Proteolytic cleavage of wild type and mutants of the F protein of human parainfluenza virus type 3 by two subtilisin-like endoproteases, furin and Kex2.

The fusion (F) protein of human parainfluenza virus type 3 contains the tribasic cleavage site R-T-K-R, which was altered by site-directed mutagenesis. Wild-type F protein and various mutants were expressed by recombinant vaccinia viruses. The endogenous endoprotease present in CV-1 cells cleaves F variants containing the furin recognition motif R-X-K/R-R but not variants containing the dibasic site K-R or a single R at the cleavage site. A similar cleavage pattern was obtained when the subtilisin-like endoproteases Kex2 and furin were coexpressed with the wild type and mutants of the F protein. Peptidylchloromethylketone inhibitors mimicking basic cleavage sites prevent cleavage of the precursor Fo by the endogenous protease only when the furin-specific motif is present in the peptidyl portion. The data support the concept that furin is a cellular protease responsible for the activation of the F protein of human parainfluenza virus type 3.     

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.     

A study of human furin specificity using synthetic peptides derived from natural substrates, and effects of potassium ions

We explored furin substrate requirements in addition to the motif R-X-K/R-R using synthetic fluorescent resonance energy transfer (FRET) decapeptides. These decapeptides were derived from furin cleavage sites in viral coat glycoproteins and human and bacterial protein precursors. The hydrolysis by furin of most substrate was activated by K⁺ ion, whereas kosmotropic anions of the Hofmeister series were inhibitors. The analysis of furin hydrolytic activity showed that its efficiency is highly dependent on the particular combinations of amino acids at different substrate positions. There is a clear interdependence of furin subsites that must be taken in account in determining its specificity and also for the design of inhibitors. However, clear preferences were detected for substrates with S at P₁′, and V at P₂′, at P₃′ the amino acids D, S, L and A are almost equally frequent. In the non-prime subsites the best substrates presented S and H at P₆; basic amino acids at P₅; and no clear tendency at P₃. Interestingly, two amino acid substitutions on the prime side of the peptide derived from H5N1 influenza hemagglutinin furin processing site highly improved its hydrolysis. These modifications are possible by single point mutations, suggesting a potential yield of a more infectious virus.     

Suppression of glycoprotein formation of Semliki Forest, influenza, and avian sarcoma virus by tunicamycin.

Tunicamycin, a new antibiotic, halts the formation of physical particles of Semliki forest and fowl plague virus, whereas avian oncornavirus particles which show a reduction in infectivity and do not contain detectable labeled glycoprotein are released in the presence of the drug. In Semliki forest virus-infected cells only the protein moieties of the glycoproteins could be labeled. In cells infected with fowl plague and avian sarcoma virus neither intact glycoproteins nor their protein moieties could be detected. By using a protease inhibitor (N-alpha-p-tosyl-L-lysin chloromethyl ketone, TLCK) it could be shown, however, that the carbohydrate-free hemagglutinin precursor of influenza virus is synthesized but is presumably degraded by intracellular proteases in the absence of TLCK as a consequence of the lack of carbohydrate.     

Membrane fusion induced by influenza virus hemagglutinin requires protein bound fatty acids

The low pH-dependent fusion of lipid membranes induced by two types of the fatty acylated influenza viral hemagglutinin has been studied by use of an energy transfer assay. When protein bound fatty acids were released from the hemagglutinin by hydroxylamine treatment viral fusion activity was inhibited. The extent of fusion inhibition correlates with the amount of fatty acids cleaved from the hemagglutinin. Virosomes prepared from fowl plague virus containing fatty acid free hemagglutinin showed a much lower fusion activity than control virosomes containing fatty acylated hemagglutinin. The hydroxylamine treatment applied has no detectable effects on the virus other than fatty acid release from its spike glycoproteins. These results support our previous hypothesis that protein bound fatty acids are involved in the induction of membrane fusion by the influenza hemagglutinin.     

Formation of Influenza Virus Proteins

Eight virus-specific proteins have been found in chicken embryo fibroblasts infected with fowl plague virus. Among them are two glycoproteins which are the constituents of the hemagglutinin on the virus particle. They are derived from a large precursor glycoprotein by cleavage of a covalent linkage. The reaction can be blocked by the protease inhibitor diisopropylfluorophosphate and the amino acid analogue fluorophenylalanine. This indicates that a peptide bond is cleaved. If infected cells are kept at 25 C, a temperature at which virus maturation is inhibited, the precursor glycoprotein is cleaved at a significantly slower rate than at 37 C. It appears, however, that a reduced synthesis of the carbohydrate-free envelope protein is responsible for the block of virus maturation at 25 C rather than the lower cleavage rate of the precursor.     

Synthesis of biologically active influenza virus hemagglutinin in insect larvae.

The hemagglutinin of influenza (fowl plague) virus was expressed in larvae of Heliothis virescens by using recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector. Animals were infected with the recombinant virus either by parenteral injection or by feeding. For oral uptake, recombinant virus occluded in polyhedra obtained from cultured Spodoptera frugiperda cells after coinfection with authentic AcNPV was used. Immunohistological analyses of infected animals revealed that the hemagglutinin was expressed only in those tissues that are also permissive for the replication of authentic AcNPV. These tissues included hypodermis, fat body, and tracheal matrix. After oral infection, hemagglutinin was also detected in individual gut cells. The amount of hemagglutinin synthesized in larvae after parenteral infection was 0.3% of the total protein, compared with 5% obtained in cultured insect cells. The hemagglutinin was transported to the cell surface and expressed in polarized cells only at the apical plasma membrane. It was processed by posttranslational proteolysis into the cleavage products HA1 and HA2. Oligosaccharides were attached by N-glycosidic linkages and were smaller than those found on hemagglutinin obtained from vertebrate cells. Hemagglutinin from larvae expressed receptor binding and cell fusion activities, but quantitation of the hemolytic capacity revealed that it was only about half as active as hemagglutinin from vertebrate or insect cell cultures. Chickens immunized with larval tissues containing hemagglutinin were protected from infection with fowl plague virus. These observations demonstrate that live insects are able to produce a recombinant membrane protein of vertebrate origin in biologically active form.     

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.     

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.     

Cleavage mechanism of the H5N1 hemagglutinin by trypsin and furin

The cleavage property of hemagglutinin (HA) by different proteases was the prime determinant for influenza A virus pathogenicity. In order to understand the cleavage mechanism, molecular modeling tools were utilized to study the coupled model systems of the proteases, i.e., trypsin and furin and peptides of the cleavage sites specific to H5N1 and H1 HAs, which constitute models of HA precursor in complex with cleavage proteases. The peptide segments 'RERRRKKR downward arrow G' and 'SIQSR downward arrow G' from the high pathogenic H5N1 H5 and the low pathogenic H1N1 H1 cleavage sites were docking to the trypsin and furin active pockets, respectively. It was observed through the docking studies that trypsin was able to recognize and cleave both the high pathogenic and low pathogenic hemagglutinin, while furin could only cleave the high pathogenic hemagglutinin. An analysis of binding energies indicated that furin got most of its selectivity due to the interactions with P(1), P(4), and P(6), while having less interaction with P(2) and little interactions with P(3), P(5), P(7), and P(8). Some mutations of H5N1 H5 cleavage sequence fitted less well into furin and would reduce high pathogenicity of the virus. These findings hint that we should focus at the subsites P(1), P(4), and P(6) for developing drugs against H5N1 viruses.     

Inhibition of proteolytic cleavage of the hemagglutinin of influenza virus by the calcium-specific ionophore A23187.

At calcium-specific ionophore A23187 concentrations of approximately 0.25 microM [which still allow assembly and release of fowl plague virus (FPV) particles] post-translational proteolytic cleavage of the viral hemagglutinin precursor HA into the fragments HA1 and HA2 is inhibited. The resulting virus particles with uncleaved hemagglutinin, that cannot be obtained under normal conditions, provide a suitable substrate for in vitro assays of the protease sensitivity of the FPV hemagglutinin. Proteolytic activation is accomplished with trypsin. Treatment with cathepsin B at low pH yields aberrant cleavage products suggesting that the cellular cleavage enzyme is not of lysosomal origin. A protease that cleaves the FPV hemagglutinin in the correct place can be detected in lysates of MDBK cells. This enzyme is calcium dependent and has a neutral pH optimum.     

Maturation of the trans-Golgi network protease furin: compartmentalization of propeptide removal,substrate cleavage,and COOH-terminal truncation

We have cloned a bovine cDNA encoding the trans-Golgi network (TGN) protease furin and expressed it via recombinant vaccinia viruses to investigate intracellular maturation. Pulse-chase labeling reveals that the 104-kD pro-furin bearing high mannose N-glycans is rapidly processed into the 98-kD protease whose N-glycans remain sensitive to endoglycosidase H for a certain period of time. Furthermore, in the presence of brefeldin A, pro-furin cleavage occurs. From these data we conclude that the ER is the compartment of propeptide removal. Studies employing the ionophore A23187 and DTT show that autocatalysis is Ca2+ dependent and that it does not occur under reducing conditions. Pro- furin produced under these conditions never gains endo H resistance indicating that it is retained in the ER. Coexpression of furin with the fowl plague virus hemagglutinin in the presence of brefeldin A and monensin reveals that furin has to enter the Golgi region to gain substrate cleaving activity. N-glycans of furin are sialylated proving its transit through the trans-Golgi network. A truncated form of furin is found in supernatants of cells. Truncation is inhibited in the absence of Ca2+ ions and in the presence of acidotropic agents indicating that it takes place in an acidic compartment of cells. Comparative analysis with furin expressed from cDNA reveals that the truncated form prevails in preparations of biologically active, endogenous furin obtained from MDBK cells. This observation supports the concept that secretion of truncated furin is a physiological event that may have important implications for the processing of extracellular substrates.     

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.     

Crimean-congo hemorrhagic fever virus glycoprotein precursor is cleaved by Furin-like and SKI-1 proteases to generate a novel 38-kilodalton glycoprotein

Crimean-Congo hemorrhagic fever virus (genus Nairovirus, family Bunyaviridae) genome M segment encodes an unusually large (in comparison to members of other genera) polyprotein (1,684 amino acids in length) containing the two major structural glycoproteins, Gn and Gc, that are posttranslationally processed from precursors PreGn and PreGc by SKI-1 and SKI-1-like proteases, respectively. The characteristics of the N-terminal 519 amino acids located upstream of the mature Gn are unknown. A highly conserved furin/proprotein convertase (PC) cleavage site motif (RSKR247) is located between the variable N-terminal region that is predicted to have mucin-like properties and the rest of PreGn. Mutational analysis of the RSKR247 motif and use of a specific furin/PC inhibitor and brefeldin A demonstrate that furin/PC cleavage occurs at the RSKR247 motif of PreGn as the protein transits the trans Golgi network and generates a novel glycoprotein designated GP38. Immunoprecipitation analysis identified two additional proteins, GP85 and GP160, which contain both mucin and GP38 domain regions, and whose generation does not involve furin/PC cleavage. Consistent with glycosylation predictions, heavy O-linked glycosylation and moderate levels of N-glycans were detected in the GP85 and GP160 proteins, both of which contain the mucin domain. GP38, GP85, and GP160 are likely soluble proteins based on the lack of predicted transmembrane domains, their detection in virus-infected cell supernatants, and the apparent absence from virions. Analogy with soluble glycoproteins and mucin-like proteins encoded by other hemorrhagic fever-associated RNA viruses suggests these proteins could play an important role in viral pathogenesis.     

Intracellular sorting and basolateral appearance of the G protein of vesicular stomatitis virus in Madin-Darby canine kidney cells

The polarity of the surface distribution of viral glycoproteins during virus infection has been studied in the Madin-Darby canine kidney epithelial cell line on nitrocellulose filters. Using a surface radioimmunoassay on Madin-Darby canine kidney strain I cells that had been infected with vesicular stomatitis virus or with avian influenza fowl plague virus, we found that the surface G protein was 97% basolateral, whereas the fowl plague virus hemagglutinin was 88% apical. Newly synthesized, pulse-labeled vesicular stomatitis virus appeared first on the basolateral plasma membrane as measured by an immunoprecipitation assay in which the anti-G protein antibody was applied to the monolayer either from the apical or the basolateral side. Labeled G protein could be accumulated inside the cell at a late stage of transport by decreasing the temperature to 20 degrees C during the chase. Reversal to 37 degrees C led to its rapid and synchronous transport to the basolateral surface at an initial rate 61-fold greater than that of transport to the apical side. These results demonstrate that the newly synthesized G protein is transported directly to the basolateral membrane and does not pass over the apical membrane en route. Since a previous study of the surface appearance of influenza virus hemagglutinins showed that the newly synthesized hemagglutinins were inserted directly from an intracellular site into the apical membrane (Matlin, K., and K. Simons, 1984, J. Cell Biol., 99:2131-2139), we conclude that the divergence of the transport pathway for the apical and basolateral viral glycoproteins has to occur intracellularly, i.e., before reaching the cell surface.     

Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture

Cultured hippocampal neurons were infected with a temperature-sensitive mutant of vesicular stomatitis virus (VSV) and a wild-type strain of the avian influenza fowl plague virus (FPV). The intracellular distribution of viral glycoproteins was monitored by immunofluorescence microscopy. In mature, fully polarized neurons the VSV glycoprotein (a basolateral protein in epithelial MDCK cells) moved from the Golgi complex to the dendritic domain, whereas the hemagglutinin protein of FPV (an apically sorted protein in MDCK cells) was targeted preferentially, but not exclusively, to the axon. The VSV glycoprotein appeared in clusters on the dendritic surface, while the hemagglutinin was distributed uniformly along the axonal membrane. Based on the finding that the same viral glycoproteins are sorted in a polarized fashion in both neuronal and epithelial cells, we propose that the molecular mechanisms of surface protein sorting share common features in the two cell types.     

The role of eukaryotic subtilisin-like endoproteases for the activation of human immunodeficiency virus glycoproteins in natural host cells.

Proteolytic activation of the precursor envelope glycoproteins gp160 of human immunodeficiency virus type 1 (HIV-1) and gp140 of HIV-2, a prerequisite for viral infection, results in the formation of gp120/gp41 and gp125/gp36, respectively. Cleavage is mediated by cellular proteases. Furin, a member of the eukaryotic subtilisin family, has been shown to be an activating protease for HIV. Here, we compared the presence of furin and other mammalian subtilisins in lymphatic cells and tissues. Northern blot analyses revealed that furin and the recently discovered protease LPC/PC7 were the only subtilisin-like enzymes transcribed in such cells. Furin was identified as an enzymatically active endoprotease present in different lymphocytic, as well as monocytic, cell lines. When expressed from vaccinia virus vectors, the proprotein convertases were correctly processed, transported, and secreted into the media and enzymatically active. Coexpression of different subtilisins with the HIV envelope precursors revealed that furin and LPC/PC7 are able to cleave HIV-1 gp160. Moreover, both enzymes proteolytically processed the envelope precursor of HIV-2. gp140 was also cleaved to some extent by PC1, which is not, however, present in lymphatic cells. Furin- and LPC/PC7-catalyzed cleavage of HIV-1 gp160 resulted in biologically active envelope protein. In conclusion, among the known members of the subtilisin family, only furin and LPC/PC7 fulfill the requirements of a protease responsible for in vivo activation of HIV envelope glycoproteins.     

The role of proteolytic cleavage of viral glycoproteins in the pathogenesis of influenza virus infections

Infectivity, tropism, spread, and pathogenicity of influenza viruses are based on the interplay between the fusogenic glycoproteins and appropriate host endoproteases. The hemagglutinin (HA) of influenza A and B viruses and the HEF (hemagglutinating, esterase, fusion) glycoprotein of influenza C virus receive their full biological activity by proteolytic cleavage of a precursor molecule at a definite cleavage site. The amino acid motifs at the cleavage site and the availability of suitable proteases are critical for the clinical manifestation of the infection. Prototype cleavage proteases, including bacterial enzymes, are described.     

Analysis of Cathepsin and Furin Proteolytic Enzymes Involved in Viral Fusion Protein Activation in Cells of the Bat Reservoir Host

Bats of different species play a major role in the emergence and transmission of highly pathogenic viruses including Ebola virus, SARS-like coronavirus and the henipaviruses. These viruses require proteolytic activation of surface envelope glycoproteins needed for entry, and cellular cathepsins have been shown to be involved in proteolysis of glycoproteins from these distinct virus families. Very little is currently known about the available proteases in bats. To determine whether the utilization of cathepsins by bat-borne viruses is related to the nature of proteases in their natural hosts, we examined proteolytic processing of several viral fusion proteins in cells derived from two fruit bat species, Pteropus alecto and Rousettus aegyptiacus. Our work shows that fruit bat cells have homologs of cathepsin and furin proteases capable of cleaving and activating both the cathepsin-dependent Hendra virus F and the furin-dependent parainfluenza virus 5 F proteins. Sequence analysis comparing Pteropus alecto furin and cathepsin L to proteases from other mammalian species showed a high degree of conservation; however significant amino acid variation occurs at the C-terminus of Pteropus alecto furin. Further analysis of furin-like proteases from fruit bats revealed that these proteases are catalytically active and resemble other mammalian furins in their response to a potent furin inhibitor. However, kinetic analysis suggests that differences may exist in the cellular localization of furin between different species. Collectively, these results indicate that the unusual role of cathepsin proteases in the life cycle of bat-borne viruses is not due to the lack of active furin-like proteases in these natural reservoir species; however, differences may exist between furin proteases present in fruit bats compared to furins in other mammalian species, and these differences may impact protease usage for viral glycoprotein processing.     

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.