Proprotein-processing endoproteases PC6 and furin both activate hemagglutinin of virulent avian influenza viruses.

Among the proprotein-processing subtilisin-related endoproteases, furin has been a leading candidate for the enzyme that activates the hemagglutinin (HA) of virulent avian influenza viruses. In the present study, we examined the cleavage activity of two other recently isolated ubiquitous subtilisin-related proteases, PACE4 and PC6, using wild-type HA of A/turkey/Ireland/1378/83 (H5N8) and a series of its mutant HAs. Vaccinia virus-expressed wild-type HA was not cleaved in human colon adenocarcinoma LoVo cells, which lack active furin. This processing defect was corrected by the expression of furin and PC6 but not of PACE4 and a control wild-type vaccinia virus. PC6 showed a sequence specificity similar to that with the endogenous proteases in cultured cells. When LoVo cells were infected with a virulent avian virus, A/turkey/Ontario/7732/66 (H5N9), only noninfectious virions were produced because of the lack of HA cleavage. However, when the cells were coinfected with vaccinia virus that expressed either furin or PC6, the avian virus underwent multiple cycles of replication, indicating that both furin and PC6 specifically cleave the virulent virus HA at the authentic site. These data suggest that PC6, as well as furin, can activate virulent avian influenza viruses in vivo, implying the presence of multiple HA cleavage enzymes in animals.     

Molecular determinants within the surface proteins involved in the pathogenicity of H5N1 influenza viruses in chickens

Although it is established that the cleavage site and glycosylation patterns in the hemagglutinin (HA) play important roles in determining the pathogenicity of H5 avian influenza viruses, some viruses exist that are not highly pathogenic despite possessing the known characteristics of high pathogenicity (i.e., their HA contains multiple basic amino acids at the cleavage site and has glycosylation patterns similar to that of the highly pathogenic H5 viruses). Currently little is known about the H5N1 viruses that fall into this intermediate category of pathogenicity. We have identified strains of H5N1 avian influenza viruses that have markers typical of high pathogenicity but distinctly differ in their ability to cause disease and death in chickens. By analyzing viruses constructed by reverse-genetic methods and containing recombinant HAs, we established that amino acids 97, 108, 126, 138, 212, and 217 of HA, in addition to those within the cleavage site, affect pathogenicity. Further investigation revealed that an additional glycosylation site within the neuraminidase (NA) protein globular head contributed to the high virulence of the H5N1 virus. Our findings are in agreement with previous observations that suggest that the activities of the HA and NA proteins are functionally linked.     

Source of high pathogenicity of an avian influenza virus H5N1: why H5 is better cleaved by furin

The origin of the high pathogenicity of an emerging avian influenza H5N1 due to the -RRRKK- insertion at the cleavage loop of the hemagglutinin H5, was studied using the molecular dynamics technique, in comparison with those of the noninserted H5 and H3 bound to the furin (FR) active site. The cleavage loop of the highly pathogenic H5 was found to bind strongly to the FR cavity, serving as a conformation suitable for the proteolytic reaction. With this configuration, the appropriate interatomic distances were found for all three reaction centers of the enzyme-substrate complex: the arrangement of the catalytic triad, attachment of the catalytic Ser³⁶⁸ to the reactive S1-Arg, and formation of the oxyanion hole. Experimentally, the -RRRKK- insertion was also found to increase in cleavage of hemagglutinin by FR. The simulated data provide a clear answer to the question of why inserted H5 is better cleaved by FR than the other subtypes, explaining the high pathogenicity of avian influenza H5N1.     

A single amino acid at the hemagglutinin cleavage site contributes to the pathogenicity and neurovirulence of H5N1 influenza virus in mice

H5 influenza viruses containing a motif of multiple basic amino acids at the hemagglutinin (HA) cleavage site (HACS) are highly pathogenic in chicken but display different virulence phenotypes in mammals. Previous studies have shown that multiple basic amino acids of H5N1 influenza virus are a prerequisite for lethality in mice. However, it remains unclear which specific residue at the cleavage site affects the pathogenicity of H5N1 in mammals. A comprehensive genetic analysis of the H5N1 HACS showed that residues at P6 (position 325, by H3 numbering) were the most polymorphic, including serine (S), arginine (R), deletion (*), glycine (G), and isoleucine (I). To determine whether a single residue at P6 could affect virulence, we introduced different mutations at P6 of an avirulent clade 7 H5N1 strain, rg325G, by reverse genetics. Among the recombinant viruses, the rg325S virus showed the highest cleavage efficiency in vitro. All these viruses were highly pathogenic in chicken but exhibited different virulences in mice. The rg325S virus exhibited the highest pathogenicity in terms of unrestricted organ tropism and neurovirulence. Remarkably, the HA-325S substitution dramatically increased the pathogenicity of H5N1 viruses of other clades, including clades 2.2, 2.3.2, and 2.3.4, indicating that this residue impacts genetically divergent H5N1 viruses. An analysis of predicted structures containing these mutations showed that the cleavage site loop with 325S was the most exposed, which might be responsible for the efficient cleavage and high virulence. Our results demonstrate that an amino acid substitution at the P6 cleavage site alone could modulate the virulence of H5N1 in mice.     

Sequence specificity of furin, a proprotein-processing endoprotease, for the hemagglutinin of a virulent avian influenza virus.

The virulence of avian influenza viruses correlates with the sensitivity of their hemagglutinin (HA) to cellular proteases. Furin, a proprotein-processing subtilisin-related endoprotease, is a leading candidate for the enzyme that cleaves the HA of virulent avian viruses. We therefore compared the specificity of furin with those of proteases in a variety of cultured cells and in a rat Golgi fraction, using the HA cleavage mutants of a virulent avian influenza virus, A/Turkey/Ireland/1378/85 (H5N8). The results indicated similar sequence specificities among the endoproteases when purified furin was used. In experiments with the vaccinia virus expression system, overexpressed furin cleaved mutant HAs that were not recognized by the endogenous proteases, resulting in an apparent broader specificity of furin. These findings authenticate the proposed role of furin as an HA-activating protease in vivo and caution against the use of expression vectors to study protease sequence specificity.     

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.     

Neuraminidase-associated plasminogen recruitment enables systemic spread of natural avian Influenza viruses H3N1

Repeated outbreaks due to H3N1 low pathogenicity avian influenza viruses (LPAIV) in Belgium were associated with unusually high mortality in chicken in 2019. Those events caused considerable economic losses and prompted restriction measures normally implemented for eradicating high pathogenicity avian influenza viruses (HPAIV). Initial pathology investigations and infection studies suggested this virus to be able to replicate systemically, being very atypical for H3 LPAIV. Here, we investigate the pathogenesis of this H3N1 virus and propose a mechanism explaining its unusual systemic replication capability. By intravenous and intracerebral inoculation in chicken, we demonstrate systemic spread of this virus, extending to the central nervous system. Endoproteolytic viral hemagglutinin (HA) protein activation by either tissue-restricted serine peptidases or ubiquitous subtilisin-like proteases is the functional hallmark distinguishing (H5 or H7) LPAIV from HPAIV. However, luciferase reporter assays show that HA cleavage in case of the H3N1 strain in contrast to the HPAIV is not processed by intracellular proteases. Yet the H3N1 virus replicates efficiently in cell culture without trypsin, unlike LPAIVs. Moreover, this trypsin-independent virus replication is inhibited by 6-aminohexanoic acid, a plasmin inhibitor. Correspondingly, in silico analysis indicates that plasminogen is recruitable by the viral neuraminidase for proteolytic activation due to the loss of a strongly conserved N-glycosylation site at position 130. This mutation was shown responsible for plasminogen recruitment and neurovirulence of the mouse brain-passaged laboratory strain A/WSN/33 (H1N1). In conclusion, our findings provide good evidence in natural chicken strains for N1 neuraminidase-operated recruitment of plasminogen, enabling systemic replication leading to an unusual high pathogenicity phenotype. Such a gain of function in naturally occurring AIVs representing an established human influenza HA-subtype raises concerns over potential zoonotic threats.     

Comment on “Cleavage mechanism of the H5N1 hemagglutinin by trypsin and furin” [Amino Acids 2008, January 31, Doi: 10.1007/s00726-007-0611-3]

Recently, Guo et al. have reported structural as well as the binding energy data of the particular interactions between the cleavage sites of hemagglutinin and serine proteases, trypsin and furin, using molecular docking approach. Due to a wrong assignment of protonation state on the histidine, one of the catalytic triad in the active site of both enzymes, their docking results are contradictory with the fundamental principle and previous theoretical studies of the known cleavage mechanism in serine proteases.     

Modification of the trypsin-dependent cleavage activation site of the human metapneumovirus fusion protein to be trypsin independent does not increase replication or spread in rodents or nonhuman primates

The contribution of cleavage activation of the fusion F protein of human metapneumovirus (HMPV) to replication and pathogenicity in rodents and nonhuman primates was investigated. Recombinant HMPVs were generated in which the naturally occurring trypsin-dependent cleavage sequence (R-Q-S-R downward arrow) was replaced by each of three sequences whose cleavage in vitro does not depend upon added trypsin. Two of these were multibasic sequences derived from avian metapneumovirus type A (R-R-R-R) or type C (R-K-A-R), with the former containing the consensus furin protease cleavage motif (R-X-R/K-R downward arrow). The third one (R-Q-P-R) was derived from a recently described trypsin independent HMPV isolate (J. H. Schickli, J. Kaur, N. Ulbrandt, R. R. Spaete, and R. S. Tang, J. Virol. 79:10678-10689, 2005). To preclude the possibility of conferring even greater virulence to this significant human pathogen, the modifications were done in an HMPV variant that was attenuated by the deletion of two of the three envelope glycoproteins, SH and G. Each of the introduced cleavage sequences conferred trypsin independent F cleavage and growth to HMPV in vitro. However, they differed in the efficiency of trypsin independent growth and plaque formation in vitro: R-R-R-R > R-K-A-R > R-Q-P-R > R-Q-S-R. The R-R-R-R mutant was the only one whose growth in vitro was not augmented by added trypsin, indicative of highly efficient trypsin independent cleavage. When inoculated intranasally into hamsters, there was essentially no difference in the magnitude of replication in the upper or lower respiratory tract between the mutants, and virus was not detected in organs outside of the respiratory tract. Evaluation of the most cleavage-efficient mutant, R-R-R-R, in African green monkeys showed that there was no detectable change in the magnitude of replication in the upper and lower respiratory tract or in immunogenicity and protective efficacy against HMPV challenge. These results suggest that cleavage activation is not a major determinant of HMPV virulence.     

Cleavage Activation of the Human-Adapted Influenza Virus Subtypes by Matriptase Reveals both Subtype and Strain Specificities

Cleavage activation of the hemagglutinin (HA) precursor is an essential step in the influenza virus replication cycle that is driven by host cell proteases. HA cleavage activation is required for virus-endosome membrane fusion and the subsequent release of the influenza virus genome into the cytoplasm. Previous studies have determined that HA cleavage is most likely driven by either membrane-bound or extracellular trypsin-like proteases that reside in the respiratory tract. However, there is still uncertainty regarding which proteases are critical for HA cleavage in vivo. Therefore, further investigation of HA cleavage activation is needed in order to gain insight into the critical proteases involved. Matriptase is a member of the type II transmembrane serine protease family that is highly expressed in a membrane-bound form throughout the respiratory tract. One feature of matriptase is that, once activated, the catalytic domain is secreted into the extracellular space and so serves as a functional extracellular protease. In this study, we have determined that the secreted, catalytic domain of matriptase has the ability to cleave and activate HA from the influenza virus H1 subtype but not the H2 and H3 subtypes. Furthermore, matriptase selectively cleaved the HA of particular strains within the H1 subtype, revealing both subtype and H1 strain specificity. Matriptase was also found to activate thrombolytic zymogens that have been shown to cleave and activate the influenza virus HA. Our data demonstrate that matriptase has the ability to cleave HA directly or indirectly by activating HA-cleaving zymogens.     

Cleavage of influenza a virus hemagglutinin in human respiratory epithelium is cell associated and sensitive to exogenous antiproteases

Proteolytic cleavage of the hemagglutinin (HA) of human influenza viruses A/Aichi/2/68 (H3N2) and A/WSN/34 (H1N1) from HA0 to HA1/HA2 was studied in primary human adenoid epithelial cells (HAEC). HAEC contain a mixture of ciliated and nonciliated secretory cells and mimic the epithelium membrane of the human respiratory tract. Pulse-chase labeling with [(35)S]methionine and Western blot analysis with anti-HA antibodies of cellular and virion polypeptides showed that HAEC cleaved newly synthesized HA0 to HA1/HA2 ("cleavage from within") and significant amounts of cleaved HA accumulated within cells. It was also shown that HAEC was able to cleave HA0 of incoming virions ("cleavage from without"), whereas the HA0 of nonabsorbed virions free in extracellular fluid were not cleaved, supporting the conclusion that HA0 cleavage in HAEC is cell associated. Low-molecular-weight inhibitors of serine proteases, aprotinin and leupeptin, when added to influenza virus-infected HAEC suppressed HA0 cleavage and reduced the amount of cleaved HA1/HA2 both in cells and in progeny virions and thus diminished the infectivity of the virus. In contrast, the addition of fetal bovine serum, containing a number of high-molecular-weight antiproteases that compete for proteases in the extracellular environment, did not inhibit influenza virus growth in HAEC. These data suggest that in human respiratory epithelium the cleavage of influenza virus HA containing a single arginine in the proteolytic site (i) is a cell-associated process accomplished by serine-type protease(s) and (ii) is sensitive to low-molecular-weight exogenous inhibitors of serine proteases.     

Substrate cleavage analysis of furin and related proprotein convertases. A comparative study

We present the data and the technology, a combination of which allows us to determine the identity of proprotein convertases (PCs) related to the processing of specific protein targets including viral and bacterial pathogens. Our results, which support and extend the data of other laboratories, are required for the design of effective inhibitors of PCs because, in general, an inhibitor design starts with a specific substrate. Seven proteinases of the human PC family cleave the multibasic motifs R-X-(R/K/X)-R downward arrow and, as a result, transform proproteins, including those from pathogens, into biologically active proteins and peptides. The precise cleavage preferences of PCs have not been known in sufficient detail; hence we were unable to determine the relative importance of the individual PCs in infectious diseases, thus making the design of specific inhibitors exceedingly difficult. To determine the cleavage preferences of PCs in more detail, we evaluated the relative efficiency of furin, PC2, PC4, PC5/6, PC7, and PACE4 in cleaving over 100 decapeptide sequences representing the R-X-(R/K/X)-R downward arrow motifs of human, bacterial, and viral proteins. Our computer analysis of the data and the follow-on cleavage analysis of the selected full-length proteins corroborated our initial results thus allowing us to determine the cleavage preferences of the PCs and to suggest which PCs are promising drug targets in infectious diseases. Our results also suggest that pathogens, including anthrax PA83 and the avian influenza A H5N1 (bird flu) hemagglutinin precursor, evolved to be as sensitive to PC proteolysis as the most sensitive normal human proteins.     

Proteolytic activation of influenza viruses by serine proteases TMPRSS2 and HAT from human airway epithelium

Host cell proteases that cleave the hemagglutinin (HA) of influenza viruses in the human respiratory tract are still not identified. Here we cloned two human type II transmembrane serine proteases with known airway localization, TMPRSS2 and HAT, into mammalian expression vector. Cotransfection of mammalian cells with plasmids encoding HA and either protease resulted in HA cleavage in situ. Transient expression of either protease in MDCK cells enabled multicycle replication of influenza viruses in these cells in the absence of exogenous trypsin. These data suggest that TMPRSS2 and HAT are candidates for proteolytic activation of influenza viruses in vivo.     

Modifications to the Hemagglutinin Cleavage Site Control the Virulence of a Neurotropic H1N1 Influenza Virus

A key determinant of influenza virus pathogenesis is mutation in the proteolytic cleavage site of the hemagglutinin (HA). Typically, low-pathogenicity forms of influenza virus are cleaved by trypsin-like proteases, whereas highly pathogenic forms are cleaved by different proteases (e.g., furin). Influenza virus A/WSN/33 (WSN) is a well-studied H1N1 strain that is trypsin independent in vitro and has the ability to replicate in mouse brain. Previous studies have indicated that mutations in the neuraminidase (NA) gene allow the recruitment of an alternate protease (plasminogen/plasmin) for HA activation. In this study we have identified an additional mutation in the P2 position of the WSN HA cleavage site (S328Y) that appears to control virus spread in a plasmin-dependent manner. We reconstructed recombinant WSN viruses containing tyrosine (Y), phenylalanine (F), or serine (S) in the P2 position of the cleavage site. The Y328 and F328 viruses allowed plaque formation in the absence of trypsin, whereas the S328 virus was unable to form plaques under these conditions. In mice, Y328 and F328 viruses were able to efficiently spread following intracranial inoculation; in contrast, the S328 virus showed only limited infection of mouse brain. Following intranasal inoculation, all viruses could replicate efficiently, but with Y328 and F328 viruses showing a limited growth defect. We also show that wild-type HA (Y328) was more efficiently cleaved by plasmin than S328 HA. Our studies form the foundation for a more complete understanding of the molecular determinants of influenza virus pathogenesis and the role of the plasminogen/plasmin system in activating HA.For all viruses, the infectious cycle begins with the penetration of the host cell (27). Enveloped viruses penetrate cells via a membrane fusion event mediated by a spike protein present in the virus envelope, with fusion triggered by conformational changes in the spike protein following exposure to low pH and/or receptor (45). In the case of influenza virus, the viral spike protein hemagglutinin (HA) mediates both receptor binding and membrane fusion (46). Many viral fusion proteins are activated following cleavage by host cell proteases (9, 20, 21), and this has been most extensively documented for influenza viruses, where cleavage is directly related to exposure of the fusion peptide and fusion activation (38). For proteases, a general nomenclature for the cleavage site positions of the substrate has been designated, with cleavage occurring between P1 and P1′ and with the position numbers increasing in the N-terminal direction relative to the cleaved peptide bond (P2, P3, P4, etc.). Low-pathogenicity influenza virus strains contain an HA cleavage site with a single arginine residue at the P1 position and are thus described as having monobasic cleavage sites. These viruses can utilize trypsin (or other trypsin-like serine proteases) for activation, with the tissue distribution of the activating protease typically restricting infection to the respiratory and/or intestinal organs. The presence of a polybasic cleavage site is critical for the systemic spread and increased virulence associated with highly pathogenic avian influenza (HPAI) viruses (33). In the case of HPAI viruses such as H5N1 and H7N7, it is well established that mutations in the region of the HA cleavage site lead to an insertion of several arginine or lysine residues in addition to the P1 arginine (specifically in the P2 to P6 cleavage site positions) that can be recognized by furin—an intracellular serine protease found in many cell types—allowing a widening of the cell tropism of the virus (18).Influenza virus is currently of major biomedical interest, both due to annual morbidity and the threat of new pandemic viruses. Influenza viruses exist as many different subtypes (H1 to H16), with H1 and H3 viruses currently infecting humans (10, 30). Normally, H1 viruses are considered to have low pathogenicity and have a monobasic cleavage site. However, two H1 isolates A/WSN/33 (WSN) and A/NWS/33 (NWS) have been selected to propagate in mouse brain and are thus considered to be highly pathogenic, neurovirulent viruses in mice (39, 40). The A/WSN/33 virus in particular has been used extensively for studies on influenza virus replication and pathogenesis, in part because this virus forms plaques in the absence of trypsin and serves as a model of highly pathogenic influenza virus. The HA of the A/WSN/33 virus was originally shown to be cleaved by plasmin, following activation of serum plasminogen in MDBK cells (22). The virulence properties of A/WSN/33 were subsequently linked to the neuraminidase (NA) gene (37) and the absence of a glycosylation site at position 130 of NA (25). In addition, the presence of a C-terminal lysine on NA was shown to be critical for the virulence properties of A/WSN/33, with the viral NA binding and sequestering of plasminogen on the cell surface, leading to increased cleavage of HA (15, 16). It has also been suggested that the HA of A/WSN/33 can be cleaved by an endosomal serine protease in MDBK cells (7).Based on the recent pandemic status of novel H1N1 viruses and the known importance of the HA cleavage site in viral pathogenicity (30), we assessed the presence of cleavage site changes, in addition to the conventional monobasic/polybasic cleavage sites, in the context of the virulence properties of H1 influenza viruses. We characterize the role of the bulky hydrophobic residues tyrosine and phenylalanine, found in the P2 cleavage positions of only A/WSN/33 and A/NWS/33 HA, respectively, and show that these residues are major virulence determinants for these viruses, allowing efficient use of plasmin for spread in vitro and in vivo.     

Insertion of a Multibasic Cleavage Motif into the Hemagglutinin of a Low-Pathogenic Avian Influenza H6N1 Virus Induces a Highly Pathogenic Phenotype

The highly pathogenic avian influenza (HPAI) virus phenotype is restricted to influenza A viruses of the H5 and H7 hemagglutinin (HA) subtypes. To obtain more information on the apparent subtype-specific nature of the HPAI virus phenotype, a low-pathogenic avian influenza (LPAI) H6N1 virus was generated, containing an HPAI H5 RRRKKR↓G multibasic cleavage site (MBCS) motif in HA (the downward arrow indicates the site of cleavage). This insertion converted the LPAI virus phenotype into an HPAI virus phenotype in vitro and in vivo. The H6N1 virus with an MBCS displayed in vitro characteristics similar to those of HPAI H5 viruses, such as cleavage of HA₀ (the HA protein of influenza A virus initially synthesized as a single polypeptide precursor) and virus replication in the absence of exogenous trypsin. Studies of chickens confirmed the HPAI phenotype of the H6N1 virus with an MBCS, with an intravenous pathogenicity index of 1.4 and systemic virus replication upon intranasal inoculation, the hallmarks of HPAI viruses. This study provides evidence that the subtype-specific nature of the emergence of HPAI viruses is not at the molecular, structural, or functional level, since the introduction of an MBCS resulted in a fully functional virus with an HPAI virus genotype and phenotype.Wild birds represent the natural reservoir of avian influenza A viruses in nature (43). Influenza A viruses are classified on the basis of the hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins. In wild birds throughout the world, influenza A viruses representing 16 HA and 9 NA antigenic subtypes have been found in numerous combinations (also called subtypes, e.g., H1N1, H6N1) (12). Besides classification based on the antigenic properties of HA and NA, avian influenza A viruses can also be classified based on their pathogenic phenotype in chickens. Highly pathogenic avian influenza (HPAI) virus, an acute generalized disease of poultry in which mortality may be as high as 100%, is restricted to subtypes H5 and H7. Other avian influenza A virus subtypes are generally low-pathogenic avian influenza (LPAI) viruses that cause much milder, primarily respiratory disease in poultry, sometimes with loss of egg production (6).The HA protein of influenza A virus is initially synthesized as a single polypeptide precursor (HA₀), which is cleaved into HA₁ and HA₂ subunits by host cell proteases. The mature HA protein mediates binding of the virus to host cells, followed by endocytosis and HA-mediated fusion with endosomal membranes (43). Influenza viruses of subtypes H5 and H7 may become highly pathogenic after introduction into poultry and cause outbreaks of HPAI. The switch from an LPAI phenotype to the HPAI phenotype of these H5 and H7 influenza A viruses is achieved by the introduction of basic amino acid residues into the HA₀ cleavage site by substitution or insertion, resulting in the so-called multibasic cleavage site (MBCS), which facilitates systemic virus replication (4, 5, 14, 44). The cleavage of the HA₀ of LPAI viruses is restricted to trypsin-like proteases which recognize the XXX(R/K)↓G cleavage motif, where the downward arrow indicates the site of cleavage. Replication of these LPAI viruses is therefore restricted to sites in the host where these enzymes are expressed, i.e., the respiratory and intestinal tract (32, 38). The introduction of an RX(R/K)R↓G or R(R/K)XR↓G minimal MBCS motif into the H5 and H7 subtype viruses facilitates the recognition and cleavage of the HA₀ by ubiquitous proprotein convertases, such as furin (20, 32, 41, 45). H5 influenza A viruses with a minimal MBCS motif only have the highly pathogenic phenotype if the masking glycosylation site at position 11 in the HA is replaced by a nonglycosylation site. Otherwise, at least one additional basic amino acid has to be inserted to allow the shift from an LPAI virus phenotype to an HPAI virus phenotype to occur (15, 18, 21, 22, 28). No information is available on the minimal prerequisites of H7 influenza A viruses to become highly pathogenic, but all HPAI H7 viruses have at least 2 basic amino acid insertions in the HA₀ cleavage site (22). HA₀ with the MBCS is activated in a broad range of different host cells and therefore enables HPAI viruses to replicate systemically in poultry (46). To date, little is known about the apparent subtype-specific nature of the introduction of the MBCS into LPAI viruses and the evolutionary processes involved in the emergence of HPAI viruses. When an MBCS was introduced in a laboratory-adapted strain of influenza virus, A/Duck/Ukraine/1/1963 (H3N8), it did not result in a dramatic change in pathogenic phenotype (35). Here, the effect of the introduction of an MBCS into a primary LPAI H6N1 virus, A/Mallard/Sweden/81/2002, is described. The introduction of an MBCS resulted in trypsin-independent replication in vitro and enhanced pathogenesis in a chicken model. Understanding the basis of the HA subtype specificity of the introduction of an MBCS into avian influenza viruses will lead to a better understanding of potential molecular restrictions involved in emergence of HPAI outbreaks.     

Targeting host cell furin proprotein convertases as a therapeutic strategy against bacterial toxins and viral pathogens

Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require processing by host proprotein convertases (PCs) to enter host cells and to cause disease. Conversely, inhibiting PCs is likely to protect host cells from multiple furin-dependent, but otherwise unrelated, pathogens. To determine if this concept is correct, we designed specific nanomolar inhibitors of PCs modeled from the extended cleavage motif TPQRERRRKKR downward arrowGL of the avian influenza H5N1 hemagglutinin. We then confirmed the efficacy of the inhibitory peptides in vitro against the fluorescent peptide, anthrax protective antigen (PA83), and influenza hemagglutinin substrates and also in mice in vivo against two unrelated toxins, anthrax and Pseudomonas exotoxin. Peptides with Phe/Tyr at P1' were more selective for furin. Peptides with P1' Thr were potent against multiple PCs. Our strategy of basing the peptide sequence on a furin cleavage motif known for an avian flu virus shows the power of starting inhibitor design with a known substrate. Our results confirm that inhibiting furin-like PCs protects the host from the distinct furin-dependent infections and lay a foundation for novel, host cell-focused therapies against acute diseases.     

A Novel Activation Mechanism of Avian Influenza Virus H9N2 by Furin

Avian influenza virus H9N2 is prevalent in waterfowl and has become endemic in poultry in Asia and the Middle East. H9N2 influenza viruses have served as a reservoir of internal genes for other avian influenza viruses that infect humans, and several cases of human infection by H9N2 influenza viruses have indicated its pandemic potential. Fortunately, an extensive surveillance program enables close monitoring of H9N2 influenza viruses worldwide and has generated a large repository of virus sequences and phylogenetic information. Despite the large quantity of sequences in different databases, very little is known about specific virus isolates and their pathogenesis. Here, we characterize a low-pathogenicity avian influenza virus, A/chicken/Israel/810/2001 (H9N2) (Israel810), which is representative of influenza virus strains that have caused severe morbidity and mortality in poultry farms. We show that under certain circumstances the Israel810 hemagglutinin (HA) can be activated by furin, a hallmark of highly pathogenic avian influenza virus. We demonstrate that Israel810 HA can be cleaved in cells with high levels of furin expression and that a mutation that eliminates a glycosylation site in HA₁ allows the Israel810 HA to gain universal cleavage in cell culture. Pseudoparticles generated from Israel810 HA, or the glycosylation mutant, transduce cells efficiently. In contrast, introduction of a polybasic cleavage site into Israel810 HA leads to pseudoviruses that are compromised for transduction. Our data indicate a mechanism for an H9N2 evolutionary pathway that may allow it to gain virulence in a distinct manner from H5 and H7 influenza viruses.     

一株鹅源H5N2亚型高致病性禽流感病毒的分离鉴定及生物学特性分析

分离到一株鹅源 H5N2亚型高致病性禽流感病毒,SPF鸡静脉接种致病指数为2.99,但鸭子对该病毒不敏感．病毒感染小鼠后不致病,但能够在肺内有效复制,表明其具有感染哺乳动物的潜在风险．血凝素(hemagglutinin, HA)蛋白裂解位点上插入有多个连续的碱性氨基酸(-RRRKKR-),从分子上证实这是一株高致病性禽流感病毒．核酸序列比较分析表明,分离的流感病毒HA基因与A/chicken/Hubei/489/2004 (H5N1)同源率达到99.4%,神经氨酸酶(neuraminidase, NA)基因与A/chicken/Jilin/53/01(H9N2)同源率达到99.8%;氨基酸水平上,HA与2004年分离到的A/chicken/Hubei/489/2004(H5N1)、A/swan/Guangxi/307/2004(H5N1)、A/wildduck/Guangdong/314/　2004(H5N1)和A/chicken/Henan/210/2004(H5N1)同源率均为99.3%,NA 与A/chicken/Jilin/53/01(H9N2)同源率为99.6%．进化树分析结果表明,该流感病毒分离株可能是由H5N1和H9N2两个亚型病毒重排而来．     

Acid stability of the hemagglutinin protein regulates H5N1 influenza virus pathogenicity

Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 Å resolution and two structures of HP HA at 2.95 and 3.10 Å resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.