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.

Restricted expression of Borna disease virus glycoprotein in brains of experimentally infected Lewis rats.

AIM: Borna disease virus (BDV) induces a persistent infection in the central nervous system (CNS) accompanied by a non-purulent meningoencephalitis. BDV-infection of Lewis rats provides an important model to investigate basic principles of neurotropism, viral persistence and resulting dysfunctions. To date, the in vivo strategies of BDV to persist in the CNS are not fully understood. Viral glycoproteins are main targets of the antiviral defence implicating a controlled expression in case of persistent infections. Therefore, we analysed the expression profiles of the BDV-glycoprotein (BDV-GP) and corresponding BDV-intron II RNA in experimentally infected rat brains, focusing on their spatio-temporal occurrence, regional, cellular and intracellular locations. METHODS: This was carried out by immunohistochemistry and in situ hybridization. The expression pattern of the most abundantly expressed BDV-nucleoprotein (BDV-N) served as a reference. RESULTS: BDV-N mRNA was detected preferentially in the cytoplasm of neurones, whereas BDV-intron II mRNA was found predominantly in the nucleus of brain cells. The genomic RNA was restricted to the nucleus. Expression of BDV-GP was significantly lower than BDV-N expression and mainly limited to cerebral cortex, hippocampus, amygdala and thalamus. BDV-GP was restricted to larger neurones; BDV-N occurred also in astrocytes, oligodendrocytes and ependymal cells. CONCLUSIONS: The expression profiles of BDV-GP, BDV-N and their mRNAs are significantly different, indicating that BDV-GP expression is regulated in vivo. This might be achieved by restricted nuclear export and/or maturation of BDV-intron II mRNA or limited translation as a viral mechanism to escape from the immune response and enable persistence in the CNS.

Host cell proteases controlling virus pathogenicity.

The majority of viral glycoproteins that undergo post-translational proteolysis are cleaved by ubiquitous intracellular proteases; however, a minority are cleaved by secreted proteases available only in a few host systems. The interplay of viral glycoproteins and cellular proteases may have a pivotal role in the spread of infection, host range and pathogenicity.

A mono phenylalanine-based motif (F790) and a leucine-dependent motif (LI760) mediate internalization of furin.

The eukaryotic endoprotease furin, a member of the subtilisin-related family of prohormone convertases, is synthesized and transported within the constitutive secretory pathway to the plasma membrane, from where it recycles to the trans-Golgi network (TGN). Previous studies showed that TGN-residence and recycling are mediated by the cytoplasmic tail. Two targeting determinants have been described so far, the acidic signal CPSDSEEDEG783 containing two casein kinase II (CKII) phosphorylation sites and the internalization signal YKGL765. Refined analyses of the cytoplasmic domain of furin, which was mutagenized and tagged to the influenza hemagglutinin and to the membrane cofactor protein (CD46) as reporter molecules reveal two additional internalization determinants, a leucine-isoleucine signal, LI760, and a mono phenylalanine-based motif at F790, which functions without any specific neighboring amino acid sequence. Both signals are capable of independently mediating internalization, as has been shown previously for the tyrosine-based signal. Thus, furin internalization is mediated by at least three independent endocytosis signals.

Multimerization potential of the cytoplasmic domain of the human cytomegalovirus glycoprotein B.

In the present study the coding sequence of the cytoplasmic tail of the human cytomegalovirus glycoprotein B (gB) was expressed. The secondary structure of the purified recombinant protein was analyzed by circular dichroism, and the quaternary structure was investigated by gel permeation chromatography, and electron microscopy. Our data indicate that the cytoplasmic gB domain contains alpha-helix structures and assembles into tetramers, suggesting that the authentic gB may represent a homotetramer.

Processing of viral glycoproteins by the subtilisin-like endoprotease furin and its inhibition by specific peptidylchloroalkylketones.

The spike glycoproteins of many enveloped viruses are proteolytically cleaved at the carboxytermini of sequences containing the basic motif R-X-K/R-R. Cleavage is often necessary for the fusion capacity of the glycoproteins and, thus, for virus infectivity. Among these viruses are pathogenic avian influenza viruses, human parainfluenza virus, human cytomegalovirus, and human immunodeficiency virus; it has been demonstrated that these viruses can be activated by furin. Indigenous furin has been identified in T-lymphocytes, which are host cells for HIV. Furin has been localized in the TGN and on the surface of cells after vectorial expression. Peptidylchloroalkylketones have been designed that inhibit with high specificity cleavage and fusion activity of viral glycoproteins, as well as virus replication.

A H1 hemagglutinin of a human influenza A virus with a carbohydrate-modulated receptor binding site and an unusual cleavage site.

Two receptor binding variants of the influenza virus A/Tübingen/12/85 (H1N1) were separated by their different plaque formation in MDCK cells. Hemagglutination of variant I was restricted to red blood cells of guinea pigs, whereas variant II also hemagglutinated chicken cells. The variants differed also in their ability to bind to alpha 2,6-linked sialic acid. Evidence is presented that this difference is determined by a complex carbohydrate side chain at asparagine131 near the receptor binding site which is absent in variant II. With both variants, the arginine found at the cleavage site of all other human isolates analyzed so far was replaced by lysine.

The carboxyterminus of the hemagglutinin-neuraminidase of Newcastle disease virus is exposed at the surface of the viral envelope.

The amino-terminal and the carboxy-terminal amino acids of the hemagglutinin-neuraminidase glycoprotein of the Ulster strain of Newcastle disease virus have been analyzed before and after proteolytic activation of the precursor HNo (Mr approximately 82K). The amino termini of HNo and of the large cleavage fragment HN (approximately 74K) obtained by in vivo and in vitro proteolysis could not be sequenced by Edman degradation. This indicates that in both instances the amino termini are blocked. The carboxy termini of HNo and HN are different as demonstrated by end-point digestion with carboxypeptidase A. Furthermore, a small cleavage fragment (approximately 9K) of HNo that was removed from the virion after trypsin treatment could be purified by HPLC. In contrast to HN, this fragment displays a free amino terminus susceptible to Edman degradation. These data indicate that conversion of HNo involves removal of a 9K glycopeptide from the carboxy-terminal end. Thus, it has to be concluded that, unlike most other viral glycoproteins, the hemagglutinin-neuraminidase is inserted in the envelope with its carboxy terminus exposed at the surface of the virus particle.

A host range mutant of Newcastle disease virus with an altered cleavage site for proteolytic activation of the F protein.

The primary structure of the F protein of a host range mutant of the Ulster strain of Newcastle Disease virus (NDV) has been determined by nucleotide sequence analysis and compared to that of the wild type and other NDV strains. The cleavage site of the mutant had the sequence Gly-Lys-Gln-Arg-Arg as compared to two isolated basic amino acids [Gly-Lys(Arg)-Gln-Gly-Arg] with the apathogenic strains and two pairs of basic amino acids [Arg-Arg-Gln-Lys(Arg)-Arg] with the pathogenic strains. The data indicate that the cleavability of the F protein of NDV increases with the number of arginine and lysine residues at the cleavage site and that the susceptibility of the pathogenic strains to ubiquitous host proteases depends on both pairs of basic amino acids.

Processing and routage of HIV glycoproteins by furin to the cell surface.

Proteolytic activation of HIV-1 and HIV-2 envelope glycoprotein precursors (gp160 and gp140, respectively) occurs at the carboxyl side of a consensus motif consisting of the highly basic amino acid sequence. We have shown previously (Hallenberger et al., 1997) and confirmed in this report, that furin and PC7 can be considered as the putative physiological enzymes involved in the proteolytic activation of the HIV-1 and HIV-2 envelope precursors. In this study, we show by cell surface biotinylation and immunoprecipitation of the cell surface associated viral glycoproteins with antibodies that the mature viral envelope glycoproteins are correctly transported to the cell. membrane. Furthermore, we show that the uncleaved forms of the glycoproteins (gp160HIV-1 and gp140HIV-2) are also highly represented at the cell surface. First, transient expression of gp160 and gp140 into CV1, a cell line known to be inefficient in the proteolytic processing of the env gene, results in the expression of gp160 and gp140 at the cell surface. Moreover, HIV-1 infection of T cells also showed that gp160 is directed to the cell surface. In addition, we show that the precursor is not incorporated in the virus particle following the budding from the cell surface. Furthermore, a gp160 mutant (deficient for three carbohydrate sites on the gp41), shown to be poorly processed with the coexpressed endoproteases, is found to be transported as an uncleaved precursor to the cell surface. In contrast to HIV envelope glycoproteins, the influenza hemagglutinin precursor (HA0), that is thought to be matured by the furin-like enzymes as well, is found to be retained within the cell and is not able to reach the cell surface. Taken together, these results show that the proteolytic maturation of the viral envelope precursors of human immunodeficiency viruses type 1 and type 2 is not a prerequisite for cell surface targeting of the HIV glycoproteins. Implications of these results for antiviral immune response are discussed.

Hemifusion activity of a chimeric influenza virus hemagglutinin with a putative fusion peptide from hepatitis B virus.

Entry of enveloped viruses is often mediated by an aminoterminal hydrophobic fusion peptide of a viral surface protein. The S domain of the hepatitis B virus surface protein contains a putative fusion peptide at position 7-18, but no systems are available to study its function directly. We tested the functionality of this peptide and a related peptide from another hepadnavirus in the context of the well-characterized influenza virus hemagglutinin H7 using gene mutation. The chimeric hemagglutinins could be expressed stably in CV 1 cells and were transported to the cell surface. The chimeras were incompletely cleaved by cellular proteases but cleavage could be completed by trypsin treatment of the cells. The chimeras did not differ in receptor binding, i.e. erythrocyte binding. Hemifusion and fusion pore formation were detected with membrane or cytosolic fluorescent dye-labeled erythrocytes as target structures of the hemagglutinin. Five of six different chimeras mediated hemifusion in 20-54% of the hemagglutinin-expressing cells, complete fusion and syncytium formation was not observed. The data suggest that the sequence 7-18 of the hepatitis B S domain may indeed initiate the first step of viral entry, i.e. hemifusion.

[Characterization of proteolytic cleavage of influenza virus nucleocapsid protein NP in infected cells]. / Kharakteristika proteoliticheskogo narezaniia nukleokapsidnogo belka NPvirusov grippa v zarazhennykh kletkakh.

Antibodies specific to the N-terminal 34-aminoacid peptide of the major nucleocapsid protein NP of human influenza A viruses were obtained. The NP proteolytic cleavage occurring in infected cells late in infection has been demonstrated using this antibody. The antibody specifically reacted with noncleaved NP of 56 kD m.w. but not with cleaved NP of 53 kD m.w. This indicates that NP56-->NP53 intracellular cleavage released the N-terminal 3 kD peptide of Np molecule. The released 3 kD peptide did not accumulate in infected cells. Since the RNA-binding and nucleus migrating signals are located in the N-terminus of NP molecule, presumably, the terminal cleavage is important for intracellular NP functions.

Activation of an influenza virus A/turkey/Oregon/71 HA insertion variant by the subtilisin-like endoprotease furin.

The hemagglutinin (HA) gene of the influenza A turkey/Oregon/71 variant Tc1 adapted to primary chicken embryo cells contains an insertion of 54 nucleotides that encodes a peptide adjacent to the HA cleavage site, which is responsible for increased cleavability by ubiquitous cellular proteases. After coexpression with human furin from cDNA by vaccinia virus vectors and by an endogenous protease, the HA of Tc1, which possesses the amino acid sequence R-T-A-R at the cleavage site, is proteolytically processed. Site-directed mutagenesis of the cleavage site indicated that the arginine in position -4 is critical for HA activation by furin. Deletion of the insert revealed that the amino acid sequence -1 to -4 predisposes the protein for furin recognition.

The major proteins of the Escherichia coli outer cell envelope membrane. Characterization of proteins II* and III, comparison of all proteins.

Protein II*, one of the major Escherichia coli outer cell envelope membrane proteins has been characterized. The protein is heat-modifiable and perhaps due to complete unfolding and/or binding of sodium dodecylsulfate only at higher temperatures the modified protein exhibits a higher apparent molecular weight (33,000) than the non-modified form (28,000). Protein-chemical evidence as well as the behavior of two mutant proteins II* very strongly suggest that this protein consists of a single polypeptide chain and that in the strains studied there is no other major protein with similar characteristics. For another outer membrane protein, protein III (molecular weight 17,000), it has not yet been established if it should be classified as a major protein. Protein III consists of one or perhaps two polypeptide chains. The possibility existed that protein III is bound covalently to lipopolysaccharide, and this has been ruled out. Also, the lipopolysaccharide of the E. coli strains studied does not carry covalently bound protein in amounts anywhere near stoichiometry. N-on-protein substituents were neither found in protein II* nor in protein III. It is concluded that in E. coli B/r and the E. coli K12 strains used there are three major proteins: I, II, and IV; protein III may also belong to this class. There are not more major proteins than these. All four proteins are compared and discussed regarding their unknown functions and their relation to E. coli outer membrane proteins studied by other authors.

The major proteins of the Escherichia coli outer cell-envelope membrane. Cyanogen bromide fragments of protein I, composition and order.

The cyanogen bromide fragments of protein I, a major protein of the Escherichia coli outer cell envelope membrane, have been isolated and characterized. There appear to be two methionine-serine or methionine-threonine sequences causing incomplete cleavage but complete conversion of methionine to homoserine. Largely due to the existence of these overlapping fragments the order of 5 of the 6 fragments present could be deduced. None of the fragments exhibits any remarkable low degree of polarity, and the tryptic fingerprint of the largest fragment (comprising about 60% of protein I) also does not show any conspicuous large fraction of lipophilic peptides. It is concluded that the domain of protein I that may be buried in the lipid phase of the outer membrane in all likelihood is not very large, and there is, in fact, no definite proof yet that protein I is a membrane protein sensu stricto.

Characterization of the carboxypeptidase involved in the proteolytic cleavage of the influenza haemagglutinin.

The arginine carboxypeptidase involved in the proteolytic cleavage of the haemagglutinin of influenza A virus has been analysed by an assay employing a Sepharose-bound peptide containing radioactive arginine as a substrate. The enzyme activity has been extracted from purified virus with non-ionic detergents and has been separated from the haemagglutinin and from the neuraminidase by isoelectric focusing and by affinity chromatography. The carboxypeptidase present in virus grown in different host cells shows variations in its isoelectric point. It can be concluded from these observations that the carboxypeptidase is a host component incorporated into the virus envelope. When the enzyme is inhibited by 2-mercaptomethyl-3-guanidinoethyl-thiopropanoic acid, haemagglutinin with the arginine attached to the carboxy terminus of HA1 can be obtained. The observation that under these conditions the haemagglutinin has retained its haemolytic activity indicates that the carboxypeptidase does not play an essential role in the activation process.

Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin.

The role of three N-linked glycans which are conserved among various hemagglutinin (HA) subtypes of influenza A viruses was investigated by eliminating the conserved glycosylation (cg) sites at asparagine residues 12 (cg1), 28 (cg2), and 478 (cg3) by site-directed mutagenesis. An additional mutant was constructed by eliminating the cg3 site and introducing a novel site 4 amino acids away, at position 482. Expression of the altered HA proteins in eukaryotic cells by a panel of recombinant vaccinia viruses revealed that rates and efficiency of intracellular transport of HA are dependent upon both the number of conserved N-linked oligosaccharides and their respective positions on the polypeptide backbone. Glycosylation at two of the three sites was sufficient for maintenance of transport of the HA protein. Conserved glycosylation at either the cg1 or cg2 site alone also promoted efficient transport of HA. However, the rates of transport of these mutants were significantly reduced compared with the wild-type protein or single-site mutants of HA. The transport of HA proteins lacking all three conserved sites or both amino-terminally located sites was temperature sensitive, implying that a polypeptide folding step had been affected. Analysis of trimer assembly by these mutants indicated that the presence of a single oligosaccharide in the stem domain of the HA molecule plays an important role in preventing aggregation of molecules in the endoplasmic reticulum, possibly by maintaining the hydrophilic properties of this domain. The conformational change observed after loss of all three conserved oligosaccharides also resulted in exposure of a normally mannose-rich oligosaccharide at the tip of the large stem helix that allowed its conversion to a complex type of structure. Evidence was also obtained suggesting that carbohydrate-carbohydrate interactions between neighboring oligosaccharides at positions 12 and 28 influence the accessibility of the cg2 oligosaccharide for processing enzymes. We also showed that terminal glycosylation of the cg3 oligosaccharide is site specific, since shifting of this site 4 amino acids away, to position 482, yielded an oligosaccharide that was arrested in the mannose-rich form. In conclusion, carbohydrates at conserved positions not only act synergistically by promoting and stabilizing a conformation compatible with transport, they also enhance trimerization and/or folding rates of the HA protein.

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.