Fowl Plague Virus Hemagglutinin Research Articles

Autocatalytic Activation of Influenza Hemagglutinin

Enveloped viruses contain surface proteins that mediate fusion between the viral and target cell membranes following an activating stimulus. Acidic pH induces the influenza virus fusion protein hemagglutinin (HA) via irreversible refolding of a trimeric conformational state leading to exposure of hydrophobic fusion peptides on each trimer subunit. Herein, we show that cells expressing fowl plague virus HA demonstrate discrete switching behavior with respect to the HA conformational change. Partially activated states do not exist at the scale of the cell, activation of HA leads to aggregation of cell surface trimers, and newly synthesized HA refold spontaneously in the presence of previously activated HA. These observations imply a feedback mechanism involving self-catalyzed refolding of HA and thus suggest a mechanism similar to the autocatalytic refolding and aggregation of prions.

Influenza M2 envelope protein augments avian influenza hemagglutinin pseudotyping of lentiviral vectors

Lentivirus-based gene transfer has the potential to efficiently deliver DNA-based therapies into non-dividing epithelial cells of the airway for the treatment of lung diseases such as cystic fibrosis. However, significant barriers both to lung-specific gene transfer and to production of lentivirus vectors must be overcome before these vectors can be routinely used for applications to the lung. In this study, we investigated whether the ability to produce lentiviral vectors pseudotyped with fowl plague virus hemagglutinin (HA) could be improved by co-expression of influenza virus M2 in vector-producing cells. We found that M2 expression led to a 10-30-fold increase in production of HA-pseudotyped lentivirus vectors based upon equine infectious anemia virus (EIAV) or human immunodeficiency virus type 1 (HIV-1). Experiments using the M2 inhibitor amantadine and a drug-resistant mutant of M2 established that the ion channel activity of M2 was important for M2-dependent augmentation of vector production. Furthermore, the neuraminidase activity necessary for particle release from producer cells could also be incorporated into producer cells by co-expression of influenza NA cDNA. Lentiviral vectors pseudotyped with influenza envelope proteins were able to efficiently transduce via the apical membrane of polarized mouse tracheal cultures in vitro as well as mouse tracheal epithelia in vivo.

Acylation-Mediated Membrane Anchoring of Avian Influenza Virus Hemagglutinin Is Essential for Fusion Pore Formation and Virus Infectivity

Attachment of palmitic acid to cysteine residues is a common modification of viral glycoproteins. The influenza virus hemagglutinin (HA) has three conserved cysteine residues at its C terminus serving as acylation sites. To analyze the structural and functional roles of acylation, we have generated by reverse genetics a series of mutants (Ac1, Ac2, and Ac3) of fowl plague virus (FPV) containing HA in which the acylation sites at positions 551, 559, and 562, respectively, have been abolished. When virus growth in CV1 and MDCK cells was analyzed, similar amounts of virus particles were observed with the mutants and the wild type. Protein patterns and lipid compositions, characterized by high cholesterol and glycolipid contents, were also indistinguishable. However, compared to wild-type virus, Ac2 and Ac3 virions were 10 and almost 1,000 times less infectious, respectively. Fluorescence transfer experiments revealed that loss of acyl chains impeded formation of fusion pores, whereas hemifusion was not affected. When the affinity to detergent-insoluble glycolipid (DIG) domains was analyzed by Triton X-100 treatment of infected cells and virions, solubilization of Ac2 and Ac3 HAs was markedly facilitated. These observations show that acylation of the cytoplasmic tail, while not necessary for targeting to DIG domains, promotes the firm anchoring and retention of FPV HA in these domains. They also indicate that tight DIG association of FPV HA is essential for formation of fusion pores and thus probably for infectivity.

N-glycans attached to hemagglutinin in the head region and the stem domain control growth of influenza viruses by different mechanisms

Background: The hemagglutinin (HA) of fowl plague virus A/FPV/Ro/34 (H7N1) contains seven N-glycans, two of which are flanking the receptor binding site and three being located in the stem domain. In the present study, we have elucidated the functional role of these five glycans in the course of virus replication in different hosts. Methods: Using a RNA polymerase I-based reverse genetics system, we generated recombinant viruses in which the HA mutants were stably incorporated in the background of the influenza reassortants WSN-HK and HK-WSN. Results: Growth of viruses lacking the glycans from the HA head region was impaired in MDCK cells as well as in embryonated chicken eggs. This restriction was due to limited release of progeny viruses from host cells as a result of the enhanced receptor affinity of the mutated HA. Accordingly, the high activity N2-subtype neuraminidase (NA) was able to partly overcome this effect, while the low activity N1-subtype NA was not. Thus, HA and NA activities need to be highly balanced to promote influenza viruses growth. When glycans were eliminated from the HA stem, the resulting viruses showed temperature sensitive growth in cell culture and in chicken eggs. The degree of growth restriction was dependent on the position from which the glycan had been removed. The viruses had a decreased pH stability, indicating that the stem glycans might maintain the HA in a fusion competent conformation. Viruses lacking the stem glycan from Asn 28 could not be rescued, indicating that this glycan is essential for the formation of replication competent viruses. Conclusions: Our results demonstrate that N-glycans linked to distinct HA domains regulate influenza virus growth at different stages of the replication cycle. Moreover, the deletion of glycan attachment sites from HA represents a powerful experimental strategy for the generation of attenuated influenza viruses.

Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics.

The hemagglutinin (HA) of fowl plague virus A/FPV/Rostock/34 (H7N1) carries two N-linked oligosaccharides attached to Asn123 and Asn149 in close vicinity to the receptor-binding pocket. In previous studies in which HA mutants lacking either one (mutants G1 and G2) or both (mutant G1,2) glycosylation sites had been expressed from a simian virus 40 vector, we showed that these glycans regulate receptor binding affinity (M. Ohuchi, R. Ohuchi, A. Feldmann, and H. D. Klenk, J. Virol. 71:8377-8384, 1997). We have now investigated the effect of these mutations on virus growth using recombinant viruses generated by an RNA polymerase I-based reverse genetics system. Two reassortants of influenza virus strain A/WSN/33 were used as helper viruses to obtain two series of HA mutant viruses differing only in the neuraminidase (NA). Studies using N1 NA viruses revealed that loss of the oligosaccharide from Asn149 (mutant G2) or loss of both oligosaccharides (mutant G1,2) has a pronounced effect on virus growth in MDCK cells. Growth of virus lacking both oligosaccharides from infected cells was retarded, and virus yields in the medium were decreased about 20-fold. Likewise, there was a reduction in plaque size that was distinct with G1,2 and less pronounced with G2. These effects could be attributed to a highly impaired release of mutant progeny viruses from host cells. In contrast, with recombinant viruses containing N2 NA, these restrictions were much less apparent. N1 recombinants showed lower neuraminidase activity than N2 recombinants, indicating that N2 NA is able to partly overrule the high-affinity binding of mutant HA to the receptor. These results demonstrate that N-glycans flanking the receptor-binding site of the HA molecule are potent regulators of influenza virus growth, with the glycan at Asn149 being dominant and that at Asn123 being less effective. In addition, we show here that HA and NA activities need to be highly balanced in order to allow productive influenza virus infection.

Incorporation of fowl plague virus hemagglutinin into murine leukemia virus particles and analysis of the infectivity of the pseudotyped retroviruses.

We describe retrovirus particles carrying the fowl plague virus (FPV) hemagglutinin (HA). When expressed in cells providing Moloney murine leukemia virus (MoMLV) Gag and Pol proteins and a lacZ retroviral vector, FPV HA was found to be efficiently expressed, correctly processed, and stably incorporated into retroviral particles. HA-bearing retroviruses were infectious with a wide host range and were only 10-fold less infectious than retroviruses carrying wild-type MLV retroviral envelopes. We also coexpressed HA proteins in retroviral particles with chimeric MoMLV-derived envelope glycoproteins that efficiently retarget virus attachment but are only weakly fusogenic. Our results suggest that HA can in some cases enhance the fusion ability of these retroviral particles, depending on the cell surface molecule that is used as a receptor.

Elongation of the cytoplasmic tail interferes with the fusion activity of influenza virus hemagglutinin.

The hemagglutinin (HA) of fowl plague virus was lengthened and shortened by site-specific mutagenesis at the cytoplasmic tail, and the effects of these modifications on HA functions were analyzed after expression from a simian virus 40 vector. Elongation of the tail by the addition of one to six histidine (His) residues did not interfere with intracellular transport, glycosylation, proteolytic cleavage, acylation, cell surface expression, and hemadsorption. However, the ability to induce syncytia at a low pH decreased dramatically depending on the number of His residues added. Partial fusion (hemifusion), assayed by fluorescence transfer from octadecylrhodamine-labeled erythrocyte membranes, was also reduced, but even with the mutant carrying six His residues, significant transfer was observed. However, when the formation of fusion pores was examined with hydrophilic fluorescent calcein, transfer from erythrocytes to HA-expressing cells was not observed with the mutant carrying six histidine residues. The addition of different amino acids to the cytoplasmic tail of HA caused an inhibitory effect similar to that caused by the addition of His. On the other hand, a mutant lacking the cytoplasmic tail was still able to fuse at a reduced level. These results demonstrate that elongation of the cytoplasmic tail interferes with the formation and enlargement of fusion pores. Thus, the length of the cytoplasmic tail plays a critical role in the fusion process.

Biosynthesis, intracellular transport and enzymatic activity of an avian influenza A virus neuraminidase: role of unpaired cysteines and individual oligosaccharides.

Intracellular transport, glycosylation, tetramerization and enzymatic activity of the neuraminidase (NA) of fowl plague virus (FPV) were analysed in vertebrate cells after expression from a vaccinia virus vector. Tetramerization occurred with a half-time of 15 min, whereas passage through the medial Golgi apparatus and transport to the plasma membrane occurred with half-times of 2 and 3 h, respectively, suggesting a step in NA maturation beyond tetramerization that limits the rate of transport to the medial Golgi. NA transport rates were about fourfold slower than those of haemagglutinin (HA). Slow transport and processing of FPV NA was not altered by coexpression of FPV HA, nor was the transport rate of HA influenced by NA. The slow transport kinetics of NA were also observed in FPV-infected CV-1 cells. As deduced from the coding sequence, FPV NA has the shortest stalk of all naturally occurring NAs described to date and contains only three potential N-glycosylation sites, which are all located in the globular head domain. Elimination of each of the three N-glycosylation sites revealed that the two oligosaccharides at positions 124 and 66 are of the complex type, whereas the one at Asn-213 remains in mannose-rich form. The glycosylation mutants showed also that oligosaccharides at positions 124 and 213 of FPV NA modulate enzymatic activity. Transport of NA is not influenced by single elimination of any of the three oligosaccharide attachment sites. Mutational analysis of the three Cys residues not involved in intrachain disulfide pairing revealed that Cys-49 in the stalk of the NA molecule is responsible for the formation of disulfide-linked dimers. Analysis of cysteine mutants of FPV NA also demonstrated that disulfide-linked dimers are not absolutely necessary for the formation of enzymatically active tetramers but may stabilize the quaternary structure of NA.

Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety.

The hemagglutinin (HA) of the fowl plague virus (FPV) strain of influenza A virus has two N-linked oligosaccharides attached to Asn123 and Asn149 in the vicinity of the receptor binding site. The effect of these carbohydrate side chains on the binding of HA to neuraminic acid-containing receptors has been analyzed. When the oligosaccharides were deleted by site-specific mutagenesis, HA expressed from a simian virus 40 vector showed enhanced hemadsorbing activity. Binding was so strong under these conditions that erythrocytes were no longer released by viral neuraminidase and that release was significantly reduced when neuraminidase from Vibrio cholerae was used. Similarly, when these oligosaccharides were removed selectively from purified viruses by N-glycosidase F, such virions were unable to elute from receptors, although they retained neuraminidase activity. Thus, release of FPV from cell receptors depends on the presence of the HA glycans at Asn123 and Asn149. On the other hand, receptor binding was abolished when these oligosaccharides were sialylated after expression in the absence of neuraminidase (M. Ohuchi, A. Feldmann, R. Ohuchi, and H.-D. Klenk, Virology 212:77-83, 1995). These observations indicate that the receptor affinity of FPV HA is controlled by oligosaccharides adjacent to the receptor binding site.

Oligosaccharides in the stem region maintain the influenza virus hemagglutinin in the metastable form required for fusion activity.

The influenza A virus hemagglutinin (HA) has three conserved oligosaccharides located in the stem region at asparagine residues 12, 28, and 478. The biological role of these oligosaccharides has been investigated by mutational analysis of HA of fowl plague virus that was expressed from a simian virus 40 vector in the presence of ammonium chloride for protection from acid denaturation in the trans-Golgi network. Resistance to endoglycosidase H and cleavage of HA into the subunits HA1 and HA2 have been analyzed as markers for intracellular transport. Cell surface exposure has been determined by hemadsorption following neuraminidase treatment, by immunofluorescence staining, and by fluorescence-activated cell sorter analysis. When all three stem oligosaccharides were removed, transport was almost completely blocked. When two of the three stem oligosaccharides, particularly those at asparagine residues 12 and 28, were missing, HA was transported to the surface but showed extremely low fusion activity. With mutants lacking one stem oligosaccharide, fusion was reduced to a lesser extent. Removal of stem oligosaccharides resulted also in an increase in the pH optimum required for fusion. On the other hand, no reduction in fusion activity was observed when oligosaccharides in the head region of the HA spike were removed. These results indicate that the conserved oligosaccharides in the stem stabilize HA in the form susceptible to the conformational change necessary for fusion.

N-acetyl-beta-glucosaminidase accounts for differences in glycosylation of influenza virus hemagglutinin expressed in insect cells from a baculovirus vector.

The hemagglutinin of fowl plague virus has been expressed in Spodoptera frugiperda (Sf9) cells and in Estigmene acrea cells by using a baculovirus vector. Structural analysis revealed that the endo-H-resistant N-glycans of HA from Sf9 cells were predominantly trimannosyl core oligosaccharides, whereas in E. acrea cells most of these cores were elongated by at least one terminal N-acetylglucosamine residue. To understand the difference in carbohydrate structures, enzymes involved in N-glycan processing have been analyzed. The results revealed that the different glycosylation patterns observed are due to an N-acetyl-beta-glucosaminidase activity that was found in Sf9 cells but not in E. acrea cells. This enzyme specifically used the GlcNAcMan(3)GlcNAc(2) oligosaccharide as a substrate. When N-acetyl-beta-glucosaminidase or alpha-mannosidase II was inhibited by specific inhibitors, the amount of terminal N-acetylglucosamine in hemagglutinin from Sf9 cells was significantly enhanced. These results demonstrate that N glycosylation in both cell lines follows the classical pathway up to the stage of GlcNAcMan(3)GlcNAc(2) oligosaccharide side chains. Whereas these structures are the end product in E. acrea cells, they are degraded in Sf9 cells to Man(3)GlcNAc(2) cores by N-acetyl-beta-glucosaminidase.

Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human beta 1,2-N-acetylglucosaminyltransferase I.

Spodoptera frugiperda (Sf9)-cells differ markedly in their protein glycosylation capacities from vertebrate cells in that they are not able to generate complex type oligosaccharide side chains. In order to improve the oligosaccharide processing properties of these cells we have used baculovirus vectors for expression of human (beta 1,2-N-acetylglucosaminyltransferase I (hGNT-I), the enzyme catalysing the crucial step in the pathway leading to complex type N-glycans in vertebrate cells. One vector (Bac/GNT) was designed to express unmodified GNT-I protein, the second vector (Bac/tagGNT) to express GNT-I protein with a tag epitope fused to its N-terminus. In Sf9-cells infected with Bac/tagGNT-virus a protein of about 50 kDa representing hGNT-I was detected with an antiserum directed against the tag epitope. HGNT-I activity was increased at least threefold in lysates of infected cells when N-acetylglucosamine (GlcNAc)-free ovalbumine was used as substrate. To monitor hGNT-I activity in intact Sf9-cells, the glycosylation of coexpressed fowl plague virus hemagglutinin (HA) was investigated employing a galactosylation assay and chromatographic analysis of isolated HA N-glycans. Coexpression of hGNT-I resulted in an at least fourfold increase of HA carrying terminal GlcNAc-residues. The only structure detectable in this fraction was GlcNAcMan3GlcNAc2. These results show that hGNT-I is functionally active in Sf9-cells and that the N-glycans of proteins expressed in the baculovirus/insect cell system are elongated by coexpression of glycosyltransferases of vertebrate origin. Complete complex type oligosaccharide side chains were not observed when hGNT-I was overexpressed, thus supporting the concept that Sf9-cells do not contain glycosyltransferases acting after hGNT-I.

Neuraminidase Is Essential for Fowl Plague Virus Hemagglutinin to Show Hemagglutinating Activity

When hemagglutinin (HA) of fowl plague virus (FPV) was expressed in CV-1 cells by a simian virus 40 vector, hemadsorption was barely detectable, although HA was exposed at the cell surface. However, treatment of HA-expressing cells with Vibrio cholerae neuraminidase (VCNA) resulted in extensive hemadsorption. VCNA treatment enhanced the electrophoretic mobility of the HA1 subunit of HA, indicating the removal of sialic acid. When two oligosaccharides in the vicinity of the receptor binding site of FPV HA were deleted by site-specific mutagenesis, VCNA treatment was not required for hemadsorption. Mutants which retained one of these oligosaccharides and mutants in which oligosaccharides not adjacent to the receptor binding site were deleted needed VCNA treatment to show hemadsorption. VCNA treatment also enhanced hemadsorption of vector-expressed HA of the WSN strain, which had a complex-type oligosaccharide in the vicinity of the receptor binding site, but had no effect on hemadsorption of Hong Kong type HA, which has a high-mannose type oligosaccharide adjacent to the receptor binding site. These results indicate that sialic acid on oligosaccharides near the receptor binding site interferes with hemadsorption. Thus, the neuraminidase is essential for FPV HA to show hemagglutinating activity.

Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin.

Furin, a subtilisin-like eukaryotic endoprotease, is responsible for proteolytic cleavage of cellular and viral proteins transported via the constitutive secretory pathway. Cleavage occurs at the C-terminus of basic amino acid sequences, such as R-X-K/R-R and R-X-X-R. Furin was found predominantly in the trans-Golgi network (TGN), but also in clathrin-coated vesicles dispatched from the TGN, on the plasma membrane as an integral membrane protein and in the medium as an anchorless enzyme. When furin was vectorially expressed in normal rat kidney (NRK) cells it accumulated in the TGN similarly to the endogenous glycoprotein TGN38, often used as a TGN marker protein. The signals determining TGN targeting of furin were investigated by mutational analysis of the cytoplasmic tail of furin and by using the hemagglutinin (HA) of fowl plague virus, a protein with cell surface destination, as a reporter molecule, in which membrane anchor and cytoplasmic tail were replaced by the respective domains of furin. The membrane-spanning domain of furin grafted to HA does not localize the chimeric molecule to the TGN, whereas the cytoplasmic domain does. Results obtained on furin mutants with substitutions and deletions of amino acids in the cytoplasmic tail indicate that wild-type furin is concentrated in the TGN by a mechanism involving two independent targeting signals, which consist of the acidic peptide CPSDSEEDEG783 and the tetrapeptide YKGL765. The acidic signal in the cytoplasmic domain of a HA-furin chimera is necessary and sufficient to localize the reporter molecule to the TGN, whereas YKGL is a determinant for targeting to the endosomes. The data support the concept that the acidic signal, which is the dominant one, retains furin in the TGN, whereas the YKGL motif acts as a retrieval signal for furin that has escaped to the cell surface.

Rescue of vector-expressed fowl plague virus hemagglutinin in biologically active form by acidotropic agents and coexpressed M2 protein

The hemagglutinin of the Rostock strain of fowl plague virus was expressed in CV-1 cells by a simian virus 40 vector, and its stability in the exocytotic transport process was examined by a fusion assay. A 50-fold increase in the fusion activity of the hemagglutinin was observed when expression occurred in the presence of ammonium chloride, Tris-HCl, or high doses of amantadine. When chloroquine, another acidotropic agent, was used, the hemagglutinin exposed at the cell surface had to be activated by trypsin, because intracellular cleavage was inhibited by this compound. Hemagglutinin mutants resistant to intracellular cleavage did not require acidotropic agents for full expression of fusion activity, when treated with trypsin after arrival at the cell surface. These results indicate that fowl plague virus hemagglutinin expressed by a simian virus 40 vector is denatured in the acidic milieu of the exocytotic pathway and that cleavage is a major factor responsible for the pH instability. Coexpression with the M2 protein also markedly enhanced the fusion activity of the hemagglutinin, and this effect was inhibited by low doses of amantadine. These results support the concept that M2, known to have ion channel function, protects the hemagglutinin from denaturation by raising the pH in the exocytotic transport system. The data also stress the importance of acidotropic agents or coexpressed M2 for the structural and functional integrity of vector-expressed hemagglutinin.

Influenza virus M2 protein ion channel activity stabilizes the native form of fowl plague virus hemagglutinin during intracellular transport

The influenza A/fowl plague virus/Rostock/34 hemagglutinin (HA), which is cleaved intracellularly and has a high pH threshold (pH 5.9) for undergoing its conformational change to the low-pH form, was expressed from cDNA in CV-1 and HeLa T4 cells in the absence of other influenza virus proteins. It was found, by biochemical assays, that the majority of the HA molecules were in a form indistinguishable from the low-pH form of HA. The acidotropic agent, ammonium chloride, stabilized the accumulation of HA in its native form. Coexpression of HA and the homotypic influenza virus M2 protein, which has ion channel activity, stabilized the accumulation of HA in its pH neutral (native) form, and the M2 protein ion channel blocker, amantadine, prevented the rescue of HA in its native form. These data provide direct evidence that the influenza virus M2 protein ion channel activity can affect the status of the conformational form of cleaved HA during intracellular transport.

Membrane traffic in polarized neurons in culture.

Fetal hippocampal neurons develop axons and dendrites in culture. To study how neurons form and maintain different plasma membrane domains, hippocampal neurons were infected with RNA viruses and the distribution of the viral glycoproteins was analyzed by light and electron microscopy. Infection of hippocampal cells with vesicular stomatitis virus (VSV) and fowl plague virus (FPV) resulted in the polarized distribution of the newly synthesized viral glycoproteins. The VSV glycoprotein appeared firstly in the Golgi apparatus and then in the dendrites. In contrast, the hemagglutinin of FPV, after accumulation in the Golgi apparatus, moved to the axons. These results suggest that the mechanism of sorting of viral glycoproteins might be similar in neurons and MDCK cells, a cell line of epithelial origin. In these cells the VSV glycoprotein and the hemagglutinin of FPV distribute to the basolateral and apical membranes, respectively. Transport of viral glycoproteins to both neuronal domains was microtubule dependent. Nocodazole treatment of infected neurons inhibited the delivery of axonal and dendritic viral glycoproteins equally. To investigate if the analogy between epithelial cells and neurons extended to include an endogenous plasma membrane protein, the distribution of Thy-1, a GPI-linked protein, was analyzed. By immunofluorescence and immunoelectron microscopy, Thy-1 was found exclusively along the axonal surface. In epithelial cells GPI-anchored proteins are located apically. The existence of a barrier on the neuronal plasma membrane that would prevent intermixing of axonal and dendritic proteins was analyzed by a liposomefusion assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Polarized distribution of the viral glycoproteins of vesicular stomatitis, fowl plague and Semliki Forest viruses in hippocampal neurons in culture: a light and electron microscopic study

We have shown previously using immunofluorescence microscopy that upon injection of polarized hippocampal cells in culture with vesicular stomatitis virus (VSV) and fowl plague virus (FPV) the VSV glycoprotein is delivered to the plasma membrane of the dendrites and of the cell body whereas the FPV hemagglutinin is transported to the axonal surface ( Cell, 62 (1990) 63–72). In this work electron microscopy of infected rat hippocampal neurons showed that VSV progeny budded from the plasma membrane of the dendrites and the cell body. The location of the budding virions corresponded to the distribution of the VSV glycoprotein which was detected over the somatodendritic plasma membrane by immunoelectron microscopy. In contrast, no FPV formation was seen in the infected neurons although the FPV hemagglutinin was localized to the axonal surface by immunoelectron microscopy. In Semliki Forest virus (SFV) infected hippocampal cells we observed that the viral glycoproteins were exclusivelly present in the dendrites and cell body but not in axons.

Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160.

The envelope glycoprotein of human immunodeficiency virus (HIV) initiates infection by mediating fusion of the viral envelope with the cell membrane. Fusion activity requires proteolytic cleavage of the gp160 protein into gp120 and gp41 at a site containing several arginine and lysine residues. Activation at basic cleavage sites is observed with many membrane proteins of cellular and viral origin. We have recently found that the enzyme activating the haemagglutinin of fowl plague virus (FPV), an avian influenza virus, is furin. Furin, a subtilisin-like eukaryotic endoprotease, has a substrate specificity for the consensus amino-acid sequence Arg-X-Lys/Arg-Arg at the cleavage site. We show here that the glycoprotein of HIV-1, which has the same protease recognition motif as the FPV haemagglutinin, is also activated by furin.

Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease.

Many viruses have membrane glycoproteins that are activated at cleavage sites containing multiple arginine and lysine residues by cellular proteases so far not identified. The proteases responsible for cleavage of the hemagglutinin of fowl plague virus, a prototype of these glycoproteins, has now been isolated from Madin-Darby bovine kidney cells. The enzyme has a mol. wt of 85,000, a pH optimum ranging from 6.5 to 7.5, is calcium dependent and recognizes the consensus sequence R-X-K/R-R at the cleavage site of the hemagglutinin. Using a specific antiserum it has been identified as furin, a subtilisin-like eukaryotic protease. The fowl plague virus hemagglutinin was also cleaved after coexpression with human furin from cDNA by vaccinia virus vectors. Peptidyl chloroalkylketones containing the R-X-K/R-R motif specifically bind to the catalytic site of furin and are therefore potent inhibitors of hemagglutinin cleavage and fusion activity.