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M J Grubman

Publications and source records attributed to M J Grubman.

At least 19 recordsLinked to original sources
PubMed1992

Identification and characterization of the structural and nonstructural proteins of African horsesickness virus and determination of the genome coding assignments.

Proteins present in purified African horsesickness virus (AHSV) and in infected cells were analyzed by SDS-polyacrylamide gel electrophoresis. Twelve viral proteins were identified, one minor and four major structural proteins, three major and two minor nonstructural proteins, as well as variable amounts of two additional structural proteins. Cell-free translation of total AHS virion RNA in a rabbit reticulocyte system resulted in the synthesis of proteins which were qualitatively and quantitatively similar to those found in infected cells. The in vivo and in vitro synthesized proteins were viral specific as demonstrated by immunoprecipitation. The coding assignments of all the purified genome segments were determined by in vitro translation and confirmed by immunoprecipitation.

African Horse Sickness Virus↗
PubMed1992

Antiviral effects of a thiol protease inhibitor on foot-and-mouth disease virus.

The thiol protease inhibitor E-64 specifically blocks autocatalytic activity of the leader protease of foot-and-mouth disease virus (FMDV) and interferes with cleavage of the structural protein precursor in an in vitro translation assay programmed with virion RNA. Experiments with FMDV-infected cells and E-64 or a membrane-permeable analog, E-64d, have confirmed these results and demonstrated interference in virus assembly, causing a reduction in virus yield. In addition, there is a lag in the appearance of virus-induced cellular morphologic alterations, a delay in cleavage of host cell protein p220 and in shutoff of host protein synthesis, and a decrease in viral protein and RNA synthesis. The implications of using E-64-based compounds as potential antiviral agents for FMDV are discussed.

Animals↗
PubMed1991

Expression, processing, and assembly of foot-and-mouth disease virus capsid structures in heterologous systems: induction of a neutralizing antibody response in guinea pigs.

Plasmids containing the foot-and-mouth disease virus structural protein precursor (P1) and 3C protease genes or the P1 gene alone were expressed in Escherichia coli. A recombinant baculovirus containing the P1 gene was also generated and expressed in Spodoptera frugiperda cells. Expression of the P1 and 3C genes in E. coli resulted in efficient synthesis and processing of the structural protein precursor and assembly into 70S empty capsids. This material reacted with neutralizing monoclonal antibodies which recognize only conformational epitopes and elicited a significant neutralizing antibody response in vaccinated guinea pigs. Expression of the P1 gene in E. coli resulted in synthesis of an insoluble product, whereas in insect cells infected with the recombinant baculovirus a soluble product was synthesized. Both soluble and insoluble P1 reacted with a 12S-specific monoclonal antibody, but only soluble P1 elicited a neutralizing antibody response in guinea pigs.

Animals↗
PubMed1990

Nucleotide and deduced amino acid sequence of the nonstructural phosphoprotein, NS2, of bluetongue virus serotype 17: comparison to two isolates of serotype 10.

The nucleotide sequence of bluetongue virus (BTV) serotype 17 segment 8 from North America (NA) coding for the nonstructural phosphoprotein, NS2, was determined. This segment contains 1125 base pairs and codes for a protein of 40,581 daltons containing 354 amino acids with a net charge of -8.5 at pH 7.0. The carboxyl terminal portion of the protein is very hydrophilic and has a high degree of potential alpha-helix. Serine is the major, if not the exclusive, phosphorylated amino acid residue and ten of the twenty serine residues present in NS2 are found in consensus phosphorylation sites. Comparison of the nucleotide sequence of BTV-17NA segment 8 with the sequence of BTV-10NA and BTV-10 South Africa (SA) revealed a greater degree of homology between different serotypes within the same geographical area, i.e., 17NA and 10NA, than between isolates of the same serotype located in different areas, i.e., 10NA and 10SA. The same homology relationship as above was found at the amino acid level.

Amino Acid Sequence↗
PubMed1990

Bluetongue virus: surface exposure of VP7.

The exposed proteins of bluetongue virus serotype 17 were determined using surface labeling and reactivity with monoclonal antibodies. Iodination of amino groups predominantly labeled VP2; however, iodination of tyrosine residues labeled both VP2 and VP5, with VP7 labeled to a significantly lesser degree. To investigate the exposure of VP7 on the intact virion further, monoclonal antibodies that reacted with this protein were used. At least two antibodies, reacting with different epitopes on VP7, bound to intact virions, as determined by adsorption of infectious particles, electron microscopic observation of antibody-bound virus, and co-sedimentation of antibody and virus. Surface iodination of viral cores was used to show that VP7 and VP3 are major exposed proteins on these particles. We conclude that a major core protein, VP7, has at least two epitopes exposed on the virus surface.

Antibodies, Monoclonal↗
PubMed1990

Expression of bluetongue virus serotype 17 NS1 protein from a cloned gene.

A full-length copy of the coding region of segment 6 from bluetongue virus (BTV) serotype 17 was constructed from five overlapping cDNA clones. The gene coding for the NS1 protein was cloned into an expression plasmid under the control of a bacteriophage T7 promoter and expressed both in vitro and in Escherichia coli BL21(DE3) cells which contain a T7 RNA polymerase gene in their chromosome. Expression in both systems resulted in the synthesis of a protein comigrating with NS1 and a minor polypeptide comigrating with another viral-induced protein, NS1a, sometimes seen in BTV-infected cells. The proteins induced in E. coli were synthesized to high levels as insoluble products.

Bluetongue virus↗
PubMed1988

Analysis of the polypeptides synthesized in rinderpest virus-infected cells.

We have identified, by [35S]methionine labeling, eight major induced proteins and a number of minor proteins in rinderpest virus-infected bovine kidney cells. The polypeptides ranged in molecular weight from 212 to 21.5 kDa. The majority of these polypeptides are virus specific, as demonstrated by immunoprecipitation with rabbit hyperimmune serum against rinderpest. Infected cells radiolabeled with glucosamine contained a 75-kDa polypeptide and a broad band migrating at 80 kDa, both identified as virus specific by immunoprecipitation. Phosphorylated virus-specific proteins of 65 kDa and a complex of polypeptides at 92.5 kDa were also identified. Monospecific and monoclonal antibodies against measles virus and canine distemper virus hemagglutinin, fusion protein, nucleocapsid protein, and phosphoproteins confirmed the identity of the corresponding rinderpest virus-specific polypeptides.

Animals↗
PubMed1988

Characterization of a nonstructural phosphoprotein of two orbiviruses.

A phosphorylated nonstructural protein, NS2, was detected in bluetongue and African horsesickness virus (BTV and AHSV) infected-radiolabeled-cell lysates by electrophoresis on SDS-polyacrylamide gels (SDS-PAGE). The NS2 proteins of both viruses have similar migration on one-dimensional (1D) 10% SDS-PAGE. Examination of infected cell lysates on two-dimensional (2D) gels (isoelectric focusing followed by SDS-PAGE) separated two phosphorylated isoelectric forms of BTV NS2 and four phosphorylated forms of AHSV NS2. The isoelectric points of both species of BTV NS2 were acidic relative to all forms of AHSV NS2. Nonphosphorylated NS2 polypeptides were not detected by 2D gels. A nonphosphorylated host protein, which comigrated with NS2 on 1D gels, could be distinguished from viral proteins by isoelectric focusing on 2D gels. High performance liquid chromatography (HPLC) elution profiles of NS2 tryptic peptides from the two orbiviruses were compared. Three 32P-labeled tryptic peptides were generated from both AHSV and BTV NS2 proteins, which had been isolated and eluted from SDS-polyacrylamide gels. The elution profile from reverse phase HPLC was very similar for the tryptic phosphopeptides; in contrast, 35S-labeled tryptic peptides displayed considerable differences in elution profiles for the two NS2 proteins. Phosphoamino acid analysis revealed only phosphoserine in hydrolysates of BTV and AHSV NS2.

African Horse Sickness Virus↗
PubMed1988

Relationship of p220 cleavage during picornavirus infection to 2A proteinase sequencing.

Infection of HeLa cells by poliovirus results in an abrupt inhibition of host cell protein synthesis. It is thought that the mechanism of this inhibition involves proteolytic cleavage of the p220 component of the cap-binding protein complex, thereby causing functional inactivation of the cap-binding protein complex and preventing capped (cellular) mRNAs from binding ribosomes. Current data suggest that the viral proteinase 2A indirectly induces p220 cleavage via alteration or activation of a second proteinase of cellular origin. We present evidence that translation of poliovirus proteinase 2A sequences in vitro activates p220 cleavage. We have also aligned published picornavirus 2A amino acid sequences for maximum homology, and we show that the picornaviruses can be divided into two classes based on the presence or absence of a highly conserved 18-amino acid sequence in the carboxy-terminal portion of 2A. This conserved 2A sequence is homologous with the active site of the cysteine proteinase 3C common to all picornaviruses. We show that picornaviruses which contain the putative 2A active site sequence (e.g., enteroviruses and rhinoviruses) will induce cleavage of p220 in vivo. Conversely, we show that two cardioviruses (encephalomyocarditis virus and Theiler's encephalomyelitis virus) do not encode this putative proteinase sequence in the 2A region and do not induce cleavage of p220 in vivo. The foot-and-mouth disease virus (FMDV) 2A sequence represents an apparent deletion and consists of only 16 amino acids, most homologous with the carboxy terminus of the cardiovirus 2A sequence. It does not contain the putative cysteine proteinase active site. However, FMDV infection induces complete cleavage of BK cell p220, and translation of FMDV RNA in vitro induces an activity that cleaves HeLa cell p220. The data predict that an alternate FMDV viral protease is responsible for the induction of p220 cleavage.

Amino Acid Sequence↗
PubMed1988

Leader protein of foot-and-mouth disease virus is required for cleavage of the p220 component of the cap-binding protein complex.

Suppression of host protein synthesis in cells infected by poliovirus and certain other picornaviruses involves inactivation of the cap-binding protein complex. Inactivation of this complex has been correlated with the proteolytic cleavage of p220, a component of the cap-binding protein complex. Since picornaviral RNA is not capped, it continues to be translated as the cap-binding protein complex is inactivated. The cleavage of p220 can be induced to occur in vitro, catalyzed by extracts from infected cells or by reticulocyte lysates translating viral RNA. Expression of polioviral protease 2A is sufficient to induce p220 cleavage, and the presence in 2A of an 18-amino-acid sequence representing a putative cysteine protease active site correlates with the ability of different picornaviruses to induce p220 cleavage. Foot-and-mouth disease virus (FMDV) infection induces complete cleavage of p220, yet the FMDV genome codes for a 2A protein of only 16 amino acids, which does not include the putative cysteine protease active site. Using cDNA plasmids encoding various regions of the FMDV genome, we have determined that the leader protein is required to initiate p220 cleavage. This is the first report of a function for the leader protein, other than that of autocatalytic cleavage from the FMDV polyprotein.

Aphthovirus↗
PubMed1987

Antigenic comparison of the polypeptides of foot-and-mouth disease virus serotypes and other picornaviruses.

The cross-reactivity of proteins coded for by the seven serotypes of foot-and-mouth disease virus (FMDV) was assessed by reaction of infected cell lysates with polyclonal and monospecific antisera against the structural and nonstructural proteins of FMDV type A12 strain 119ab. It was shown that the homologous polypeptides from most serotypes are antigenically related. The least cross-reactivity occurred between VP1, VP3, and the protease (3C) of type A12 and South African Territories types 1 and 3. There was also a reduced degree of reactivity of A12 VP1 serum with VP1 from some A subtypes and the other serotypes. Comparison of FMDV proteins with polypeptides from other picornaviruses by a radioimmune binding assay revealed a low level of reactivity of antisera against some A12 polypeptides with encephalomyocarditis virus (EMCV) infected cell lysates but no reactivity with bovine enterovirus type 1 and swine vesicular disease virus infected cells. The same EMCV proteins were immunoprecipitated by the various reactive A12 antisera, but the reaction was abolished if the lysate from EMCV infected cells was denatured prior to immunoprecipitation.

Animals↗
PubMed1987

Proteolytic processing of foot-and-mouth disease virus polyproteins expressed in a cell-free system from clone-derived transcripts.

All picornaviral genes are expressed as a single, large polyprotein, which is proteolytically processed into the system produces functional proteins, including viral protease 3C, which plays a major role in processing the precursor proteins. To study the function of the two putative proteases 3C and leader (L) in processing, we constructed several cDNA plasmids encoding various regions of the FMDV type A12 genome. These plasmids, containing FMDV cDNA segments under the control of the T7 promoter, were transcribed in vitro by using T7 RNA polymerase and then translated in rabbit reticulocyte lysates. The expressed FMDV gene products were identified by immunoprecipitation with specific antisera and analyzed by gel electrophoresis. The results demonstrate the following: (i) the leader protein, L, is processed from the structural protein precursor, P1, in the absence of any P2 or P3 region proteins; (ii) protein 2A remains associated with the structural protein precursor, P1, rather than the precursor, P2; (iii) the processing of the P1-2A/P2 junction is not catalyzed by 3C or L; (iv) the proteolytic processing of polyproteins from the structural P1 region (except VP4/VP2) and the nonstructural P2 and P3 region is catalyzed by 3C.

Animals↗
PubMed1986

Antigenic comparison of foot-and-mouth disease virus serotypes with monoclonal antibodies.

The capsid structures of the 7 serotypes of foot-and-mouth disease virus have been compared utilizing a series of neutralizing monoclonal antibodies which were previously shown to recognize at least 4 distinct epitopes on type A12 virus. A radioimmune binding assay using radioactively labeled antigens and the monoclonal antibodies revealed that certain conformation dependent epitopes are conserved among A subtypes, while some continuous epitopes are conserved among A subtypes as well as other FMDV serotypes. On the basis of differential reactivity among other FMDV subtypes and serotypes two additional epitopes have been defined on the A12 particle. Binding and neutralization assays revealed that the presence and function of an epitope are not necessarily correlated.

Animals↗
PubMed1985

Sequence variation in the gene for the immunogenic capsid protein VP1 of foot-and-mouth disease virus type A.

The nucleotide sequences have been determined and compared from cloned cDNA genes coding for the foot-and-mouth disease virus (FMDV) immunogenic capsid protein, VP1, from eight different A subtypes: A5 Westerwald/58, A12 119ab (large plaque variant), A22 550 USSR/65, A24 Cruzeiro Brazil/55, A27 Cundinamarca Colombia/76, A32 Venezuela/70, A Venceslau Brazil/76, and A Argentina/79. We have also found sequence variations among different cDNA clones of the A5 and A24 subtypes. There are regions of nucleotide sequence within the VP1 gene that vary considerably among the subtypes as well as other regions that remain relatively constant. One highly variable region (codons 130-171) encodes amino acids previously identified as being exposed on the virus surface and constituting an important immunogenic site of the virus. There potentially exist secondary structures within the viral RNA sequences that code for this immunogenic site that could decrease the fidelity of replication at this sequence. The rapid generation of FMDV variants encouraged by such structures in the RNA could work together with various selective pressures to explain the observed accumulation of immunologically distinct viruses of the FMDV A type.

Aphthovirus↗
PubMed1985

Nucleotide and amino acid sequence coding for polypeptides of foot-and-mouth disease virus type A12.

The coding region for the structural and nonstructural polypeptides of the type A12 foot-and-mouth disease virus genome has been identified by nucleotide sequencing of cloned DNA derived from the viral RNA. In addition, 704 nucleotides in the 5' untranslated region between the polycytidylic acid tract and the probable initiation codon of the first translated gene, P16-L, have been sequenced. This region has several potential initiation codons, one of which appears to be a low-frequency alternate initiation site. The coding region encompasses 6,912 nucleotides and ends in a single termination codon, UAA, located 96 nucleotides upstream from a 3'-terminal polyadenylic acid tract. Microsequencing of radiolabeled in vivo and in vitro translation products identified the genome position of the major foot-and-mouth disease virus proteins and the cleavage sites recognized by the putative viral protease and an additional protease(s), probably of cellular origin, to generate primary and functional foot-and-mouth disease virus polypeptides.

Amino Acid Sequence↗
PubMed1985

Capsid intermediates assembled in a foot-and-mouth disease virus genome RNA-programmed cell-free translation system and in infected cells.

Structural protein complexes sedimenting at 140S, 70S (empty capsids), and 14S were isolated from foot-and-mouth disease virus-infected cells. The empty capsids were stable, while 14S complexes were relatively short-lived. Radioimmune binding assays involving the use of neutralizing monoclonal antibodies to six distinct epitopes on type A12 virus and polyclonal antisera to A12 structural proteins demonstrated that native empty capsids were indistinguishable from virus. Infected cell 14S particles possessed all the neutralizing epitopes and reacted with VP2 antiserum. Cell-free structural protein complexes sedimenting at 110S, 60S, and 14S containing capsid proteins VP0, VP3, and VP1 are assembled in a rabbit reticulocyte lysate programmed with foot-and-mouth viral RNA. These structures also contain the six epitopes, and cell-free 14S structures like their in vivo counterparts reacted with VP2 antiserum. Capsid structures from infected cells and the cell-free complexes adsorbed to susceptible cells, and this binding was inhibited, to various degrees, by saturating levels of unlabeled virus. These assays and other biochemical evidence indicate that capsid assembly in the cell-free system resembles viral morphogenesis in infected cells. In addition, epitopes on the virus surface possibly involved in interaction with cellular receptor sites are found early in virion morphogenesis.

Animals↗