Biomedical subjects

E Wimmer

Publications and source records attributed to E Wimmer.

At least 127 records · Page 7Linked to original sources

PubMed1991

Poliovirus can enter and infect mammalian cells by way of an intercellular adhesion molecule 1 pathway.

Mouse fibroblast cell lines were transfected with truncated forms of the human poliovirus receptor (PVR) cDNA and tested for the expression of functional receptors for poliovirus. Several receptor constructs, all containing the coding region of the first 143 amino acids of PVR, were able to render mouse cells susceptible to poliovirus infection. A deletion of 65 amino acids in the first extracellular domain of PVR prevented virus attachment and infection. These data suggest that domain 1 is necessary and sufficient for the virus-receptor interaction. A PVR/intercellular adhesion molecule 1 hybrid receptor, expressing the PVR variable domain on a truncated receptor molecule for human rhinovirus 14, was shown to be a functional receptor for poliovirus. This observation indicates that, subsequent to attachment to the PVR-binding domain, poliovirus can use the same pathway as the major receptor group rhinoviruses to enter cells.

Animals↗

PubMed1991

Site-directed mutagenesis of the putative catalytic triad of poliovirus 3C proteinase.

Based on predictions of the structure of proteinase 3C of poliovirus, mutations have been made at residues that are supposed to constitute the catalytic triad. Wild-type and mutant 3C were expressed in Escherichia coli, purified to homogeneity, and characterized by the ability to cleave a synthetic peptide substrate or an in vitro translated polypeptide consisting of part of the polyprotein of poliovirus. Additionally, the ability of autocatalytic processing of a precursor harboring wild-type or mutant 3C sequences was tested. Single substitutions of the residues His-40, Glu-71, and Cys-147 by Tyr, Gln, and Ser, respectively, resulted in an inactive enzyme. Replacement of Asp-85 by Asn resulted in an enzyme that was as active as wild-type enzyme in trans cleavage assays but whose autoprocessing ability was impaired. Our results are consistent with the proposal that residues His-40, Glu-71, and Cys-147 constitute the catalytic triad of poliovirus 3C proteinase. Furthermore, residue Asp-85 is not required for proper proteolytic activity despite being highly conserved between different picornaviruses. This indicates that Asp-85 might be involved in a different function of 3C.

Amino Acid Sequence↗

PubMed1991

Poliovirus proteinase 3C converts an active form of transcription factor IIIC to an inactive form: a mechanism for inhibition of host cell polymerase III transcription by poliovirus.

In HeLa cells, RNA polymerase III (pol III)-mediated transcription is severely inhibited by poliovirus infection. This is due primarily to a reduction in the transcriptional activity of TFIIIC, a transcription factor which binds in a sequence specific manner to the internal promoter of pol III genes. Using gel retardation assays, we have shown previously that inhibition of pol III transcription by poliovirus is correlated with disappearance of a transcriptionally active form of TFIIIC (complex I) concomitant with the appearance of a faster mobility, transcriptionally inactive form of TFIIIC (complex III). We show here that a poliovirus with a point mutation in the proteinase 3C (3Cpro) region failed to produce complex III and is limited in its ability to inhibit pol III transcription compared with the wild-type virus. Incubation of purified 3Cpro, expressed in Escherichia coli, with transcriptionally active TFIIIC (complex I) in vitro resulted in generation of the transcriptionally inactive complex III form of TFIIIC. In an in vitro transcription assay, treatment of the complex I form of TFIIIC with 3Cpro almost completely inhibited pol III transcription. Finally expression of the 3Cpro gene in transfected HeLa cells resulted in significant inhibition of pol III-mediated transcription. The results presented here suggest that proteolysis of the transcriptionally active form of TFIIIC by poliovirus 3Cpro is a mechanism by which poliovirus inhibits host cell RNA pol III transcription.

3C Viral Proteases↗

PubMed1991

Mutational analysis of a native substrate of the HIV-1 proteinase.

The purpose of this study was to define further the determinants of substrate specificity of HIV-1 PR. Rather than using small peptides, we used an in vitro system which permitted us to evaluate the effect of a mutated site within the context of its natural precursor. We made single-amino-acid substitutions around two sites which are processed by the HIV-1 PR. The Tyr/Pro site within gag appears to encode highly specific determinants which direct proteinase processing between MA and CA. The Phe/Pro site in pol, however, appears to be far more tolerant to amino acid substitutions, as none of our single-amino-acid substitutions blocked cleavage at or around this site. The increased tolerance of the Phe/Pro site may indicate that at this site, structural features are more important determinants of cleavage than primary amino acid sequence. We have shown that sequences outside of those encoding mature PR can inhibit proteolytic processing in this system. By preventing PR from cleaving itself from the polyprotein prematurely, p6* sequences would regulate morphogenesis and infectious particle formation. Late in infection, when the protein concentration of gag and gag/pol polyproteins at the cell surface becomes very high, cooperative protein-protein interactions may cause alterations of a p6*-PR interaction, relieving repression and permitting autocatalysis.

Amino Acid Sequence↗

PubMed1991

Construction and characterization of a poliovirus/rhinovirus antigenic hybrid.

In order to study the properties of foreign antigenic sites expressed on poliovirus a hybrid was constructed in which neutralization antigenic site IA of poliovirus type 1 strain Mahoney [PV1(M)] was replaced by neutralization immunogenic site IA (NImIA) of human rhinovirus 14 (HRV14). The resulting hybrid was viable, but growth was impaired in comparison to PV1(M). The hybrid expressed both PV1(M) and HRV14 antigenic determinants. When inoculated into rabbits it elicited neutralizing antibodies against both PV1(M) and HRV14. Furthermore, the hybrid was efficiently neutralized by polyclonal antisera specific for either PV1(M) or HRV14 and by three out of five monoclonal antibodies directed to NImIA. The monoclonal antibodies also blocked binding of the hybrid to the cellular receptor for poliovirus. One of them is thought to neutralize rhinovirus in this manner, and it appears that NImIA is expressed in a sufficiently favorable context on the hybrid for the same mechanism to be effective. This can be interpreted to mean that the interactions between the parental viruses and their respective cellular receptors are very similar.

Amino Acid Sequence↗

PubMed1991

Vaccinia virus-mediated expression and identification of the human poliovirus receptor.

We have used cDNA encoding the cellular receptor for poliovirus (PVR) to prepare polyclonal antisera against beta-galactosidase PVR fusion proteins. One of these antisera allowed identification of a glycoprotein doublet band of about 67 kDa in membrane preparations of HeLa cells and in a PVR cosmid-bearing mouse cell line. In vitro translation of PVR-specific transcripts gave rise to a protein of 46 kDa; the product had a molecular weight of 67 kDa when microsomal membranes were added to the cell-free extract. Overexpression of PVR cDNA in mouse L-cells by means of a recombinant vaccinia virus led to the synthesis of a glycoprotein having a molecular weight identical to that of the glycosylated in vitro product. The vaccinia virus-mediated protein was also recognized by a monoclonal antibody that blocks poliovirus infection. Its biological activity was demonstrated by poliovirus binding and infectivity assays. The data show that PVR is a glycoprotein of 67 kDa and that this protein is sufficient to confer poliovirus susceptibility to mouse cells.

Animals↗

PubMed1991

The effect of site and mode of expression of a heterologous antigenic determinant on the properties of poliovirus hybrids.

The antigenic site normally situated in the EF loop of VP2 (2EF) of poliovirus type 2 (Lansing) [PV2 (L)] was expressed in 2EF or in the BC loop of VP1 (1BC) of PV1 (Mahoney) [PV1 (M)]. A hybrid virus expressing the site in 2EF of PV1 (M) is known to be neutralizable by PV2 (L)-specific antisera and to induce neutralizing antibodies against PV-2 (L). In contrast, a hybrid expressing a related sequence in 1BC of PV1 (M), which maintained the length of the native BC loop, was not neutralizable by PV2 (L)-specific antisera and did not induce PV2 (L)-neutralizing antibodies. However, when 1BC was extended, so that it was longer than the native 1BC, the resulting hybrids induced low titers of PV2 (L)-neutralizing antibody although they were still not neutralizable by PV2 (L)-specific antisera. Synthetic peptides copying the extended sequences raised neutralizing antibodies which were able to distinguish between the sequence expressed in the extended 1BC, in the native-length 1BC and in 2EF. The growth rates of the hybrids with modifications to 1BC depended upon the nature of the modifications. The hybrid with the native length 1BC grew poorly, but extending 1BC tended to improve the growth rate, and extending 1BC with PV1 (M)-specific sequences nearly restored wild-type growth rates. Thus, the size, precise sequence and location of a heterologous antigenic site expressed on poliovirus have significant effects on the properties of that determinant and of the hybrid expressing it. Antigenicity of hybrids can be modified and their growth rate can be enhanced by appropriate choices of these parameters.

Amino Acid Sequence↗

PubMed1991

Phenotypic characterization of antigenic hybrids of poliovirus.

Three poliovirus hybrids, modified in neutralization antigenic sites (NAgs) I or II, were characterized for several phenotypic traits. The modifications to the capsid interfered with some stage of the life-cycle of the virus, since all three hybrids were growth-impaired in comparison to poliovirus type 1 (Mahoney) [PV1 (M)], the wild-type parent virus. All hybrids exhibited a reduced growth rate and a small-plaque phenotype, but they were not temperature sensitive. Furthermore, only one hybrid was slightly less stable to heating than the parent virus; the other two were as stable as the parent. Therefore, decreased thermal stability of the capsid is not an important cause of the poor growth characteristics of these hybrids.

Amino Acid Sequence↗

PubMed1991

Antibodies may act synergistically or additively with interferon to inhibit poliovirus.

We investigated the protective effects of combinations of interferon-alpha (IFN-alpha) and neutralizing monoclonal antibodies (mAbs) and serum antibodies against poliovirus type 1 (PV-1) in vitro. Our results indicate that the antiviral effects of IFN-alpha and most neutralizing mAbs to PV-1 act synergistically to inhibit PV-1. However, the antiviral effects of IFN-alpha and one type specific mAb to PV-1 were additive. Further, the protective effects observed with combinations of IFN-alpha and rabbit, monkey or human serum containing effects observed with combinations strains Mahoney (Mah) and Sabin (Sab) were similar to those observed with combinations of IFN-alpha and mixtures of mAbs with synergistic and additive activities. Our studies suggest that the antiviral activity of neutralizing antibody acts with the antiviral activity of IFN to inhibit virus infection synergistically or additively and that the different antibody activities are associated with the mechanism of neutralization.

Animals↗

PubMed1991

Specificity of the polioviral proteinase 3C towards genetically engineered cleavage sites in the viral capsid.

In a study of the cleavage specificity of poliovirus proteinase 3Cpro, two mutant polioviruses were constructed to include putative 3Cpro cleavage sites in the BC loop of VP1. The BC loop of VP1 in the wild-type virus is the neutralization antigenic site IA, consisting of a continuous chain of nine amino acids (ASTTNKDKL). The first mutant, W1-1D-BC1, has four altered amino acids in the BC loop (ASTQGPGKL); the second mutant, W1-1D-BC2, has an insertion of nine amino acids in the BC loop (ASTGTAKVQGPGNKDKL). W1-1D-BC1 and W1-1D-BC2 were viable, grew to high titre and produced plaques of normal size. W1-1D-BC1 virions were resistant to proteolytic cleavage of the BC loop in vivo as well as upon incubation with a large excess of 3Cpro in vitro, although a synthetic decapeptide (PASTQGPGKL) containing the amino acids of the BC loop in W1-1D-BC1 was cleaved by 3Cpro. In contrast, W1-1D-BC2 yielded virus the VP1 of which was cleaved partially in vivo and completely when incubated with 3Cpro in vitro. Our results showed that an insertion of nine amino acids into the antigenic loop of poliovirus, representing a synthetic 3Cpro cleavage site, renders the loop susceptible to cleavage by proteinase 3Cpro, but that this cleavage is restricted if the loop is the length of that in the native virion. This result implies that, in this case, structural restrictions override sequence determinants for cleavage of the BC loop by 3Cpro.

3C Viral Proteases↗

PubMed1991

Catalysis of poliovirus VP0 maturation cleavage is not mediated by serine 10 of VP2.

The maturation of the poliovirus capsid occurs as the result of a single unexplained proteolytic event during which 58 to 59 copies of the 60 VP0 capsid protein precursors are cleaved. An autocatalytic mechanism for cleavage of VP0 to VP4 and VP2 was proposed by Arnold et al. (E. Arnold, M. Luo, G. Vriend, M. G. Rossman, A. C. Palmenberg, G. D. Parks, M. J. Nicklin, and E. Wimmer, Proc. Natl. Acad. Sci. USA 84:21-25, 1987) in which serine 10 of VP2 is activated by virion RNA to catalyze VP4-VP2 processing. The hypothesis rests on the observation that a hydrogen bond was observed between serine 10 of VP2 (S2010) and the carboxyl terminus of VP4 in three mature picornaviral atomic structures: rhinovirus 14, mengovirus, and poliovirus type 1 (Mahoney). We constructed mutant viruses with cysteine (S2010C) or alanine (S2010A) replacing serine 10 of VP2; these exhibited normal proteolytic processing of VP0. While our results do not exclude an autocatalytic mechanism for the maturation cleavage, they do eliminate the conserved S2010 residue as the catalytic amino acid.

Amino Acid Sequence↗

PubMed1991

Translation of poliovirus RNA: role of an essential cis-acting oligopyrimidine element within the 5' nontranslated region and involvement of a cellular 57-kilodalton protein.

Translation of poliovirus RNA is initiated by cap-independent internal entry of ribosomes into the 5' nontranslated region. This process is dependent on elements within the 5' nontranslated region (the internal ribosomal entry site) and may involve novel translation factors. Systematic mutation of a conserved oligopyrimidine tract has revealed a cis-acting element that is essential for translation in vitro. The function of this element is related to its position relative to other cis-acting domains. This element is part of a more complex structure that interacts with several cellular factors, but changes in protein binding after mutation of this element were not detected in a UV cross-linking assay. A 57-kDa protein from the ribosomal salt wash fraction of HeLa cells was identified that binds upstream of the oligopyrimidine tract. Translation of poliovirus mRNA in vitro was strongly and specifically inhibited by competition with the p57-binding domain (nucleotides 260 to 488) of the 5' nontranslated region of encephalomyocarditis virus, indicating a probable role for p57 in poliovirus translation. p57 is likely to be identical to the ribosome-associated factor that binds to and is necessary for the function of the internal ribosomal entry site of encephalomyocarditis virus RNA.

Base Sequence↗

PubMed1991

Characterization of poliovirus 2A proteinase by mutational analysis: residues required for autocatalytic activity are essential for induction of cleavage of eukaryotic initiation factor 4F polypeptide p220.

The poliovirus proteinase 2A is autocatalytically released from the poliovirus polyprotein by cotranslational cleavage at its own amino terminus, resulting in separation of structural and nonstructural protein precursors. Cleavage is a prerequisite for further processing of the structural protein precursor and consequently for poliovirus encapsidation. A second function of 2Apro is in the rapid shutoff of host cell protein synthesis that occurs upon infection with poliovirus. This is associated with proteolytic cleavage of the p220 component of eukaryotic initiation factor eIF-4F, which is induced but not directly catalyzed by 2Apro. We introduced single-amino-acid substitutions in the 2Apro-coding region of larger poliovirus precursors that were subsequently translated in vitro and thus demonstrated that His-20, Asp-38, and Cys-109 (which constitute the putative catalytic triad) are essential for, and that His-117 is an important determinant of, the autocatalytic activity of 2Apro. This is consistent with the proposal that 2Apro is structurally related to a subclass of trypsinlike serine proteinases. Moreover, 2Apro containing a Cys109Ser substitution retained a small but significant autocatalytic activity. Cleavage of p220 was not induced by those mutants that had reduced proteolytic activity, indicating that the cellular factor that cleaves p220 is probably activated by 2Apro-catalyzed proteolytic cleavage.

DNA Mutational Analysis↗

PubMed1991

[CA-5000 (Sysmex). A contribution to the complete automation in blood coagulation analysis].

The Sysmex CA-5000 is a completely automated analysis device for the determination of various coagulation tests. The measurement is based on the scattered light measurement in the 90 degree angle. The apparatus is characterized by simple operation, great flexibility in the selection of reagents and by excellent precision. The comparability with globular coagulometry is described.

Autoanalysis↗

PubMed1990

Expression in Escherichia coli and purification of human immunodeficiency virus type 1 capsid protein (p24).

Capsid protein (p24;CA) of human immunodeficiency virus type 1 (HIV-1) was synthesized in Escherichia coli strain BL21 (DE3) using a plasmid encoding a truncated HIV-1 gag/pol gene. The plasmid, which contained a mutation in the frameshift region, expressed viral proteinase (PR), a pol gene product, in the gag reading frame, resulting in efficient processing of mature CA and other gag-related products. The expressed CA is soluble, recognized by monoclonal antibodies directed against HIV CA and has an N-terminal sequence identical to that of CA purified from HIV. Purification was done under mild conditions where coexpressed HIV PR retained enzymatic activity. Milligram quantities of 90% pure CA protein were obtained after chromatography on DEAE cellulose followed by facilitated aggregation of the CA in the unbound fraction. The precipitated CA was readily dissolved in low ionic strength aqueous buffer. Gel exclusion chromatography results indicated that, in solution, CA existed in oligomeric form.

Base Sequence↗

PubMed1990

Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein.

Translation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal internal entry into the 5'-nontranslated region (5' NTR) rather than by ribosomal scanning. The internal ribosomal entry site (IRES) in the EMCV 5' NTR was determined by in vitro translation with RNAs that were generated by in vitro transcription of EMCV cDNAs containing serial deletions from either the 5' or 3' end of the EMCV 5' NTR. Regions downstream of nucleotide 403 and upstream of nucleotide 811 of EMCV were required for efficient translation. Site-directed mutagenesis revealed that a stem-loop structure (400 nucleotides upstream of the initiation codon) was essential for IRES function. We discovered a 57-kD cellular protein whose specific interaction with this stem-loop appears to be prerequisite for IRES function. A A pyrimidine-rich stretch proximal to the initiation codon was also crucial for efficient translation of EMCV mRNA. We propose that ribosomes bind directly to the initiating AUG without scanning.

Animals↗

PubMed1990

Improved distribution of antigenic site specificity of poliovirus-neutralizing antibodies induced by a protease-cleaved immunogen in mice.

Previous studies showed that the distribution of antigenic site specificity of neutralizing antibodies to type 3 poliovirus obtained with the inactivated poliovirus vaccine can be deficient as compared with that obtained following poliovirus infection. This observation was shown by the relatively low capacity of sera from inactivated-poliovirus-vaccine-immunized persons to neutralize poliovirus cleaved at antigenic site 1. We investigated possibilities for improving the situation in a mouse model. Balb/c mice were immunized with intact or trypsin-cleaved type 3 poliovirus (Saukett strain). Sera from mice immunized with the intact virus readily neutralized the intact virus but neutralized the cleaved virus only rarely. In contrast, cleaved-virus-immunized mice produced antibodies that were able to neutralize the cleaved virus as well as the intact one. Mice immunized with a 100-fold-higher dose of the intact virus produced significant levels of antibodies to the cleaved virus, too. Somewhat surprisingly, mice immunized with high doses of the cleaved virus produced antibodies specific for the intact loop between beta sheets B and C of VP1 (virion protein 1), which should be cleaved in the immunogen. This was shown by a higher titer of antibodies to intact Saukett virus than to the corresponding cleaved virus, as well as to a type 1/type 3 hybrid poliovirus in which only the BC loop amino acids were derived from type 3 poliovirus. The cleavage-induced enhanced availability of antigenic determinants residing outside the BC loop was also shown by increased neutralization titers of monoclonal antibodies specific for some of these other determinants. These results indicate that by using a trypsin-cleaved type 3 poliovirus as a parenteral immunogen, it is possible to change the distribution of antigenic site specificities of neutralizing antibodies to resemble that following poliovirus infection.

Animals↗

PubMed1990

Very high frequency of reversion to guanidine resistance in clonal pools of guanidine-dependent type 1 poliovirus.

We have carefully examined the frequency of guanidine-resistant revertants in six different clonal pools of guanidine-dependent mutants of type 1 poliovirus. The mutation frequency was (6.5 +/- 6.3) x 10(-4) (with all amino acid substitutions occurring at position 227). The minimal corrected base substitution frequency per single nucleotide site in the codon for amino acid 227 was (2.1 +/- 1.9) x 10(-4).

Base Sequence↗