49K Protein Research Articles

Complete genome sequence of a novel varicosavirus infecting tall morning glory (Ipomoea purpurea).

A novel varicosa-like virus was identified in a tall morning glory (Ipomoea purpurea) plant by high-throughput sequencing and tentatively named "morning glory varicosavirus" (MGVV). The complete genome of MGVV contains two segments of negative-sense single-stranded RNA of 6409 (RNA1) and 5288 (RNA2) nucleotides. RNA1 encodes a 224.3-kDa large protein (224K), and RNA2 encodes four putative proteins of 48.6 kDa (49K), 46.4 kDa (46K), 35.7 kDa (36K), and 36.8 kDa (37K), respectively. The 224K and 49K proteins show amino acid sequence similarity to the large protein (39.4%) and the 49K protein (22.6%), respectively, of red clover-associated varicosavirus, and the 36K protein shares 19.6% amino acid sequence similarity with protein 3 of lettuce big-vein associated virus. The 46K and 37K proteins share no significant sequence similarity to known functional viral sequences. Phylogenetic analysis based on the large protein of MGVV and other rhabdoviruses showed that MGVV clustered with the varicosaviruses. These analyses indicate that MGVV is a novel member of the genus Varicosavirus in the family Rhabdoviridae.

Characterization of E3/49K, a novel, highly glycosylated E3 protein of the epidemic keratoconjunctivitis-causing adenovirus type 19a.

The early transcription unit 3 (E3) of human adenoviruses (Ads) encodes proteins with various immunomodulatory functions. Ads from different subgenera differ considerably in their E3 coding capacity, suggesting that distinct sets of immunomodulatory E3 proteins may influence the disease pattern associated with different Ad subgenera. Interestingly, the E3 region of Ads classified in subgenus D, which are often isolated from AIDS patients and have the propensity to cause eye infections, contains a unique gene, named E3/49K, that may encode a protein with a calculated molecular weight of 48,984 that might be implicated in diseases caused by this subgenus. The 49K sequence predicts a highly glycosylated type I transmembrane protein with a short cytoplasmic tail containing two motifs, YXXPhi and LL, potentially involved in targeting the protein to endosomal or lysosomal compartments. Remarkably, the 49K protein is predicted to contain an unusual immunoglobulin-like fold. Here we have characterized the E3/49K protein of Ad type 19a, an Ad of subgenus D which causes epidemic keratoconjunctivitis. E3/49K was synthesized as an 80- to 100-kDa protein, which is unusually large for an E3 protein. In contrast to another early protein, E3/19K, the expression of E3/49K started early but continued throughout the infection cycle. Analysis of the 49K glycosylation revealed that the majority of 49K molecules contained only 12 of the predicted 14 N-glycans. Furthermore, we provide evidence that 49K is O-glycosylated. At steady state, E3/49K was localized in the Golgi-trans-Golgi network and in early endosomes. Interestingly, the 49K protein has a rather short half-life and seems to be proteolytically cleaved. A processing pattern similar to that in the early stages of infection is seen in transfected cells, constitutively expressing 49K in the absence of other Ad proteins. Together, our data provide the first biochemical and cell biological characterization of an unique E3 protein of subgenus D Ads.

Direct Demonstration of the Bifunctional Property ofTetrahymena14-nm Filament Protein/Citrate Synthase Following Expression of the Gene inEscherichia coli

Tetrahymena14-nm filament protein/citrate synthase (49K protein) is a bifunctional protein with roles in the cytoskeleton and as a citrate synthase. Though previous studies have shown that the 49K protein is derived from a single transcript of a single gene, direct demonstration of the 49K protein's bifunctional property remained to be elucidated. In this study, a recombinant 49K protein was expressed inEscherichia coli,purified and characterized. The citrate synthase activity of the recombinant 49K protein was comparable to that of the 49K protein purified fromTetrahymena.The recombinant 49K protein formed 14-nm filaments, but only of short length. The filaments were elongated in the presence of a soluble fraction ofTetrahymena.These results suggest that the 49K protein itself is bifunctional, but some co-factor(s) is necessary for elongation of filaments.

Tetrahymena 14-nm filament protein (49K protein) and citrate synthase are expressed by the same gene

Summary The Tetrahymena 14-nm filament protein (designated as 49K protein) is a cytoskeletal protein involved in oral morphogenesis and in pronuclear behavior during fertilization. From the previous gene analysis, the 49K protein was shown to share a high sequence identity with porcine heart citrate synthase, and in fact, the 49K protein possessed citrate synthase activity and was detected in mitochondria. These results led to the hypothesis that the 49K protein has dual functions. Since the 49K protein and citrate synthase are clearly different in function and localization, it is necessary to ascertain strictly whether the gene and its transcript are the same or not. We first performed Southern blot analysis in the genomic DNA using three probes, probe 1 corresponding to full length cDNA, probe 2 corresponding to the sequence of 49K protein except for the signal sequence, and probe 3 corresponding to the mitochondrial signal sequence. The Southern hybridizations revealed a reasonable correspondence between the digestion patterns using several restriction enzymes and the patterns deduced from the restriction maps of the genomic 49K protein gene. Neither band suggesting another type of gene was detected in the Southern hybridization under the low stringency conditions. Secondly, Northern hybridizations using the three probes revealed that the transcript of the 49K protein gene was also the same single type. These results indicate that the 14-nm filament protein in the cytoskeleton and citrate synthase in mitochondria are derived from the same gene and the same transcript.

Early region 3 of adenovirus type 19 (subgroup D) encodes an HLA-binding protein distinct from that of subgroups B and C

Early region 3 (E3) of human adenoviruses (Ads) codes for proteins that appear to control viral interactions with the host. For example, the most abundant E3 protein, E3/19K, inhibits the transport of newly synthesized class I major histocompatibility molecules to the cell surface, thereby interfering with antigen presentation. So far, the E3 regions of Ad subgroups A, B, C, and F have been characterized. We have cloned the E3A region of Ad type 19a (Ad19a), which belongs to the largest subgroup, D, and causes epidemic keratoconjunctivitis in humans. The sequence reveals five open reading frames (ORFs) with the potential to encode the Ad19 equivalent of pVIII, as well as proteins 12.2K, 16.2K, and 18.6K. The last ORF predicts a novel 49K protein which has no counterpart in other subgroups. Both the sequence and the overall organization of the E3 region from Ad19a shows a closer relationship to group B than to group C Ads. The 18.6K ORF represents the Ad19 homolog of the Ad2 E3/19K protein. By using 293 cells stably transfected with the Adl9a E3A region, we showed by immunoprecipitation, pulse-chase experiments, and fluorescence-activated cell sorter analysis that the Ad19 E3/19K protein binds to and prevents the transport of major histocompatibility complex molecules to the cell surface. The similar but distinct functional activity of the Ad19 E3/19K protein, combined with the new sequence which differs from those of subgroup B and C proteins, allows a more precise definition of amino acids essential for HLA binding.

Polymerization of highly purified Tetrahymena 14-nm filament protein/citrate synthase into filaments and its possible role in regulation of enzymatic activity.

Tetrahymena 14-nm filament protein (49K protein) is a bifunctional protein with roles in the cytoskeleton and as citrate synthase. Previous studies in our laboratory showed that elongation factor 1 alpha (EF-1 alpha) copurifies with the 49K protein upon polymerization and depolymerization of the 49K protein. In this study, the 49K protein was isolated from partially purified 49K protein fraction containing EF-1 alpha. Using the purified 49K protein and/or purified EF-1 alpha, the interaction between 49K protein and EF-1 alpha in filament formation was investigated electronmicroscopically and it was demonstrated that purified 49K protein was capable of forming 14-nm filaments without EF-1 alpha. The 49K protein/citrate synthase has been suggested to form filaments in mitochondria. Here we show that the citrate synthase activity of 49K protein is decreased by polymerization and increased by depolymerization, suggesting a possible modulating mechanism of citrate synthase activity by monomer-polymer conversion in mitochondria in situ.

Antibodies against Tetrahymena 14-nm filament-forming protein recognize the replication band in Euplotes

Tetrahymena 14-nm filament-forming protein (49K protein) is a structural protein which is involved in activity of the pronuclei during conjugation (O. Numata, T. Sugai, and Y. Watanabe (1985)Nature (London) 314, 192–194). Using monoclonal and polyclonal antibodies, we here demonstrate the presence of a cross-reactive protein (CRP-49) within the macronuclear replication bands of Euplotes harpa and E. eurystomus which is recognized by anti-49K protein antibodies. Immunoblotting reveals that both monoclonal and polyclonal antibodies cross-react to a protein with an apparent molecular mass of 50 kDa in an E. harpa cell extract and to a protein of 49 kDa in a macronuclear extract of E. eurystomus. The antibodies used in this study have no effect upon in vitro DNA synthesis in the replication band of E. eurystomus.

Tetrahymena 14-NM filament-forming protein has citrate synthase activity

The Tetrahymena 14-nm filament-forming protein (49K protein) is a structural protein involved in oral morphogenesis and in pronuclear behavior during conjugation. Cloning the 49K protein gene from a Tetrahymena thermophila cDNA library, we found that its primary structure exhibits a high sequence identity (51.5%) with porcine heart citrate synthase and retains functional domains. The 49K protein actually possesses citrate synthase activity, and is detected in mitochondria. These results suggest that the 49K protein has dual functions as both a respiratory enzyme and a structural protein in the cytoskeleton.

Properties and in vitro Translation of Maize Dwarf Mosaic Virus RNA

SUMMARY The RNA of maize dwarf mosaic virus strain A (MDMV-A) was characterized and compared with that of strain B (MDMV-B). Glyoxal-treated MDMV-A RNA has an M r of 3·32 × 106 measured in agarose gels, compared with that of MDMV-B which is 3·41 × 106 under the same conditions. MDMV-A RNA has a Tm of 50·7 °C and a hyperchromicity of 23·3%, which are higher than those reported for MDMV-B RNA. Translation products of both RNAs in a rabbit reticulocyte cell-free system ranged from 23K to 121K. Four major polypeptide products of 57K, 91K, 100K and 117K were produced by MDMV-A RNA-directed translation, and four major products of 39K, 55K, 86K and 121K by MDMV-B RNA. Protein profiles from the two RNAs were distinct including translation products that comigrated with authentic coat protein at 37K (MDMV-A) or 39K (MDMV-B) for each strain. Anti-virion sera immunoprecipitated polypeptide products from each strain. Distinct subsets of translation products were immunoprecipitated with antisera to tobacco etch virus 49K and 54K nuclear inclusion proteins. No cell-free translation products were recognized by antisera to potato virus Y (PVY) or tobacco vein mottling virus (TVMV) helper components or antisera to the cylindrical inclusion proteins of PVY or TVMV.

Intracellular localization of TEV capsid and inclusion proteins by immunogold labeling

Thin sections of tobacco etch virus (TEV)-infected leaves were treated with goat anti-rabbit antibodies conjugated to 15-nm gold particles after the sections had been exposed to rabbit antisera specific to virus particles, virus capsid, 70K CI protein, 54K NI protein, or 49K NI protein. Anti-TEV or anti-TEV capsid sera specifically detected virus particles and capsid protein in the cytoplasm and capsid protein which was also found arrayed in a regular fashion adjacent to the cell wall. The NI 54K and 49K inclusions were found primarily in the nucleus and surprisingly in the cytoplasm. Free NI 54K and 49K proteins were also found in regions adjacent to the nuclear membrane and in association with the nucleolus. The 70K CI protein was found in the pinwheel inclusions and laminated aggregates as well as filamentous structures associated with the cell membrane. The latter observation is significant in view of the hypothesis that the 70K CI protein represents the membrane binding component of the viral replication complex.

Analysis of in vitro Translation of Bean Yellow Mosaic Virus RNA: Inhibition of Proteolytic Processing by Antiserum to the 49K Nuclear Inclusion Protein

Summary In vitro translation of bean yellow mosaic virus (BYMV) RNA in rabbit reticulocyte lysate without dithiothreitol (DTT) resulted in the accumulation of high M r products. These were readily processed to low M r mature proteins after the addition of DTT followed by a 2 h incubation. Immunoprecipitation analyses of the partially processed high M r products with antisera to the 49K and 54K nuclear inclusion (NI) proteins, cylindrical inclusion (CI) protein, tobacco vein mottling virus helper component (HC) protein and capsid protein (CP) resolved the following BYMV RNA genome map: 5′ end, 32K unidentified protein, 48K HC, 42K unidentified protein, 73K CI, 49K NI, 54K NI, 32K CP, 3′ end. The addition of the 49K NI protein antiserum inhibited the proteolytic processing of the high M r translation products. The inhibition of processing was diminished by diluting the 49K NI antiserum or by adding purified 49K NI protein. Preimmune serum or the antisera to the 54K NI, CI and capsid proteins were not as effective in inhibiting the proteolytic processing. These results provide evidence that the 49K NI protein or a related protein may have a protease function in the processing of the potyvirus polyproteins.

Processing of the tobacco etch virus 49K protease requires autoproteolysis

The final products encoded by the tobacco etch virus genome arise by proteolytic cleavage of a single large polyprotein precursor. Processing of the polyprotein at several sites requires the activity of a viral protease of 49,000 molecular weight (49K). We have examined the excision of the 49K protease from polyproteins translated from defined RNA transcripts. Polyproteins containing an intact 49K protein were efficiently processed after synthesis in a rabbit reticulocyte lysate to yield the 49K product. Introduction of a single amino acid substitution (cysteine to alanine) at the putative active site of the 49K protease abolished processing, indicating that the protease was excised from the polyprotein via an autocatalytic mechanism. Release of the 49K protease was determined to require autoproteolysis, since synthetic polyproteins which contained either or both 49K cleavage sites were processed poorly, if at all, in trans reactions. Protein microsequence analysis revealed that processing in vitro occurred between a glutamine-glycine dipeptide to generate the 49K amino terminus.

Small Nuclear Inclusion Protein Encoded by a Plant Potyvirus Genome Is a Protease

Tobacco etch virus, a plant potyvirus, expresses its RNA genome as a large polyprotein precursor which undergoes extensive proteolytic processing to yield seven or more mature products. Two of these products, proteins with apparent molecular weights of 49,000 and 54,000 (49K and 54K proteins), aggregate in the form of crystalline inclusions within the nuclei of infected cells. Cell-free translation of synthetic transcripts was used to map the genes for these two products on the viral genome and to express an enzymatically active protein. The 49K protein was determined to be a viral protease responsible for several cleavages of the polyprotein, including its own autocatalytic excision. Analyses of products expressed from the 49K protein genes which were altered by deletion revealed that only the carboxyl-terminal half was required for proteolytic activity.

Physicochemical Characterization of Festuca Leaf Streak Virus

SUMMARY Particles of festuca leaf streak virus (FLSV) contain three major proteins. One of these [mol. wt. 49000 (49K)] is the main constituent of the nucleocapsid, whereas the other two (mol. wt. 58K and 20K) were released from the nucleocapsid when particles were treated with non-ionic detergent. The 58K protein is glycosylated. The 58K, 49K and 20K proteins correspond to the G, N and M proteins of rhabdoviruses, respectively. Four minor proteins associated with the virus particles have mol. wt. of 189K, 117K, 101K and 41K. The 189K and 101K proteins are associated with the nucleocapsid, whereas the 117K protein was found in the soluble fraction after detergent treatment. Nucleic acid isolated from virus particles is probably RNA with an estimated mol. wt. of 4·3 × 106. The buoyant density of virus particles in sucrose was estimated to be 1·194 g/ml and the s 20 w, be 704S. The present results, together with previous information, make FLSV a definitive member of subgroup A of the plant rhabdovirus group of the family Rhabdoviridae.

Purification and characterization of a liver cell membrane specific antigen.

Antibodies against human liver cell membranes were raised in rabbits by immunizing 105,000 g precipitate of normal human liver homogenates (insoluble fraction). After absorption with non-liver antigens such as human spleen, kidney, lung homogenates and serum, the anti-liver cell membrane specific antigen (LCM) IgG fraction was prepared. The antibody reacted with both 105,000 g supernatant of the liver homogenate (soluble fraction) and Triton X-100 extract of the insoluble fraction, and with the surface of Chang liver cells. Using affinity columns to conjugate this antibody and other antibodies against non-liver antigens, LCM was prepared from the soluble fraction composed of 70K, 59K, 49K, and 31K proteins. These proteins of LCM were included in liver specific lipoprotein (LSP). The number of LCM proteins was limited to 4, although that of LSP was 8 or more. Furthermore, circulating antibodies against "LCM" were detected in patients with chronic liver diseases type B and autoimmune hepatitis by an 125I-LCM-IgG-Protein A method. Therefore, LCM may be more useful than LSP to study the immunological responses involved in liver diseases.

Control of germ cell nuclear behaviour at fertilization by Tetrahymena intermediate filament protein.

Intermediate filament protein [relative molecular mass (Mr) 49,000 (49K)] from the ciliated protozoan Tetrahymena has been shown to resemble intermediate filament proteins from mammalian cells in several respects, and to have a possible role in the oral morphogenesis preceding binary fission in Tetrahymena. Here, based on immunofluorescence localization of the 49K protein in Tetrahymena during the early stages of conjugation, we suggest that the protein is involved in some nuclear events, such as the production of four haploid nuclei by prezygotic divisions (meiosis), selection of one of the four meiotic products, formation of the gametic pronucleus by the mitotic division of the selected meiotic product, transfer of the gametic pronucleus across a cell-cell junction, and zygote formation by pronuclear fusion.

Induction of hepatic microsomal cytochrome P-450 and inhibition of brain, liver, and plasma esterases by an acute dose of S,S,S-tri- n-butyl phosphorotrithioate (DEF) in the adult hen

The differential effects of oral and dermal administration of single doses of 100 to 1000 mg/kg S,S,S-tri- n-butyl phosphorotrithioate (DEF) on nonspecific esterases and liver metabolism enzymes were investigated one day following administration. O,O-Diethyl O-(4-nitrophenyl) phosphorothioate (parathion) and tri- o-cresyl phosphate (TOCP) were used as negative and positive controls for organophosphorus-induced delayed neurotoxicity (OPIDN). Brain acetylcholinesterase was significantly inhibited with topical doses of 500 and 1,000 mg/kg of DEF and with orally and dermally applied parathion. Plasma cholinesterase and liver microsomal carboxylesterase activities were significantly reduced from control in all treatment groups. Neurotoxic esterase (NTE) was significantly decreased from control with topical dosing of 200, 500, and 1000 mg/kg DEF and with TOCP treatments. Oral doses of DEF increased cytochrome P-450 content by 70 to 200% while dermal application caused a 200 to 325% increase over control. p-Chloro- N-methylaniline demethylase was also increased by DEF treatments but to a lesser extent than that of aniline hydroxylase or cytochrome P-450 content. TOCP and parathion had no significant effect on liver microsomal oxidative enzymes. Liver microsomal proteins from hens treated with phenobarbital (PB), 3-methylcholanthrene (3MC), or DEF were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A striking increase in a 49K protein band in microsomes from PB and DEF (616 and 338%, respectively) treated hens was seen, while the 55K protein band showed an 861% increase in microsomes from 3MC-treated hens. In conclusion, dermally applied DEF was more effective in inhibiting esterases and inducing cytochrome P-450 than orally administered DEF; toxicity was directly related to the dose and route of administration.

Involvement of Tetrahymena intermediate filament protein, a 49K protein, in the oral morphogenesis

To study the biological function of Tetrahymena intermediate-type filament protein (a 49K protein), we examined the immunofluorescence localization of 49K protein within Tetrahymena cells. The results showed that the immunofluorescence was localized in the oral apparatus, mitochondria and mucocysts. Among them, the fluorescence in the oral apparatus was of high interest in its unique region and vicissitude in the cell cycle: a τ-shaped region of the oral apparatus intensely fluoresced during interphase, but the fluorescence completely disappeared during dividing phase. The τ-shaped region corresponded to ‘posterior connectives’ and the root part of ‘deep fiber’, to the conjunction parts of microtubule bundles. In the those parts, there was electron-dense material in the microtubule bundles. Hence, it is conceivable that 49K protein corresponds to the dense material and has a function of microtubule bundle conjunction. On the other hand, disappearance of immunofluorescence from the old oral apparatus of most dividing cells reflected the oral apparatus regression and remodelling which have been known as necessary sequential events in the cell cycle. We observed that oral fluorescence disappeared concurrently with the onset of oral regression and of constriction of division furrow, whereas at a late dividing stage immunofluorescence began to appear simultaneously in both new and old oral apparatus. Thus, the 49K protein may play a crucial role(s) not only in the morphogenesis of oral primordia but also in the transient morphogenesis in the old oral system.

Changes in electrophoretic patterns of aqueous buffer-insoluble proteins during the cultivation of guinea pig epidermal cells.

AbstractThe process of formation of aqueous buffer‐insoluble proteins, which are tentatively regarded as keratins or their precursors, was investigated using epidermal cells cultured from guinea pig ear skin. It was found that cells in the early stages of the culture (proliferation stage) contained a small number of aqueous buffer‐insoluble proteins with molecular weights of 65K, 58K, 57K and 49K. After six days of culture (differentiation stage), cells rapidly formed these proteins, mainly consisting of 58K, 57K, and 49K proteins. In these cultured cells, 70K protein, which has been observed in the whole epidermis (in vivo), was not detected. Addition of the retinoid Ro 10‐1670, an activated form of Ro 10‐9359, into the culture medium accelerated the starting of the formation of these proteins, while inhibiting the formation of 65K and 45K polypeptides.

Isolation of two lipoamide dehydrogenases from [formula omitted] grown on valine

Pseudomonas putida PPG2 grown on valine as the principal carbon and energy source produces two species of lipoamide dehydrogenase, molecular weights 56k and 49k. Pseudomonas putida grown on glucose as the carbon source produces only the 56k lipoamide dehydrogenase. The 56k proteins from P. putida grown on glucose and valine were not separated by SDS-Page and may be the same proteins. Pyruvate dehydrogenase of mutant JS94 lacking lipoamide dehydrogenase was restored by the 56k protein but not by the 49k protein. Branched chain keto acid dehydrogenase was partially restored by the 49k protein, but not the 56k protein.