sequence with a single arginine at the cleavage site suitable for processing by the trypsin-like serine proteases human airway trypsin-like protease (HAT) and transmembrane protease serine 2 (TMPRSS2), albeit a minority of viruses possesses HA cleavage site motifs that are processed by other proteases. TMPRSS2-deficient mice demonstrated the relevance of TMPRSS2 for pneumotropism and pathogenicity of H1N1 and H7N9 virus infections. In contrast, H3N2 virus infections are promoted by an additional not yet identified protease. Highly pathogenic avian H5 and H7 viruses are characterized by an enlarged cleavage site loop containing a multibasic amino acid motif, where the eukaryotic subtilases furin or PC5/6 cleave. Their ubiquitous presence in the organism allows a systemic virus infection. Peptidomimetic inhibitors derived from the HA cleavage site inhibit the HA-activating proteases and thus virus propagation.

Peptidomimetic furin inhibitor MI-701 in combination with oseltamivir and ribavirin efficiently blocks propagation of highly pathogenic avian influenza viruses and delays high level oseltamivir resistance in MDCK cells.

Antiviral medication is used for the treatment of severe influenza infections, of which the neuraminidase inhibitors (NAIs) are the most effective drugs, approved so far. Here, we investigated the antiviral efficacy of the peptidomimetic furin inhibitor MI-701 in combination with oseltamivir carboxylate and ribavirin against the infection of highly pathogenic avian influenza viruses (HPAIV) that are activated by the host protease furin. Cell cultures infected with the strains A/Thailand/1(KAN-1)/2004 (H5N1) and A/FPV/Rostock/1934 (H7N1) were treated with each agent alone, or in double and triple combinations. MI-701 alone achieved a concentration-dependent reduction of virus propagation. Double treatment of MI-701 with oseltamivir carboxylate and triple combination with ribavirin showed synergistic inhibition and a pronounced delay of virus propagation. MI-701 resistant mutants were not observed. Emergence of NA mutation H275Y conferring high oseltamivir resistance was significantly delayed in the presence of MI-701. Our data indicate that combination with a potent furin inhibitor significantly enhances the therapeutic efficacy of conventional antivirals drugs against HPAIV infection.

Novel Furin Inhibitors with Potent Anti-infectious Activity.

New peptidomimetic furin inhibitors with unnatural amino acid residues in the P3 position were synthesized. The most potent compound 4-guanidinomethyl-phenylacteyl-Arg-Tle-Arg-4-amidinobenzylamide (MI-1148) inhibits furin with a Ki value of 5.5 pM. The derivatives also strongly inhibit PC1/3, whereas PC2 is less affected. Selected inhibitors were tested in cell culture for antibacterial and antiviral activity against infectious agents known to be dependent on furin activity. A significant protective effect against anthrax and diphtheria toxin was observed in the presence of the furin inhibitors. Furthermore, the spread of the highly pathogenic H5N1 and H7N1 avian influenza viruses and propagation of canine distemper virus was strongly inhibited. Inhibitor MI-1148 was crystallized in complex with human furin. Its N-terminal guanidinomethyl group in the para position of the P5 phenyl ring occupies the same position as that found previously for a structurally related inhibitor containing this substitution in the meta position, thereby maintaining all of the important P5 interactions. Our results confirm that the inhibition of furin is a promising strategy for a short-term treatment of acute infectious diseases.

Taxonomic reorganization of the family Bornaviridae.

Knowledge of bornaviruses has expanded considerably during the last decade. A possible reservoir of mammalian Borna disease virus has been identified, divergent bornaviruses have been detected in birds and reptiles, and endogenous bornavirus-like elements have been discovered in the genomes of vertebrates of several species. Previous sequence comparisons and alignments have indicated that the members of the current family Bornaviridae are phylogenetically diverse and are not adequately classified in the existing bornavirus taxonomy supported by the International Committee on Taxonomy of Viruses (ICTV). We provide an update of these analyses and describe their implications for taxonomy. We propose retaining the family name Bornaviridae and the genus Bornavirus but reorganizing species classification. PAirwise Sequence Comparison (PASC) of bornavirus genomes and Basic Local Alignment Search Tool (BLAST) comparison of genomic and protein sequences, in combination with other already published phylogenetic analyses and known biological characteristics of bornaviruses, indicate that this genus should include at least five species: Mammalian 1 bornavirus (classical Borna disease virus and divergent Borna disease virus isolate No/98), Psittaciform 1 bornavirus (avian/psittacine bornaviruses 1, 2, 3, 4, 7), Passeriform 1 bornavirus (avian/canary bornaviruses C1, C2, C3, LS), Passeriform 2 bornavirus (estrildid finch bornavirus EF), and Waterbird 1 bornavirus (avian bornavirus 062CG). This classification is also in line with biological characteristics of these viruses and their vertebrate hosts. A snake bornavirus, proposed to be named Loveridge's garter snake virus 1, should be classified as a member of an additional species (Elapid 1 bornavirus), unassigned to a genus, in the family Bornaviridae. Avian bornaviruses 5, 6, MALL, and another "reptile bornavirus" ("Gaboon viper virus") should stay unclassified until further information becomes available. Finally, we propose new virus names and abbreviations when necessary to achieve clear differentiation and unique identification.

The hemagglutinin: a determinant of pathogenicity.

The hemagglutinin (HA) is a prime determinant of the pathogenicity of influenza A viruses. It initiates infection by binding to cell surface receptors and by inducing membrane fusion. The fusion capacity of HA depends on cleavage activation by host proteases, and it has long been known that highly pathogenic avian influenza viruses displaying a multibasic cleavage site differ in protease sensitivity from low pathogenic avian and mammalian influenza viruses with a monobasic cleavage site. Evidence is increasing that there are also variations in proteolytic activation among the viruses with a monobasic cleavage site, and several proteases have been identified recently that activate these viruses in a natural setting. Differences in protease sensitivity of HA and in tissue specificity of the enzymes are important determinants for virus tropism in the respiratory tract and for systemic spread of infection. Protease inhibitors that interfere with cleavage activation have the potential to be used for antiviral therapy and attenuated viruses have been generated by mutation of the cleavage site that can be used for the development of inactivated and live vaccines. It has long been known that human and avian influenza viruses differ in their specificity for sialic acid-containing cell receptors, and it is now clear that human tissues contain also receptors for avian viruses. Differences in receptor-binding specificity of seasonal and zoonotic viruses and differential expression of receptors for these viruses in the human respiratory tract account, at least partially, for the severity of disease. Receptor binding and fusion activation are modulated by HA glycosylation, and interaction of the glycans of HA with cellular lectins also affects virus infectivity. Interestingly, some of the mechanisms underlying pathogenicity are determinants of host range and transmissibility, as well.

TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice.

UNLABELLED: Cleavage of the hemagglutinin (HA) by host proteases is essential for the infectivity of influenza viruses. Here, we analyzed the role of the serine protease TMPRSS2, which activates HA in the human respiratory tract, in pathogenesis in a mouse model. Replication of the human H7N9 isolate A/Anhui/1/13 and of human H1N1 and H3N2 viruses was compared in TMPRSS2 knockout (TMPRSS2(-/-)) and wild-type (WT) mice. Knockout of TMPRSS2 expression inhibited H7N9 influenza virus replication in explants of murine tracheas, bronchi, and lungs. H1N1 virus replication was also strongly suppressed in airway explants of TMPRSS2(-/-) mice, while H3N2 virus replication was only marginally affected. H7N9 and H1N1 viruses were apathogenic in TMPRSS2(-/-) mice, whereas WT mice developed severe disease with mortality rates of 100% and 20%, respectively. In contrast, all H3N2 infected TMPRSS2(-/-) and WT mice succumbed to lethal infection. Cleavage analysis showed that H7 and H1 are efficiently activated by TMPRSS2, whereas H3 is less susceptible to the protease. Our data demonstrate that TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 and H1N1 influenza virus in mice. In contrast, replication of H3N2 virus appears to depend on another, not yet identified protease, supporting the concept that human influenza viruses differ in protease specificity. IMPORTANCE: Cleavage of the hemagglutinin (HA) by host proteases is essential for the infectivity of influenza virus, but little is known about its relevance for pathogenesis in mammals. Here, we show that knockout mice that do not express the HA-activating protease TMPRSS2 are resistant to pulmonary disease with lethal outcome when infected with influenza A viruses of subtypes H7N9 and H1N1, whereas they are not protected from lethal H3N2 virus infection. These findings demonstrate that human influenza viruses differ in protease specificity, and that expression of the appropriate protease in respiratory tissues is essential for pneumotropism and pathogenicity. Our observations also demonstrate that HA-activating proteases and in particular TMPRSS2 are promising targets for influenza therapy.

Activation of influenza A viruses by host proteases from swine airway epithelium.

Pigs are important natural hosts of influenza A viruses, and due to their susceptibility to swine, avian, and human viruses, they may serve as intermediate hosts supporting adaptation and genetic reassortment. Cleavage of the influenza virus surface glycoprotein hemagglutinin (HA) by host cell proteases is essential for viral infectivity. Most influenza viruses, including human and swine viruses, are activated at a monobasic HA cleavage site, and we previously identified TMPRSS2 and HAT to be relevant proteases present in human airways. We investigated the proteolytic activation of influenza viruses in primary porcine tracheal and bronchial epithelial cells (PTEC and PBEC, respectively). Human H1N1 and H3N2 viruses replicated efficiently in PTECs and PBECs, and viruses containing cleaved HA were released from infected cells. Moreover, the cells supported the proteolytic activation of HA at the stage of entry. We found that swine proteases homologous to TMPRSS2 and HAT, designated swTMPRSS2 and swAT, respectively, were expressed in several parts of the porcine respiratory tract. Both proteases cloned from primary PBECs were shown to activate HA with a monobasic cleavage site upon coexpression and support multicycle replication of influenza viruses. swAT was predominantly localized at the plasma membrane, where it was present as an active protease that mediated activation of incoming virus. In contrast, swTMPRSS2 accumulated in the trans-Golgi network, suggesting that it cleaves HA in this compartment. In conclusion, our data show that HA activation in porcine airways may occur by similar proteases and at similar stages of the viral life cycle as in human airways.

Activation of influenza viruses by proteases from host cells and bacteria in the human airway epithelium.

Influenza is an acute infection of the respiratory tract, which affects each year millions of people. Influenza virus infection is initiated by the surface glycoprotein hemagglutinin (HA) through receptor binding and fusion of viral and endosomal membranes. HA is synthesized as a precursor protein and requires cleavage by host cell proteases to gain its fusion capacity. Although cleavage of HA is crucial for virus infectivity, little was known about relevant proteases in the human airways for a long time. Recent progress in the identification and characterization of HA-activating host cell proteases has been considerable however and supports the idea of targeting HA cleavage as a novel approach for influenza treatment. Interestingly, certain bacteria have been demonstrated to support HA activation either by secreting proteases that cleave HA or due to activation of cellular proteases and thereby may contribute to virus spread and enhanced pathogenicity. In this review, we give an overview on activation of influenza viruses by proteases from host cells and bacteria with the main focus on recent progress on HA cleavage by proteases HAT and TMPRSS2 in the human airway epithelium. In addition, we outline investigations of HA-activating proteases as potential drug targets for influenza treatment.

The role of oligomerization for the biological functions of the arenavirus nucleoprotein.

The Lassa virus nucleoprotein (NP) is a multifunctional protein that plays an essential role in many aspects of the viral life cycle, including RNA encapsidation, viral transcription and replication, recruitment of ribonucleoprotein complexes to viral budding sites, and inhibition of the host cell interferon response. While it is known that NP is capable of forming oligomers, both the oligomeric state of NP in mammalian cells and the significance of NP oligomerization for its various functions remain unclear. Here, we demonstrate that Lassa virus NP solely forms trimers upon expression in mammalian cells. Using a minigenome assay we show that mutants that are not able to form stable trimers are no longer functional during transcription and/or replication of the minigenome, indicating that NP trimerization is essential for transcription and/or replication of the viral genome. However, mutations leading to destabilization of the NP trimer did not impact the incorporation of NP into virus-like particles or its ability to suppress interferon-induced gene expression, two important functions of arenavirus NP.

Identification of the first synthetic inhibitors of the type II transmembrane serine protease TMPRSS2 suitable for inhibition of influenza virus activation.

TMPRSS2 (transmembrane serine proteinase 2) is a multidomain type II transmembrane serine protease that cleaves the surface glycoprotein HA (haemagglutinin) of influenza viruses with a monobasic cleavage site, which is a prerequisite for virus fusion and propagation. Furthermore, it activates the fusion protein F of the human metapneumovirus and the spike protein S of the SARS-CoV (severe acute respiratory syndrome coronavirus). Increased TMPRSS2 expression was also described in several tumour entities. Therefore TMPRSS2 emerged as a potential target for drug design. The catalytic domain of TMPRSS2 was expressed in Escherichia coli and used for an inhibitor screen with previously synthesized inhibitors of various trypsin-like serine proteases. Two inhibitor types were identified which inhibit TMPRSS2 in the nanomolar range. The first series comprises substrate analogue inhibitors containing a 4-amidinobenzylamide moiety at the P1 position, whereby some of these analogues possess inhibition constants of approximately 20 nM. An improved potency was found for a second type derived from sulfonylated 3-amindinophenylalanylamide derivatives. The most potent derivative of this series inhibits TMPRSS2 with a K(i) value of 0.9 nM and showed an efficient blockage of influenza virus propagation in human airway epithelial cells. On the basis of the inhibitor studies, a series of new fluorogenic substrates containing a D-arginine residue at the P3 position was synthesized, some of them were efficiently cleaved by TMPRSS2.

The microtubule motor protein KIF13A is involved in intracellular trafficking of the Lassa virus matrix protein Z.

The small matrix protein Z of arenaviruses has been identified as the main driving force to promote viral particle production at the plasma membrane. Although multiple functions of Z in the arenaviral life cycle have been uncovered, the mechanism of intracellular transport of Z to the site of virus budding is poorly understood and cellular motor proteins that mediate Z trafficking remain to be identified. In the present study, we report that the Z protein of the Old World arenavirus Lassa virus (LASV) interacts with the kinesin family member 13A (KIF13A), a plus-end-directed microtubule-dependent motor protein. Plasmid-driven overexpression of KIF13A results in relocalization of Z to the cell periphery, while functional blockage of endogenous KIF13A by overexpression of a dominant-negative mutant or KIF13A-specific siRNA causes a perinuclearaccumulation and decreased production of both Z-induced virus-like particles and infectious LASV. The interaction of KIF13A with Z proteins from both Old and New World arenaviruses suggests a conserved intracellular transport mechanism. In contrast, the intracellular distribution of the matrix proteins of prototypic members of the paramyxo- and rhabdovirus family is independent of KIF13A. In summary, our studies identify for the first time a molecular motor protein as a critical mediator for intracellular microtubule-dependent transport of arenavirus matrix proteins.

Matriptase, HAT, and TMPRSS2 activate the hemagglutinin of H9N2 influenza A viruses.

Influenza A viruses of the subtype H9N2 circulate worldwide and have become highly prevalent in poultry in many countries. Moreover, they are occasionally transmitted to humans, raising concern about their pandemic potential. Influenza virus infectivity requires cleavage of the surface glycoprotein hemagglutinin (HA) at a distinct cleavage site by host cell proteases. H9N2 viruses vary remarkably in the amino acid sequence at the cleavage site, and many isolates from Asia and the Middle East possess the multibasic motifs R-S-S-R and R-S-R-R, but are not activated by furin. Here, we investigated proteolytic activation of the early H9N2 isolate A/turkey/Wisconsin/1/66 (H9-Wisc) and two recent Asian isolates, A/quail/Shantou/782/00 (H9-782) and A/quail/Shantou/2061/00 (H9-2061), containing mono-, di-, and tribasic HA cleavage sites, respectively. All H9N2 isolates were activated by human proteases TMPRSS2 (transmembrane protease, serine S1 member 2) and HAT (human airway trypsin-like protease). Interestingly, H9-782 and H9-2061 were also activated by matriptase, a protease widely expressed in most epithelia with high expression levels in the kidney. Nephrotropism of H9N2 viruses has been observed in chickens, and here we found that H9-782 and H9-2061 were proteolytically activated in canine kidney (MDCK-II) and chicken embryo kidney (CEK) cells, whereas H9-Wisc was not. Virus activation was inhibited by peptide-mimetic inhibitors of matriptase, strongly suggesting that matriptase is responsible for HA cleavage in these kidney cells. Our data demonstrate that H9N2 viruses with R-S-S-R or R-S-R-R cleavage sites are activated by matriptase in addition to HAT and TMPRSS2 and, therefore, can be activated in a wide range of tissues what may affect virus spread, tissue tropism and pathogenicity.

Hemagglutinin activating host cell proteases provide promising drug targets for the treatment of influenza A and B virus infections.

Cleavage of the influenza virus hemagglutinin (HA) by host cell proteases is crucial for infectivity and spread of the virus. Some years ago, we identified TMPRSS2 and HAT from human airways as activating proteases of influenza A viruses containing a monobasic HA cleavage site. Therefore, these proteases are considered as potential drug targets. In this report, first we show that HA of influenza B virus is activated by TMPRSS2 and HAT, too. We further demonstrate that benzylsulfonyl-d-arginine-proline-4-amidinobenzylamide (BAPA), which is a potent inhibitor of HAT and TMPRSS2, efficiently suppresses virus propagation in TMPRSS2-expressing human airway epithelial cells by inhibition of HA cleavage. BAPA treatment reduced virus titers of different influenza A and B viruses more than 1000-fold and delayed virus propagation by 24-48 h at non-cytotoxic concentrations. A combination of BAPA with the neuraminidase (NA) inhibitor oseltamivir carboxylate efficiently blocked influenza virus replication in airway epithelial cells at remarkable lower concentrations for each compound than treatment with either inhibitor alone. Our studies provide a novel and potent approach for influenza chemotherapy that should be considered for influenza treatment.

Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases.

Optimization of our previously described peptidomimetic furin inhibitors was performed and yielded several analogs with a significantly improved activity. The most potent compounds containing an N-terminal 4- or 3-(guanidinomethyl)phenylacetyl residue inhibit furin with K(i) values of 16 and 8 pM, respectively. These analogs inhibit other proprotein convertases, such as PC1/3, PC4, PACE4, and PC5/6, with similar potency, whereas PC2, PC7, and trypsin-like serine proteases are poorly affected. Incubation of selected compounds with Madin-Darby canine kidney cells over a period of 96 h revealed that they exhibit great stability, making them suitable candidates for further studies in cell culture. Two of the most potent derivatives were used to inhibit the hemagglutinin cleavage and viral propagation of a highly pathogenic avian H7N1 influenza virus strain. The treatment with inhibitor 24 (4-(guanidinomethyl)phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide) resulted in significantly delayed virus propagation compared with an inhibitor-free control. The same analog was also effective in inhibiting Shiga toxin activation in HEp-2 cells. This antiviral effect, as well as the protective effect against a bacterial toxin, suggests that inhibitors of furin or furin-like proprotein convertases could represent promising lead structures for future drug development, in particular for the treatment of infectious diseases.

Isolation of influenza viruses in MDCK 33016PF cells and clearance of contaminating respiratory viruses.

This paper summarizes results obtained by multiplex PCR screening of human clinical samples for respiratory viruses and corresponding data obtained after passaging of virus-positive samples in MDCK 33016PF cells. Using the ResPlexII v2.0 (Qiagen) multiplex PCR, 393 positive results were obtained in 468 clinical samples collected during an influenza season in Germany. The overall distribution of positive results was influenza A 42.0%, influenza B 38.7%, adenovirus 1.5%, bocavirus 0.5%, coronavirus 3.3%, enterovirus 5.6%, metapneumovirus 1.0%, parainfluenza virus 0.8%, rhinovirus 4.1%, and respiratory syncytial virus (RSV) 2.5%. Double infections of influenza virus together with another virus were found for adenovirus B and E, bocavirus, coronavirus, enterovirus and for rhinovirus. These other viruses were rapidly lost upon passages in MDCK 33016PF cells and under conditions as applied to influenza virus passaging. Clinical samples, in which no influenza virus but other viruses were found, were also subject to passages in MDCK 33016PF cells. Using lower inoculum dilutions than those normally applied for preparations containing influenza virus (total dilution of the original sample of ∼10(4)), the positive results for the different viruses turned negative already after 2 or 3 passages in MDCK 33016PF cells. These results demonstrate that, under practical conditions as applied to grow influenza viruses, contaminating viruses can be effectively removed by passages in MDCK cells. In combination with their superior isolation efficiency, MDCK cells appear highly suitable to be used as an alternative to embryonated eggs to isolate and propagate influenza vaccine candidate viruses.

Development of substrate analogue inhibitors for the human airway trypsin-like protease HAT.

A series of substrate analogue inhibitors of the serine protease HAT, containing a 4-amidinobenzylamide moiety as the P1 residue, was prepared. The most potent compounds possess a basic amino acid in the d-configuration as P3 residue. Whereas inhibitor 4 (K(i) 13 nM) containing proline as the P2 residue completely lacks selectivity, incorporation of norvaline leads to a potent inhibitor (15, K(i) 15 nM) with improved selectivity for HAT in comparison to the coagulation proteases thrombin and factor Xa or the fibrinolytic plasmin. Selected inhibitors were able to suppress influenza virus replication in a HAT-expressing MDCK cell model.

Molecular mechanisms of interspecies transmission and pathogenicity of influenza viruses: Lessons from the 2009 pandemic.

The emergence of the 2009 H1N1 virus pandemic was unexpected, since it had been predicted that the next pandemic would be caused by subtype H5N1. We also had to learn that a pandemic does not necessarily require the introduction of a new virus subtype into the human population, but that it may result from antigenic shift within the same subtype. The new variant was derived from human and animal viruses by genetic reassortment in the pig, supporting the concept that this animal is the mixing vessel for the generation of new human influenza viruses. Although it is generally believed that the 2009 outbreak was mild, there have been severe cases particularly among the young and the middle-aged. Pathogenicity and host range are determined to a large extent by the polymerase, the haemagglutinin and the NS1 protein of influenza A viruses. There is evidence that mutations of these proteins may change the pathogenicity of the new virus.