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Biomedical subjects

H D Klenk

Publications and source records attributed to H D Klenk.

At least 19 recordsLinked to original sources
PubMed2003

Intracellular cleavage of human influenza a virus hemagglutinin and its inhibition.

Replication of human influenza A viruses and proteolytic cleavage of the viral glycoprotein HA0 -->HA1/2 were studied in passaged cultures of epithelial cells of the mucosal membrane of human large intestine (CACO-2 line), dog kidney cells (MDCK), and monkey kidney cells (CV-1). Cleavage of the viral glycoprotein HA0, synthesis of activated virions, multicycle virus infection, and effective production of viral foci under an agarose overlayer were found in CACO-2 cells. By pulse-chase labeling of viral glycoproteins, testing the sensitivity to endoglycosidase-H of the viral glycoproteins HA0 and HA1/2 synthesized, and inhibiting the HA0 proteolysis with brefeldin A, the HA0 -->HA1/2 proteolysis was established to occur in the late stages of intracellular transport in the trans-Golgi and plasma membrane areas of the cells. Proteolysis of the viral glycoprotein HA0 in CACO-2 cells was suppressed by aprotinin, a natural inhibitor of serine proteinases. Unlike MDCK and CV-1 cells resistant to apoptosis induced by influenza virus, CACO-2 cells retained their viability for 2-3 days after infection with human influenza A virus.

Animals↗
PubMed2002

Interaction of influenza A virus M1 matrix protein with caspases.

In this investigation, an ability of influenza A virus M1 matrix protein to bind intracellular caspases, the key enzymes of cell apoptosis, has been examined. Protein-protein binding on polystyrene plates and polyvinyl pyrrolidone membrane was employed for this purpose. Under a comparative study of caspases-3, -6, -7, -8 influenza virus M1 protein specifically bound caspase-8 and weakly bound caspase-7. Using a computer analysis of the N-terminal region of M1 protein, a site similar to the anti-caspase site of baculovirus p35 protein, which inhibits caspases and displays antiapoptotic activity, was identified. These results are in good agreement with the supposition that influenza virus M1 protein is involved in a caspase-8-mediated apoptosis pathway in influenza virus infected cells.

Amino Acid Sequence↗
PubMed2001

The Hantaan virus glycoprotein precursor is cleaved at the conserved pentapeptide WAASA.

The medium segment of the tripartite negative-stranded RNA genome of hantaviruses encodes for the predicted glycoprotein precursor GPC. We have demonstrated here the expression of the glycoprotein precursor of Hantaan virus following transfection of mammalian cells. The cleavage of the precursor into the glycoproteins G1 and G2 followed the rules for signal peptides and seemed to occur directly at the pentapeptide motif "WAASA." Our data indicate that the signal peptidase complex is responsible for the proteolytic processing of the precursor GPC of Hantaan virus. The comparison of this region of the glycoprotein precursor, including the absolutely conserved WAASA motif, suggests a similar cleavage event for all hantavirus glycoproteins.

Amino Acid Sequence↗
PubMed2001

The Lassa virus glycoprotein precursor GP-C is proteolytically processed by subtilase SKI-1/S1P.

The surface glycoprotein of the Lassa virus, a member of the arenaviridae family, is synthesized as a 76-kDa precursor (GP-C) that is posttranslationally cleaved into an N-terminal 44-kDa subunit and a C-terminal membrane-anchored 36-kDa subunit. Cleavage occurs at the C-terminal end of the unusual recognition motif R-R-L-L. We show here that GP-C is cleaved in the endoplasmic reticulum by the cellular subtilase SKI-1/S1P, an enzyme that has so far been observed to be involved in cholesterol metabolism. Furthermore, we present evidence that only cleaved glycoprotein is incorporated into virions and that this is necessary for the formation of infectious virus. To our knowledge, there have been no previous reports of this type of viral glycoprotein processing, one that may be an interesting target for antiviral therapy.

Animals↗
PubMed2001

A single amino acid change in the cytoplasmic domains of measles virus glycoproteins H and F alters targeting, endocytosis, and cell fusion in polarized Madin-Darby canine kidney cells.

As we have shown previously, release of measles virus (MV) from polarized epithelial cells is not determined by the viral envelope proteins H and F. Although virus budding is restricted to the apical surfaces, both proteins were abundantly expressed on the basolateral surface of Madin-Darby canine kidney cells. In this report, we provide evidence that the basolateral expression of the viral proteins is of biological importance for the MV infection of polarized epithelial cells. We demonstrate that both MV glycoproteins possess a basolateral targeting signal that is dependent upon the unique tyrosine in the cytoplasmic tails. These tyrosines are shown to be also part of an endocytosis signal. In MV-infected cells, internalization of the glycoproteins was not observed, indicating that recognition of the endocytosis signals is disturbed by viral factors. In contrast, basolateral transport was not substantially hindered, resulting in efficient cell-to-cell fusion of polarized Madin-Darby canine kidney cells. Thus, recognition of the signals for endocytosis and polarized transport is differently regulated in infected cells. Mutation of the basolateral sorting signal in one of the MV glycoproteins prevented fusion of polarized cells. These results suggest that basolateral expression of the MV glycoproteins favors virus spread in epithelia.

Amino Acid Sequence↗
PubMed2001

Recovery of infectious Ebola virus from complementary DNA: RNA editing of the GP gene and viral cytotoxicity.

To study the mechanisms underlying the high pathogenicity of Ebola virus, we have established a system that allows the recovery of infectious virus from cloned cDNA and thus permits genetic manipulation. We created a mutant in which the editing site of the gene encoding envelope glycoprotein (GP) was eliminated. This mutant no longer expressed the nonstructural glycoprotein sGP. Synthesis of GP increased, but most of it accumulated in the endoplasmic reticulum as immature precursor. The mutant was significantly more cytotoxic than wild-type virus, indicating that cytotoxicity caused by GP is down-regulated by the virus through transcriptional RNA editing and expression of sGP.

Animals↗
PubMed2001

Adverse effects of MVA-T7 on the transport of Marburg virus glycoprotein.

Expression of glycoproteins has been carried out successfully using recombinant vaccinia virus vectors. Especially attractive is the use of recombinant vaccinia viruses which express the DNA-dependent RNA polymerase of the phage T7 (T7-polymerase). The T7-polynerase drives the transcription of plasmid-based genes under the control of the T7 RNA polymerase promoter transfected into the infected cell. Comparison of two different recombinant vaccinia viruses, vTF7-3 and MVA-T7, revealed that post-translational processing of Marburg virus surface glycoprotein (GP) is impaired in the MVA-T7 but not in the vTF7-3 system. Influenza virus hemagglutinin, however, was transported and processed like the authentic protein in both systems. It is shown that transport of GP in the MVA-T7 system is not completely blocked, but the vast majority of molecules remained Endo H-sensitive. Only trace amounts evaded the endoplasmatic reticulum and reached the plasma membrane. Thus, the adverse effects of MVA-T7 on the processing of recombinant glycoproteins cannot be predicted, and correct processing has to be investigated for every expressed glycoprotein.

Cell Membrane↗
PubMed2001

A novel nonnucleoside inhibitor specifically targets cytomegalovirus DNA maturation via the UL89 and UL56 gene products.

3-Hydroxy-2,2-dimethyl-N-[4([[5-(dimethylamino)-1-naphthyl]sulfonyl]amino)-phenyl]propanamide (BAY 38-4766) is a novel selective nonnucleoside inhibitor of cytomegalovirus (CMV) replication with an excellent safety profile. This compound and structural analogues inhibit neither viral DNA synthesis nor viral transcription and translation. Accumulation of dense bodies and noninfectious enveloped particles coincides with inhibition of both concatemer processing and functional cleavage at intergenomic transitions, pointing to interference with viral DNA maturation and packaging of monomeric genome lengths. Resistant virus populations, including a murine CMV (MCMV) isolate with 566-fold-decreased drug sensitivity, were selected in vitro. Sequencing of the six open reading frames (ORFs) known to be essentially involved in viral DNA cleavage and packaging identified mutations in ORFs UL56, UL89, and UL104. Construction of MCMV recombinants expressing different combinations of murine homologues of mutant UL56, UL89, and UL104 and analysis of drug susceptibilities clearly demonstrated that mutant ORFs UL89 exon II (M360I) and M56 (P202A I208N) individually confer resistance to BAY 38-4766. A combination of both mutant proteins exhibited a strong synergistic effect on resistance, reconstituting the high-resistance phenotype of the in vitro mutant. These findings are consistent with genetic mapping of resistance to TCRB (2,5,6-trichloro-1-beta-D-ribofuranosyl benzimidazole) (P. M. Krosky et al., J. Virol. 72:4721-4728, 1998) and provide further indirect evidence that proteins encoded by UL89 and UL56 function as two subunits of the CMV terminase. While these studies also suggest that the molecular mechanism of BAY 38-4766 is distinct from that of benzimidazole ribonucleosides, they also offer an explanation for the excellent specificity and tolerability of BAY 38-4766, since mammalian DNA does not undergo comparable maturation steps.

Animals↗
PubMed2001

Infection and activation of monocytes by Marburg and Ebola viruses.

In this study we investigated the effects of Marburg virus and Ebola virus (species Zaire and Reston) infections on freshly isolated suspended monocytes in comparison to adherent macrophages under culture conditions. Our data showed that monocytes are permissive for both filoviruses. As is the case in macrophages, infection resulted in the activation of monocytes which was largely independent of virus replication. The activation was triggered similarly by Marburg and Ebola viruses, species Zaire and Reston, as indicated by the release of the proinflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and IL-6 as well as the chemokines IL-8 and gro-alpha. Our data suggest that infected monocytes may play an important role in the spread of filoviruses and in the pathogenesis of filoviral hemorrhagic disease.

Animals↗
PubMed2001

Sorting of Marburg virus surface protein and virus release take place at opposite surfaces of infected polarized epithelial cells.

Marburg virus, a filovirus, causes severe hemorrhagic fever with hitherto poorly understood molecular pathogenesis. We have investigated here the vectorial transport of the surface protein GP of Marburg virus in polarized epithelial cells. To this end, we established an MDCKII cell line that was able to express GP permanently (MDCK-GP). The functional integrity of GP expressed in these cells was analyzed using vesicular stomatitis virus pseudotypes. Further experiments revealed that GP is transported in MDCK-GP cells mainly to the apical membrane and is released exclusively into the culture medium facing the apical membrane. When MDCKII cells were infected with Marburg virus, the majority of GP was also transported to the apical membrane, suggesting that the protein contains an autonomous apical transport signal. Release of infectious progeny virions, however, took place exclusively at the basolateral membrane of the cells. Thus, vectorial budding of Marburg virus is presumably determined by factors other than the surface protein.

Animals↗
PubMed2000

Membrane association induces a conformational change in the Ebola virus matrix protein.

The matrix protein VP40 from Ebola virus is targeted to the plasma membrane, where it is thought to induce assembly and budding of virions through its association with the lipid bilayer. Ebola virus VP40 is expressed as a monomeric molecule in solution, consisting of two loosely associated domains. Here we show that a C-terminal truncation of seven residues destabilizes the monomeric closed conformation and induces spontaneous hexamerization in solution, as indicated by chemical cross-linking and electron microscopy. Three-dimensional reconstruction of electron microscopy images shows ring-like structures consisting of the N-terminal domain along with evidence for flexibly attached C-terminal domains. In vitro destabilization of the monomer by urea treatment results in similar hexameric molecules in solution. In addition, we demonstrate that membrane association of wild-type VP40 also induces the conformational switch from monomeric to hexameric molecules that may form the building blocks for initiation of virus assembly and budding. Such a conformational change induced by bilayer targeting may be a common feature of many viral matrix proteins and its potential inhibition may result in new anti-viral therapies.

Cell Membrane↗
PubMed2000

Molecular characterization of guinea pig-adapted variants of Ebola virus.

Serial passage of initially nonlethal Ebola virus (EBOV) in outbred guinea pigs resulted in the selection of variants with high pathogenicity. Nucleotide sequence analysis of the complete genome of the guinea pig-adapted variant 8mc revealed that it differed from wild-type virus by eight mutations. No mutations were identified in nontranscribed regions, including leader, trailer, and intragenic sequences. Among noncoding regions the only base change was found in the VP30 gene. Two silent base changes were found in the open reading frame (ORF) encoding NP protein. Nucleotide changes resulting in single-amino-acid exchanges were identified in both NP and L genes. Three other mutations found in VP24 caused amino acid substitutions, which are responsible for larger structural changes of this protein, as indicated by an alteration in electrophoretic mobility. A highly pathogenic EBOV variant K5 from another passaging series showed an amino acid substitution at nearly the same location in the VP24 gene, suggesting the importance of this protein in the adaptation process. In addition, sequence variability of the GP gene was found when plaque-purified clones of EBOV-8mc were analyzed. Three of five viral clones showed insertion of one uridine residue at the GP gene-editing site, which led to a significant change in the expression of virus glycoproteins. This observation suggests that the editing site is a hot spot for insertion and deletion of nucleotides, not only at the level of transcription but also of genome replication. Irrespective of the number of uridine residues at the editing site, all plaque-purified clones of EBOV variant 8mc resembled each other in their pathogenicity for guinea pigs, indicating either the absence or only supportive role of mutations in the GP gene on the adaptation process.

Amino Acid Substitution↗
PubMed2000

The Ebola virus VP35 protein functions as a type I IFN antagonist.

An assay has been developed that allows the identification of molecules that function as type I IFN antagonists. Using this assay, we have identified an Ebola virus-encoded inhibitor of the type I IFN response, the Ebola virus VP35 protein. The assay relies on the properties of an influenza virus mutant, influenza delNS1 virus, which lacks the NS1 ORF and, therefore, does not produce the NS1 protein. When cells are infected with influenza delNS1 virus, large amounts of type I IFN are produced. As a consequence, influenza delNS1 virus replicates poorly. However, high-efficiency transient transfection of a plasmid encoding a protein that interferes with type I IFN-induced antiviral functions, such as the influenza A virus NS1 protein or the herpes simplex virus protein ICP34.5, rescues growth of influenza delNS1 virus. When plasmids expressing individual Ebola virus proteins were transfected into Madin Darby canine kidney cells, the Ebola virus VP35 protein enhanced influenza delNS1 virus growth more than 100-fold. VP35 subsequently was shown to block double-stranded RNA- and virus-mediated induction of an IFN-stimulated response element reporter gene and to block double-stranded RNA- and virus-mediated induction of the IFN-beta promoter. The Ebola virus VP35 therefore is likely to inhibit induction of type I IFN in Ebola virus-infected cells and may be an important determinant of Ebola virus virulence in vivo.

Animals↗
PubMed2000

Crystal structure of the matrix protein VP40 from Ebola virus.

Ebola virus maturation occurs at the plasma membrane of infected cells and involves the clustering of the viral matrix protein VP40 at the assembly site as well as its interaction with the lipid bilayer. Here we report the X-ray crystal structure of VP40 from Ebola virus at 2.0 A resolution. The crystal structure reveals that Ebola virus VP40 is topologically distinct from all other known viral matrix proteins, consisting of two domains with unique folds, connected by a flexible linker. The C-terminal domain, which is absolutely required for membrane binding, contains large hydrophobic patches that may be involved in the interaction with lipid bilayers. Likewise, a highly basic region is shared between the two domains. The crystal structure reveals how the molecule may be able to switch from a monomeric conformation to a hexameric form, as observed in vitro. Its implications for the assembly process are discussed.

Amino Acid Sequence↗
PubMed2000

Structural characterization and membrane binding properties of the matrix protein VP40 of Ebola virus.

The matrix protein VP40 of Ebola virus is believed to play a central role in viral assembly as it targets the plasma membrane of infected cells and subsequently forms a tightly packed layer on the inner side of the viral envelope. Expression of VP40 in Escherichia coli and subsequent proteolysis yielded two structural variants differing by a C-terminal truncation 114 amino acid residues long. As indicated by chemical cross-linking studies and electron microscopy, the larger polypeptide was present in a monomeric form, whereas the truncated one formed hexamers. When analyzed for their in vitro binding properties, both constructs showed that only monomeric VP40 efficiently associated with membranes containing negatively charged lipids. Membrane association of truncated, hexameric VP40 was inefficient, indicating a membrane-recognition role for the C-terminal part. Based on these observations we propose that assembly of Ebola virus involves the formation of VP40 hexamers that is mediated by the N-terminal part of the polypeptide.

Amino Acid Sequence↗
PubMed2000

Proteolytic processing of Marburg virus glycoprotein.

Processing of the transmembrane glycoprotein (GP) of Marburg virus involved the conversion of an endo H-sensitive, ER-specific form into an endo H-resistant, Golgi-specific precursor that was cleaved into GP(1) and GP(2). Cleavage was mediated by furin or another subtilisin-like endoprotease with similar substrate specificity as indicated by mutational analysis of the cleavage site and inhibition using peptidyl chloromethylketones. Mature GP consisted of disulfide-linked GP(1) and GP(2) subunits.

Animals↗
PubMed2000

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.

Amino Acid Sequence↗