28K Polypeptide Research Articles

Identification and characterization of the protein product of gene 67 in equine herpesvirus type 1 strain Ab4

Equine herpesvirus type 1 (EHV-1) strain Ab4 gene 67 has no counterpart in any herpesvirus sequenced to date. To identify and characterize the product of EHV-1 gene 67, we have expressed the putative amino acids 11 to 260 encoded by gene 67 as a beta-galactosidase fusion protein in Escherichia coli. The expressed fusion protein has been used to generate an antiserum raised against the gene 67 product. Immunoblotting and immunoprecipitation experiments have revealed that the anti-67 serum specifically recognizes a polypeptide with an M(r) of 36,000 (the 36K polypeptide) in infected cell extracts. The gene 67 protein is regulated as an early polypeptide in EHV-1 strain Ab4 infected cells and post-translational modification experiments have revealed that the protein is phosphorylated, but not glycosylated. The gene 67 protein has been transiently expressed in BHK-21/C13 cells using plasmid pCMV67, which contains the putative gene 67 ORF under the control of the cytomegalovirus immediate early promoter. Immunoblotting experiments with anti-67 have shown that the 36K protein is expressed at high levels in transfected cells. From both immunofluorescence and cellular fractionation experiments it is concluded that the gene 67 protein is associated with intracellular membranes and produces novel ribbon or filament-like structures within the cytoplasm of infected cells. We have demonstrated that the gene 67 product is a component of the virion nucleocapsid/tegument.

Nucleotide sequence, genomic organization and synthesis of infectious transcripts from a full-length clone of artichoke mottle crinkle virus

The complete nucleotide sequence of the genome of artichoke mottle crinkle virus (AMCV), a member of the tombusvirus group, has been determined. The genome is 4790 nucleotides (nt) in length. A full-length cDNA of the AMCV genome has been cloned in pUC9 downstream of the T7 RNA polymerase promoter. Transcripts were infective when inoculated onto Nicotiana clevelandii and N. benthamiana plants. The AMCV genome contains five open reading frames (ORFs). The first ORF from the 5' terminus (ORF1) encodes a protein with a predicted M(r) of 33K. ORF2 extends through the amber termination codon of ORF1 to yield a polypeptide of predicted M(r) 92K and which is the putative RNA-dependent RNA polymerase. ORF3 codes for the coat protein (41K). Two nested ORFs in different reading frames (ORFs 4 and 5) code for a 22K and a 19K polypeptide respectively. Sequence homologies suggest that the 22K protein could be involved in cell-to-cell movement of virus. ORFs 3, 4 and 5 are translated from two 3' coterminal subgenomic (sg) RNAs, the 5' termini of which have been mapped. The two sg RNAs are 2155 (sg1) and 934 (sg2) nt in length. ORF3 is expressed from sg1 RNA whereas ORFs 4 and 5 are potentially expressed from sg2 RNA. Time course experiments with Cynara scolymus protoplasts indicate that during AMCV infection both positive and negative strands of genomic and sg RNAs are produced and that sg2 RNA is produced before and at a higher level than sg1 RNA.

Comparative analysis of the conserved region of the orthopoxvirus genome encoding the 36K and 12K proteins

Genes encoding virus-specific proteins with molecular masses of 36 kDa and 12 kDa were mapped in HindIII-P and HindIII-U DNA fragments of vaccinia strain LIVP and ectromelia strain K-1 viruses, respectively, by hybrid selection of RNA to cloned DNA fragments followed by in vitro translation. The 36K translation initiation codon was detected in the HindIII-J fragment. The nucleotide sequences of corresponding genes from vaccinia, ectromelia, cowpox and variola virus genomes were determined. The 12K protein has similarity to mammalian glutaredoxins. The derived amino acid sequence of the 36K polypeptide was compared with the protein bank PIR. No homology was found between the 36K protein and known structures of proteins. The 36K protein genes of vaccinia and ectromelia viruses were cloned in pUR290, which led to the production of E. coli chimeric proteins, consisting of the sequence of β-galactosidase and the viral protein on their C-ends. The chimeric proteins were shown to possess viral antigenic specificity. To identify the protein product of the 36K gene monospecific antisera to chimeric proteins were obtained. The late 36K protein is associated with virosomes but is not incorporated into the virions of orthopoxviruses.

Separation of gate- and channel-forming domains in the pore-forming protein of the outer membrane of Pseudomonas aeruginosa

The domains of the pore-forming protein responsible for the gate and channel formations were separated and identified in the outer membrane of Pseudomonas aeruginosa. The proteolytic cleavage of the 46K channel protein, protein D2, yielded two major domains with apparent M r of 27K and 19K. We identified the 27K polypeptide to be the channel-forming domain by an in vitro permeability assay. The channel size of purified 27K domain was indistinguishable from that of native protein D2. Degradation of the 19K domain into small subfragments increases the channel activity about ten times suggesting that the 19K polypeptide forms the gate or cap.

The 36K polypeptide synthesized in Newcastle disease virus-infected cells possesses properties predicted for the hypothesized 'V' protein

Newcastle disease virus (NDV) virions possess two proteins which react strongly with monoclonal antibody 688 following separation by high resolution two-dimensional (isoelectric focusing/SDS) PAGE and detection by Western blotting. One is the phosphorylated nucleocapsid-associated 53K [P (NAP)] protein, the other comigrates with the 36K protein detected by radiolabelling NDV-infected chick embryo fibroblasts. [35S]Cysteine/[3H]leucine dual-labelling experiments show that the 36K protein is very rich in cysteine compared to the P (NAP) protein. In the Beaudette C strain it comigrates on one-dimensional SDS-polyacrylamide gels with the matrix protein (M); however, it is resolved from the slower migrating M protein from the Ulster strain of NDV. The size, strain-specific isoelectric point, high cysteine content and antigenic relatedness to the P (NAP) protein suggest that the 36K protein is the 'V' protein of NDV, the counterpart of which is found in other Paramyxoviridae.

The Genome Organization of Potato Virus M RNA

The 8534 nucleotide sequence of the genome of the carlavirus, potato virus M (PVM), has been determined. The sequence contains six large open reading frames (ORFs) and non-coding regions consisting of 75 nucleotides at the 5' end, 70 nucleotides followed by a poly(A) tail at the 3' end and 38 and 21 nucleotides between three large blocks of coding sequences. The ORF beginning at the first initiation codon at nucleotide 76 encodes a polypeptide of 223K which, according to its primary sequence analysis, seems to be a virus RNA replicase. The next coding block consists of three ORFs encoding polypeptides of 25K, 12K and 7K. The third block consists of two ORFs encoding polypeptides of 34K (PVM coat protein) and 11K. The 11K polypeptide contains a pattern resembling the consensus for a metal-binding nucleic acid-binding 'finger'.

A synthetic peptide elicits antibody reactive with the native duck hepatitis B virus pre-S protein

Synthetic peptides P37-49 and P63-79, derived from the pre-S region of duck hepatitis B virus (DHBV), have been synthesized. Only P37-49 was reactive with rabbit anti-DHBs/pre-S antibodies by radioimmunoprecipitation. Antiserum prepared against P37-47 reacted with a 35K polypeptide of native DHBs/pre-S by immunoblotting. It is concluded that P37-49 (MGQHPAKSMDVRR) mimics one of the epitopes of the DHBV pre-S antigen.

The Expression of the Adenovirus 12 Early Region 1B 19K Protein Using a Recombinant Simian Virus 40 System

Using a simian virus 40/adenovirus 12 (Ad12)-recombinant virus, the Ad12 early region 1B (E1B) 19K protein has been produced at high levels after infection of Cos 1 cells. Expression of the 19K polypeptide reaches a maximum at about 48 h post-infection, declining at later times as host cell death occurs. Using two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis, we have shown that the Ad12 E1B protein is a major species following infection of Cos 1 cells with recombinant virus. Two forms of 19K polypeptide can be distinguished following isoelectric focusing. Using subcellular fractionation of infected cells, it was found that a similar distribution of 19K protein occurred after recombinant virus and Ad12 infection, with the polypeptide being most abundant in nuclear and membranous fractions. Similarly, as in Ad12-infected cells, a certain proportion of the protein is located on the outside surface of the cell after recombinant viral infection. Immunohistochemical studies suggest that, at early times post-infection, the E1B 19K protein is located in the nuclear membrane, the Golgi apparatus and the endoplasmic reticulum. At later times, it can be seen to have spread to the cytoplasm as well as to the other organelles. These results are discussed in relation to the known functions of the 19K E1B protein.

In vitro Translation of Apple Chlorotic Leaf Spot Virus RNA

Summary Apple chlorotic leaf spot virus (ACLSV) RNA was translated in a rabbit reticulocyte lysate. Two major polypeptides of M r 105000 (105K) and 51 K, and some less prominent polypeptides (88K, 66K, 28K and 23K) were produced. The 23K polypeptide was identified as the coat protein on the basis of its electrophoretic mobility and serological reaction to virus antiserum. Time course and immuno-precipitation experiments suggested that the 23K polypeptide might be generated from the 28K polypeptide by post-translational cleavage.

Characterization of the mammalian toxicity of the crystal polypeptides of Bacillus thuringiensis subsp. israelensis

Solubilized crystal polypeptide preparations of Bacillus thuringiensis subsp. israelensis (BTI) were fractionated by immunoaffinity chromatography using a bound monoclonal antibody formed against the 28K crystal polypeptide. The 28K polypeptide was confirmed to be hemolytic and to possess low mosquitocidal activity against Aedes aegypti larvae. By comparison, the 28K polypeptide was more potent than the solubilized BTI crystals in male Swiss Webster mice, as the LD50 values were ( p < 0.05) 0.77 and 2.33 mg protein/kg body wt, respectively. Acute administration of the 28K polypeptide (mg/kg, ip) produced severe hypothermia and bradycardia in the mouse. No evidence for cooperativity between the 28K and other crystal polypeptides was observed. Preliminary histological examination of the mouse hearts exposed to the 28K polypeptide did not reveal any specific lesion, suggesting that the deficient cardiac performance might be a secondary physiological response. Gross pathological examination of mice as well as Sprague-Dawley rats acutely treated with equivalent doses of solubilized BTI crystal preparations revealed focal to segmental reddened and edematous areas within the small intestine. Histopathology indicated that the major lesion was in the jejunum. Contrary to expectations from in vitro hemolysis assays, cytolysis of mouse red and white blood cells was not detectable after in vitro exposure to the BTI solubilized proteins. The present results indicate that the 28K polypeptide is the mammalian toxic component of BTI crystals.

Role of Adenovirus Type 2 Early Region 1B 19K Protein Stability in Expression of the cyt and deg Phenotypes

The cell line KB18 constitutively expresses adenovirus type 2 (Ad2) early region 1B (E1B) genes in the absence of E1A expression and thus is useful for understanding the function and properties of E1B gene products. We report here that KB18 cells complement the cyt and deg phenotypes of Ad2 cytocidal (cyt) mutants and an Ad12 cyt mutant. Thus the E1B 19K polypeptide in KB18 is functional. Expression of E1B mRNA and synthesis of the 19K polypeptide were studied in wild-type Ad2- and Ad2cyt15-infected KB cells at 15 h post-infection, and in KB18 cells. Although E1B mRNA synthesized in KB18 cells at 5 to 7% of the level in Ad2-infected KB cells, the amount of E1B 19K polypeptide formed was similar. In contrast, the amount of 19K protein in Ad2cyt15-infected KB cells was about one-tenth of that in Ad2-infected cells, although expression of E1B mRNA was comparable to that during Ad2 infection. The half-lives of the 19K polypeptide in wild-type-infected KB cells, in Ad2cyt15-infected KB cells and in KB18 were approximately 90 min, 25 min and 22 h, respectively. The cyt phenotype was expressed at 30 to 35 h post-infection, thus showing that the instability of the 19K polypeptide in Ad2cyt15-infected cells is not due to alteration of cell morphology or cell destruction.

Retention and Expression of the Left End Subfragment of the Herpes Simplex Virus Type 2 BglII N DNA Fragment Do Not Correlate with Tumorigenic Conversion of NIH 3T3 Cells

Cotransfection experiments have been carried out using recombinant plasmids pAG60, conferring resistance to antibiotic G418, and pXho3 which contains the left end subfragment (map coordinates 0.583 to 0.596) of the transforming herpes simplex virus type 2 BglII N DNA fragment and encodes the 36K polypeptide associated with the viral ribonucleotide reductase activity. Several NIH 3T3 cell clones resistant to G418 and having morphological changes commonly observed for transformed NIH 3T3 cells were isolated and examined for the presence and stable retention of the viral sequences. Seven of the clones that retained the transfected viral sequences were analysed for the expression of the 36K polypeptide and the tumorigenic phenotype. The results gathered from these studies show that neither the retention of the viral DNA nor the expression of the 36K polypeptide correlated with tumorigenic conversion of these cells.

Characterization of the Mammalian Toxicity of the Crystal Polypeptides of Bacillus thuringiensis subsp. israelensis

Solubilized crystal polypeptide preparations of Bacillus thuringiensrs subsp. israelensis (BTI) were fractionated by immunoaffinity chromatography using a bound monoclonal antibody formed against the 28K crystal polypeptide. The 28K polypeptide was confirmed to be hemolytic and to possess low mosquitocidal activity against Aedes aegypti larvae. By comparison, the 28K polypeptide was more potent than the solubilized BTI crystals in male Swiss Webester mice, as the LD5O values were ( p < 0.05) 0.77 and 2.33 mg protein/kg body wt, respectively. Acute administration of the 28K polypeptide (mg/kg, ip) produced severe hypothermia and bradycardia in the mouse. No evidence for cooperativity between the 28K and other crystal polypeptides was observed. Preliminary histological examination of the mouse hearts exposed to the 28K polypeptide did not reveal any specific lesion, suggesting that the deficient cardiac performance might be a secondary physiological response. Gross pathological examination of mice as well as Sprague-Dawley rats acutely treated with equivalent doses of solubilized BTI crystal preparations revealed focal to segmental reddened and edematous areas within the small intestine. His topathology indicated that the major lesion was in the jejunum. Contrary to expectations from in vitro hemolysis assays, cytolysis of mouse red and white blood cells was not detectable after in vivo exposure to the BTI solubilized proteins. The present results indicate that the 28K polypeptide is the mammalian toxic component of BTI crystals.

Identification and Characterization of a Bacteroid-Specific Dehydrogenase Complex in Rhizobium leguminosarum PRE

In membranes of Rhizobium leguminosarum bacteroids isolated from nitrogen-fixing pea root nodules, two different protein complexes with NADH dehydrogenase activity were detected. One of these complexes, with a molecular mass of 110 kilodaltons, was also found in membranes of free-living rhizobia, but the other, with a molecular mass of 550 kilodaltons, appeared to be present only in bacteroids. The bacteroid-specific complex, referred to as DH1, probably consists of at least four different subunits. Using antibodies raised against the separate polypeptides, we found that a 35,000-molecular-weight polypeptide (35K polypeptide) in the DH1 complex is bacteroid specific, while the other proposed subunits were also detectable in cytoplasmic membranes of free-living bacteria. Dehydrogenase complex DH1 is also present in bacteroids of a R. leguminosarum nifA mutant, indicating that the synthesis of the dehydrogenase is not dependent on the gene product of this nif-regulatory gene. A possible involvement of the bacteroid-specific DH1 complex in electron transport to nitrogenase is discussed.

Characterization of papillomavirus polypeptides from bovine cutaneous fibropapillomas.

Virions of bovine papilloma virus (BPV) were isolated from a pool of cutaneous bovine fibropapillomas and purified by CsCl gradient centrifugation. SDS-PAGE revealed several polypeptides with an Mr ranging from 76K to 19K. Western blot analysis of the viral isolate identified additional polypeptides when a rabbit anti-BPV serum was used, but only the main capsid component of 57K when a rabbit antiserum raised against human papillomavirus was used. The viral preparation was then 125I-labelled and further purified by gel filtration. SDS-PAGE of immunoprecipitates of the anti-BPV serum with different fractions from the chromatographic column revealed the polypeptides of 76K, 57K and 28K to be viral structural components. The 28K polypeptide, not previously characterized, was shown to be composed of several molecular forms, migrating over a pH range of 3.5 to 4.6 when analysed by two-dimensional PAGE. Following SDS-PAGE performed under non-reducing conditions, the 28K and 76K polypeptides and the main capsid component of 57K appeared to be linked by disulphide bridges to form hetero- or homopolymers.

Intracellular precursors and secretion of alkaline extracellular protease of Yarrowia lipolytica.

Processing and secretion of the alkaline extracellular protease (AEP) from the yeast Yarrowia lipolytica was studied by pulse-chase and immunoprecipitation experiments. Over half of newly synthesized AEP was secreted by 6 min. Over 99% of AEP activity which was external to the cytoplasmic membrane was located in the supernatant medium. Polypeptides of 55, 52, 44, 36, and 32 kilodaltons (55K, 52K, 44K, 36K, and 32K polypeptides) were immunoprecipitated from [3H]leucine-labeled cell extracts by rabbit antibodies raised against mature, secreted AEP (32K polypeptide). Experiments with tunicamycin and endoglycosidase H indicated that the 55K, 52K, and 44K polypeptides contained about 2 kilodaltons of N-linked oligosaccharide and that the 36K and 32K polypeptides contained none. Results of pulse-chase experiments did not fit a simple precursor-product relationship of 55K----52K----44K----36K----32K. In fact, maximum labeling intensity of the 52K polypeptide occurred later than for the 44K and 36K polypeptides. Secretion of polypeptides of 19 and 20 kilodaltons derived from the proregion of AEP indicated that one major processing pathway was 55K----52K----32K. The gene coding for AEP (XPR2) was cloned and sequenced. The sequence and the immunoprecipitation results suggest that AEP is originally synthesized with an additional preproI-proII-proIII amino-terminal region. Processing definitely involves cleavage(s) after pairs of basic amino acids and the addition of one N-linked oligosaccharide. Signal peptidase cleavage, dipeptidyl aminopeptidase cleavages, and at least one additional proteolytic cleavage may also be involved.

Intracellular precursors and secretion of alkaline extracellular protease of Yarrowia lipolytica.

Processing and secretion of the alkaline extracellular protease (AEP) from the yeast Yarrowia lipolytica was studied by pulse-chase and immunoprecipitation experiments. Over half of newly synthesized AEP was secreted by 6 min. Over 99% of AEP activity which was external to the cytoplasmic membrane was located in the supernatant medium. Polypeptides of 55, 52, 44, 36, and 32 kilodaltons (55K, 52K, 44K, 36K, and 32K polypeptides) were immunoprecipitated from [3H]leucine-labeled cell extracts by rabbit antibodies raised against mature, secreted AEP (32K polypeptide). Experiments with tunicamycin and endoglycosidase H indicated that the 55K, 52K, and 44K polypeptides contained about 2 kilodaltons of N-linked oligosaccharide and that the 36K and 32K polypeptides contained none. Results of pulse-chase experiments did not fit a simple precursor-product relationship of 55K----52K----44K----36K----32K. In fact, maximum labeling intensity of the 52K polypeptide occurred later than for the 44K and 36K polypeptides. Secretion of polypeptides of 19 and 20 kilodaltons derived from the proregion of AEP indicated that one major processing pathway was 55K----52K----32K. The gene coding for AEP (XPR2) was cloned and sequenced. The sequence and the immunoprecipitation results suggest that AEP is originally synthesized with an additional preproI-proII-proIII amino-terminal region. Processing definitely involves cleavage(s) after pairs of basic amino acids and the addition of one N-linked oligosaccharide. Signal peptidase cleavage, dipeptidyl aminopeptidase cleavages, and at least one additional proteolytic cleavage may also be involved.

Biochemical and cytological analysis of the complex periplasmic flagella from Spirochaeta aurantia.

The periplasmic flagella of Spirochaeta aurantia were isolated and were found to be ultrastructurally and biochemically complex. Generally, flagellar filaments were 18 to 20 nm in diameter and appeared to consist of an 11 to 13-nm-wide inner region and an outer layer. The hook-basal body region consisted of two closely apposed disks connected to a hook by a rod. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified flagella together with a Western blot analysis of a motility mutant that produces hooks and basal bodies but not flagellar filaments revealed that the filaments were composed of three major polypeptides of 37,500, 34,000, and 31,500 apparent molecular weight (37.5K, 34K, and 31.5K polypeptides) and three minor polypeptides of 36,000, 33,000, and 32,000 apparent molecular weight (36K, 33K, and 32K polypeptides). Purified hook-basal body preparations were greatly enriched in three polypeptides in the range of 62,000 to 66,000 apparent molecular weight. Immunogold labeling experiments with a monoclonal antibody specific for the 37.5K flagellin and one that reacts with an epitope common to the 36K, 34K, 33K, 32K, and 31.5K flagellins revealed that the 37.5K major polypeptide was a component of the outer layer, whereas one or more of the other polypeptides constituted the core.

Infectious RNA transcripts derived from full-length DNA copies of the genomic RNAs of cowpea mosaic virus

A set of full-length DNA copies of both M and B RNA of Cowpea mosaic virus (CPMV) was cloned downstream of a phage T7 promoter. Upon in vitro transcription using T7 RNA polymerase, M and B RNA-like transcripts were obtained from these DNA copies with only two additional nucleotides at the 5′ end and five extra nucleotides at the 3′ end in comparison to natural viral RNA. In Cowpea protoplasts the transcripts of several cDNA clones of B RNA were able to replicate leading to detectable synthesis of viral RNA and proteins. Transcripts of M cDNA clones inoculated together with these B RNA transcripts were also expressed, although the number of protoplasts in which both transcripts were expressed was very low. Preliminary infectivity tests with mutagenized RNA transcripts indicate essential roles of the B RNA-encoded 24K and 32K polypeptides in viral RNA replication.

Identification of the gene coding for the precursor of adenovirus core protein X.

Nucleoprotein complexes extracted from infected cells (J. Weber and L. Philipson, Virology 136:321-327, 1984) or ts1 virions grown at 39 degrees C (J. M. Weber, G. Khitto, and A. R. Bhatti, Can. J. Microbiol. 29:235-241, 1983) contain, in addition to known virion proteins, a polypeptide with a molecular weight of 11,000 (11K polypeptide). We identified the gene for this polypeptide by sequence analysis of the radioactively labeled 11K polypeptide isolated from ts1 virions and comparison of this sequence with the nucleotide sequence of the adenovirus type 2 genome. The 11K polypeptide was encoded by an open reading frame of 80 residues located between nucleotides 17,676 and 17,915 in late transcription region 2 of adenovirus type 2; the initiating methionine residue was removed leaving a 79-residue product. The late transcription region 2 that encoded the 79-residue polypeptide (11K) was arginine rich (21%) and had a predicted molecular weight of 8,715. It was cleaved by the viral endoprotease to give two products which comigrated on sodium dodecyl sulfate-polyacrylamide gels as virion polypeptide X. Our data suggest that additional cleavage of the carboxy-terminal 48-residue fragment generates a 19-amino-acid fragment with the amino acid composition of mu (K. Hosokawa and M. T. Sung, J. Virol. 17:924-934, 1976).