52K Protein Research Articles

Genome organization of grapevine fanleaf nepovirus RNA2 deduced from the 122K polyprotein P2 in vitro cleavage products

The full-length transcript of grapevine fanleaf virus (GFLV) RNA2 produces a primary product of 122K when translated in the rabbit reticulocyte system. This 122K polyprotein is completely processed in vitro by the RNA1-encoded 24K proteinase. The positions of the cleavage sites within the polyprotein have been mapped and the genome organization of GFLV-F13 RNA2 has been established. The order of mature proteins in the 122K polyprotein is the amino-terminal 28K protein, the 38K protein followed by the 56K coat protein at the carboxy terminus. These proteins represent the final cleavage products of the 122K polyprotein. A 66K protein which yields 28K and 38K proteins constitutes the major maturation intermediate. Microsequencing of the amino extremity of radioactively labelled 38K protein allowed identification of the Cys257/Ala258 site as the cleavage site recognized by the GFLV proteinase between the 28K and the 38K proteins in the 66K protein in addition to the Arg605/Gly606 site between the 38K protein and the coat protein.

The cytokeratin filament-aggregating protein filaggrin is the target of the so-called "antikeratin antibodies," autoantibodies specific for rheumatoid arthritis.

In rheumatoid arthritis (RA), the high diagnostic value of serum antibodies to the stratum corneum of rat esophagus epithelium has been widely reported. These so-called "antikeratin antibodies," detected by indirect immunofluorescence, were found to be autoantibodies since they also labeled human epidermis. Despite their name, the actual target of these autoantibodies was not known. In this study, a 40-kD protein (designated as 40K), extracted from human epidermis and specifically immunodetected by 75% of RA sera, was purified and identified as a neutral/acidic isoform of basic filaggrin, a cytokeratin filament-aggregating protein, by peptide mapping studies and by the following evidences: (a) mAbs specific for filaggrin reacted with the 40K protein; (b) the autoantibodies, affinity-purified from RA sera on the 40K protein, immunodetected purified filaggrin; (c) the reactivity of RA sera to the 40K protein was abolished after immunoadsorption with purified filaggrin; (d) the 40K protein and filaggrin had similar amino acid compositions. Furthermore, autoantibodies against the 40K protein and the so-called "antikeratin antibodies" were shown, by immunoadsorption experiments, to be largely the same. The identification of filaggrin as a RA-specific autoantigen could contribute to the understanding of the pathogenesis of this disease and, ultimately, to the development of methods for preventing the autoimmune response.

Characteristics of a retrovirus associated with Jembrana disease in Bali cattle.

A virus causing Jembrana disease in Bali cattle (Bos javanicus) was demonstrated to have characteristics of a retrovirus. Reverse transcriptase activity was detected in virus purified by sucrose gradient centrifugation. Electron microscopic examination of tissue from the affected cattle indicated that the virus matured by C-type budding through the plasma membrane and into intracytoplasmic vacuoles of cells in lymphoid tissue, with the formation of circular enveloped virus particles ranging in diameter from 96 to 124 nm with an eccentric nucleoid. Western immunoblotting using sera from recovered animals demonstrated virus proteins of M(r) 100K, 45K, 42K, 33K, 26K, 16K and 14K. The 26K protein of Jembrana disease virus cross-reacted in Western blots with the 26K capsid protein of bovine immunodeficiency virus (BIV). The apparent morphogenesis, protein structure and antigenic relationship with BIV suggested the virus was a lentivirus.

The involvement of cowpea mosaic virus M RNA-encoded proteins in tubule formation

On the surface of cowpea protoplasts inoculated with cowpea mosaic virus (CPMV), tubular structures containing virus particles have been found. Such tubular structures are thought to be involved in cell-to-cell movement of CPMV in cowpea plants. To study the involvement of the 58K/48K and capsid proteins of CPMV in the formation of the tubular structures, mutations were introduced into M cDNA clones from which infectious transcripts could be derived. No tubules were found on protoplasts inoculated with a mutant that fails to produce the 48K protein nor with a mutant that has a deletion in the 48K coding region, suggesting that the 48K protein is essential for this process. However, a possible role of the 58K protein in tubule formation could not be excluded. A mutant that fails to produce the capsid proteins did produce tubules and therefore the capsid proteins are not involved in the formation of the tubular structures. Electron microscopic analysis revealed that the tubules produced by this mutant are, apart from the absence of virus particles, morphologically identical to the tubules formed by the wild-type virus.

Complete Nucleotide Sequence and Organization of the Bipartite RNA Genome of Soil-Borne Wheat Mosaic Virus

The complete nucleotide sequences of RNAs 1 and 2 of soil-borne wheat mosaic virus (SBWMV), type member of the furovirus group, were determined. RNA 1 is 7099 nucleotides (nt) and encodes a 150-kDa protein from the 5′ end region, the UGA termination codon of which can be partially read through to produce a 209-kDa protein, and a 37-kDa protein in the 3′ end region. The C-terminal region of the 150-kDa protein contains an NTP-binding helicase motif and the readthrough region, an RNA polymerase motif, indicating that these two overlapping proteins may form an RNA replication complex similar to those of tobamo- and tobraviruses. The 37-kDa protein has sequence similarity with the cell-to-cell transport protein of dianthoviruses. RNA 2 is 3593 nt and, from the 5′ end region, encodes the 19-kDa capsid protein, whose UGA termination codon can be partially suppressed to produce an 84-kDa readthrough protein and, at the 3′ proximity, a 19-kDa protein which is rich in cysteine residues. The 28K (kilodaltons, as estimated by SDS-PAGE) protein, previously considered as another capsid readthrough product, is apparently initiated at an in-frame non-AUG codon upstream from the capsid protein gene. In both RNAs 1 and 2, the 5′ terminus is capped, and the 3′ untranslated region possibly forms internal consecutive pseudoknots as found in tobamovirus RNA as well as a terminal tRNA-like structure similar to tymovirus RNA. An amino acid sequence comparison of RNA replicase genes indicates that, phylogenetically, SBWMV belongs to a cluster formed by tobamo-, tobra-, and hordeiviruses. Differences in the 3′ end structure and in the cell-to-cell movement protein, and the distant phylogeny of the RNA replicase genes of SBWMV and beet necrotic yellow vein virus, suggest that the furoviruses should be divided into at least two groups.

Efficient Replication of the in Vitro Transcripts from Cloned cDNA of Tomato Black Ring Virus Satellite RNA Requires the 48K Satellite RNA-Encoded Protein

Tomato black ring virus isolate L supports the multiplication of a large satellite RNA of 1376 nt which has no common features with the two genomic RNAs except for the terminal motif 5′ VPg UUGAAAA and a 3′ poly(A) tail. The TBRV sat-RNA contains an ORF for a protein of 48K which is translated both in vitro and in vivo. To determine the function of the 48K protein we have studied the effect of different mutations introduced in the ORF of the cDNA clone on the capacity of transcripts to multiply in Chenopodium quinoa plants or protoplasts when inoculated along with the genomic RNAs. Transcripts in which nucleotides have been substituted within the 5′ proximal region of the ORF multiplied poorly even when the modification conserved the 48K protein sequence, suggesting that this portion of the ORF contains cis-acting RNA sequences. Transcripts with alterations in the internal region of the ORF retained their multiplication capacity provided the mutation did not destroy the ORF or modify the length of the protein expressed. The absence of multiplication in plants of transcripts unable to express the 48K protein and their inability to replicate in protoplasts suggest strongly that the sat-RNA translation product itself is implicated in the replication of sat-RNA.

Enteric Adenovirus Type 40: Expression of E1B Proteins in Vitro and in Vivo

The genes encoding the enteric adenovirus type 40 E1B proteins designated 19K, 55K, and 15K (55K related) have been cloned into the pET3a expression vector and synthesized by in vitro transcription and translation and by in vivo expression after induction in bacteria. The 19K product expressed in bacteria is recognized by anti-peptide sera specific for the C-terminal region of the open reading frame and has the same M r as 19K protein immunoprecipitated from virus-infected cells. The 55K protein synthesized in bacteria is insoluble except under extreme denaturing conditions, but after in vitro transcription followed by translation, a polypeptide of the predicted size is obtained. The 15K protein, equivalent to the first 73 and last 29 of the 476-residue 55K protein with an internal deletion of 374 amino acids, is expressed to a high level in bacteria in a soluble form and interacts weakly but specifically with N- and C-terminal anti-peptide sera. The bacterially expressed 15K protein was used to raise antibodies in rabbits. This serum precipitates the 55K protein expressed by in vitro translation, but only the 15K product can be immunoprecipitated from virus-infected cells. The same antiserum, however, detects the 55K protein in infected cells by Western blotting, at a time broadly coinciding with the onset of DNA replication. This is the first identification of Ad40 55K protein in infected cells and confirms that the Ad40 22S mRNA can be utilized in vivo. The question of whether this protein is functional can now be addressed.

Binding of an intrinsic ATPase inhibitor to the interface between alpha- and beta-subunits of F1FoATPase upon de-energization of mitochondria.

Yeast mitochondrial F1FoATPase has three regulatory subunit proteins: ATPase inhibitor, 9K protein, and 15K protein. Mutant yeasts lacking one or more of these protein factors were constructed by gene disruption [Ichikawa, N. et al. (1990) J. Biol. Chem. 265, 6274-6278; Yoshida, Y. et al. (1990) Eur. J. Biochem. 193, 49-53]. Dissipation of the electrochemical potential of protons of the mitochondrial inner membrane by an uncoupler or by a combination of valinomycin and potassium ions induced ATP-hydrolyzing activity of F1FoATPase in mitochondria of all the mutants, as in those of wild-type cells. However, the ATPase activity was inactivated within a minute in normal mitochondria, but was not suppressed in inhibitor-deficient mitochondria, and in mitochondria lacking either 9K or 15K protein, the inactivation of ATPase was slow and incomplete. Covalent binding of inhibitor protein to the enzyme was achieved with a zero length cross-linker, EEDQ, in uncoupled normal mitochondria, in which the inhibitor linked directly to both the alpha- and beta-subunits. This result strongly suggests that the binding site of the inhibitor protein is located at the interface between the two subunits.

The trans-spliceosomal U2 snRNP protein 40K of Trypanosoma brucei: cloning and analysis of functional domains reveals homology to a mammalian snRNP protein.

Through immunoscreening we have isolated a cDNA encoding the trans-spliceosomal U2 snRNP-specific 40 kDa protein of Trypanosoma brucei. The protein has a predicted molecular weight of 36.6 kDa and shows 31% amino acid identity with the human U2 snRNP A' protein of 28.4 kDa. The homology between the trypanosome and human protein sequences is restricted to the N-terminal half where they share a series of six leucine repeat motifs. Sequence alignment revealed three 40K-specific regions: a C-terminal extension and two insertions, one of which makes up a seventh leucine repeat. Bacterially expressed 40K protein efficiently bound RNA by itself in a nonspecific manner; this general RNA binding activity was located to a region in the C-terminal half overlapping with the leucine repeat domain. U2 RNA-specific interaction required the presence of other trypanosome proteins and depended upon the loop IV sequence of U2 RNA. Deletion analysis of the 40K protein demonstrated the leucine repeats, including the 40K-specific, seventh repeat, to be essential for specific U2 RNP assembly, most likely through their role as an interface for protein-protein interaction.

Amplified Expression of Intercellular Adhesion Molecule-1 (ICAM-1) and M R 40K Protein by DLD-1 Colon Tumor Cells by Interferon-γ

Inflammatory bowel disease, in particular ulcerative colitis, is characterized by localization of leukocytes in close proximity to the colonic epithelium, which may be mediated by the expression of intercellular adhesion molecules (ICAM-1; CD 54). We previously reported the presence of an organ-specific M r , 40K colonic protein that acts as an autoantigen in ulcerative colitis and is present on the surface of colonic epithelial cells and also in DLD-1 colon cancer cells. Using the colon tumor cell line DLD-1 and flow cytometry, ICAM-1 antibody binding by untreated cultured DLD-1 cells was similar to background antibody binding (mean channel number, MCN = 9.77 ± 2.13). Interferon-γ (100 U) induced a 1-2 log increase in anti-ICAM-1 antibody binding from as early as 12 hr after exposure up to 72 hr and a moderate increase (up to about 100%) in the binding of anti- M r 40K antibody. Additional studies showed that anti-ICAM-1 and anti- M r 40K antibodies bound to DLD-1 cells regardless of the presence of the other antibody. Taken together, the present observations suggest that the epitopes of ICAM-1 and M r 40K molecules are coexpressed by colonic epithelial cells, regardless of the presence of the other molecule. Furthermore, lymphocytes in the colonic mucosa that are activated to produce interferon-γ may upregulate the expression of both of these molecules.

Properties and sequence of a female-specific, juvenile hormone-induced protein from locust hemolymph.

In the fat body of Locusta migratoria, an RNA transcript of about 800 nucleotides has been detected that is specific to the adult female and dependent on induction by juvenile hormone (JH) or an analog. The corresponding cDNA has been cloned (lambda 21) and a 718-base pair sequence determined. It encodes a 196-amino acid polypeptide, including a signal peptide. An NH2-terminal sequence has 24 out of 28 amino acids identical with those of a previously described 19K locust hemolymph protein, but the remainder of the sequence shows no similarity. From adult female hemolymph, a 21-kDa protein, designated 21K protein, has been purified, with an NH2-terminal sequence exactly matching that deduced from clone lambda 21. This 21K protein is found only in the adult female, is dependent on induction by JH, and is assumed to represent the product of the lambda 21 gene. It shows no immunochemical cross-reaction with locust 19K protein, apolipophorin III, nor with vitellogenin (Vg). Its isoelectric point is pH 5.4; it contains some carbohydrate. 21K protein is synthesized in adult female fat body, accumulates in hemolymph, and is taken up into the developing oocytes in parallel with Vg. In locusts deprived of JH with precocene, production of 21K protein and of lambda 21-hybridizing transcripts is induced by the JH analog, methoprene, in parallel with Vg and its mRNA. Because of its sex-, stage-, and JH-dependent regulation, coordinate with Vg, the 21K protein will be valuable for analysis of gene expression.

Ulcerative Colitis Serum Recognizes the M<sub>r</sub> 40K Protein on Colonic Adenocarcinoma Cells for Antibody-Dependent Cellular Cytotoxicity

Antibodies in ulcerative colitis (UC) serum promote antibody-dependent cellular cytotoxicity (ADCC) against colon cancer cells. The target molecules on the cells recognized by the antibody are unknown. This study examines the role of the M(r) 40K colon epithelial protein in this process. Serum from 29 UC patients, but not from 19 patients with Crohn's disease, significantly (p < 0.001) induced more cytotoxicity than did normal serum against DLD-1 colon cancer cells which are known to express the M(r) 40K protein. Peripheral blood lymphocytes but not polymorphonuclear leukocytes showed ADCC. Anti-M(r) 40K monoclonal antibodies significantly (p < 0.001) inhibited the UC-serum-induced ADCC, but not the spontaneous cytotoxicity. These results suggest the involvement of the M(r) 40K protein in ADCC induced by UC serum.

Cloning and characterization of the ferric enterobactin receptor gene (pfeA) of Pseudomonas aeruginosa

Pseudomonas aeruginosa K407, a mutant lacking a high-affinity 80,000-molecular-weight ferric enterobactin receptor protein (80K protein), exhibited poor growth (small colonies) on iron-deficient succinate minimal medium containing ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and enterobactin. The gene encoding the ferric enterobactin receptor was cloned by complementation of this growth defect. The complementing DNA was subsequently localized to a 7.1-kilobase-pair (kb) SstI-HindIII fragment which was able to restore synthesis of the 80K protein in strain K407 and also to direct the synthesis of high levels of a protein of the same molecular weight in the outer membranes of Escherichia coli fepA strains MT912 and IR20. Moreover, the fragment complemented the fepA mutation in MT912, restoring both growth in EDDHA-containing medium and enterobactin-dependent uptake of 55Fe3+. Expression of the P. aeruginosa receptor in E. coli IR20 was shown to be regulated by both iron and enterobactin. The complementing DNA was further localized to a 5.3-kb SphI-SstI fragment which was then subjected to deletion analysis to obtain the smallest fragment capable of directing the synthesis of the 80K protein in the outer membrane of strain K407. A 3.2-kb DNA fragment that restored production of the receptor in strain K407 was subsequently isolated. The fragment also directed synthesis of the protein in E. coli MT912 but at levels much lower than those previously observed. Nucleotide sequencing of the fragment revealed an open reading frame (designated pfeA for Pseudomonas ferric enterobactin) of 2,241 bp capable of encoding a 746-amino-acid protein with a molecular weight of 80,967. The PfeA protein showed more than 60% homology to the E. coli FepA protein. Consistent with this, the two proteins showed significant immunological cross-reactivity.

High density lipoproteins stimulate molecular weight 80K protein phosphorylation in human trophoblast cells: evidence for a protein kinase-C-dependent pathway in human placental lactogen release.

Previous studies from our laboratory have demonstrated that high density lipoprotein (HDL) HDL and apolipoprotein-AI (apoAI) stimulate human placental lactogen (hPL) release from human trophoblast cells. To determine whether protein kinase-C (PKC) activation is involved in the mechanism of HDL- and apoAI-mediated hPL release, we examined the effects of these factors on the phosphorylation of cytosolic proteins known to be phosphorylated in response to PKC activation by phorbol myristate acetate (PMA). HDL and apoAI each caused a dose- and time-dependent increase in phosphorylation of a PMA-inducible 80K mol wt acidic cytosolic protein in a manner similar to that observed in many other cell types. Stimulation of 80K protein phosphorylation was apparent 5 min after the addition of HDL, apoAI, or PMA and was maximal at 15 min. Maximal 80K protein phosphorylation in cells exposed to PMA (1.6 microM), HDL (1500 micrograms/ml), and apoAI (600 micrograms/ml) was 284%, 206%, and 239% that in untreated cells, respectively. The increase in both 80K protein phosphorylation and hPL release in response to apoAI was prevented by pretreatment of the cells with the PKC inhibitor staurosporine (10 microM) or by down-regulation of PKC after extended preincubation of the cells with 16 microM PMA. (Bu)2cAMP and the adenylate cyclase activator forskolin, which stimulate hPL release, had no effect on 80K protein phosphorylation. These results strongly suggest that HDL- and apoAI-stimulated hPL release involves a PKC-dependent pathway. Since earlier studies also implicate a cAMP-mediated pathway in the stimulation of hPL release by these agents, it appears that multiple intracellular pathways are involved in the stimulation of hPL release.

High density lipoproteins stimulate molecular weight 80K protein phosphorylation in human trophoblast cells: evidence for a protein kinase-C-dependent pathway in human placental lactogen release

Previous studies from our laboratory have demonstrated that high density lipoprotein (HDL) HDL and apolipoprotein-AI (apoAI) stimulate human placental lactogen (hPL) release from human trophoblast cells. To determine whether protein kinase-C (PKC) activation is involved in the mechanism of HDL- and apoAI-mediated hPL release, we examined the effects of these factors on the phosphorylation of cytosolic proteins known to be phosphorylated in response to PKC activation by phorbol myristate acetate (PMA). HDL and apoAI each caused a dose- and time-dependent increase in phosphorylation of a PMA-inducible 80K mol wt acidic cytosolic protein in a manner similar to that observed in many other cell types. Stimulation of 80K protein phosphorylation was apparent 5 min after the addition of HDL, apoAI, or PMA and was maximal at 15 min. Maximal 80K protein phosphorylation in cells exposed to PMA (1.6 microM), HDL (1500 micrograms/ml), and apoAI (600 micrograms/ml) was 284%, 206%, and 239% that in untreated cells, respectively. The increase in both 80K protein phosphorylation and hPL release in response to apoAI was prevented by pretreatment of the cells with the PKC inhibitor staurosporine (10 microM) or by down-regulation of PKC after extended preincubation of the cells with 16 microM PMA. (Bu)2cAMP and the adenylate cyclase activator forskolin, which stimulate hPL release, had no effect on 80K protein phosphorylation. These results strongly suggest that HDL- and apoAI-stimulated hPL release involves a PKC-dependent pathway. Since earlier studies also implicate a cAMP-mediated pathway in the stimulation of hPL release by these agents, it appears that multiple intracellular pathways are involved in the stimulation of hPL release.

Evidence for a novel insulin-like growth factor (IGF)-dependent protease regulating IGF-binding protein-4 in dermal fibroblasts.

The mechanisms by which insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP-4) levels in cellular conditioned media are poorly understood. The effect of IGFs on IGFBP-4 levels in fibroblast conditioned media is not mediated via the type 1 or type 2 cellular IGF receptors, and the IGFs exert little or no effects on IGFBP-4 messenger RNA levels in human adult fibroblasts or in rat neuroblastoma cells. To determine whether the effects of IGFs on IGFBP-4 might be exerted through alterations in IGFBP-4 degradation, we incubated cell-free, fibroblast-conditioned media from either sheep or human dermal fibroblasts with or without IGF-I, IGF-II (each 1 microgram/ml), or insulin (10 micrograms/ml) for 72 h at 37 C. Samples were then analyzed by Western ligand blot using radiolabeled IGFs and by immunoblotting using a polyclonal antisera to human IGFBP-4. In the absence of IGFs, no apparent changes in the basal concentrations of the various IGFBPs were observed. In contrast, incubation of media with IGFs caused a 70-80% reduction in levels of both sheep and human IGFBP-4, whereas incubation with insulin was without effect. Similarly, incubation of cell-free conditioned media containing recombinant human IGFBP-4 with IGF-I caused a reduction in detectable levels of the 28K protein. The decrease in IGFBP-4 levels was accompanied by the appearance of an immunoreactive approximate 17-20K fragment that did not bind radiolabeled IGFs by ligand blot. The IGF-dependent decrease in IGFBP-4 was prevented by coincubation of the media with serine protease inhibitors, EDTA, or 1,10-phenanthrolene, suggesting that IGFs may activate an IGFBP-4 specific metallo-serine protease present in fibroblast conditioned media. Alternatively, binding of IGF-I or -II to IGFBP-4 may enhance the susceptibility of IGFBP-4 to proteolytic degradation. The demonstration that IGF-I and IGF-II can promote directly the proteolytic degradation of IGFBP-4 into fragments that do not bind IGFs provides a novel mechanism by which the IGFs may increase their own availability and/or activity in biological fluids.

Activation of bovine rod outer segment phospholipase C by arrestin

Phospholipase C (PLC) enzyme activity in rod outer segment (ROS) membranes bleached in the presence of ATP and GTP was assayed using exogenously added [3H]phosphatidylinositol 4,5-bisphosphate vesicles as substrate. The addition of the soluble ROS protein arrestin (also known as S-antigen or 48K protein) to ROS membranes activated PLC 2-3.4-fold. This activation was dose-dependent, and maximal activation was observed at an arrestin concentration of congruent to 110-220 nM. PLC activation by arrestin was dependent on ROS protein concentration and free Ca2+. Soluble PLC (s-PLC) enzyme activity present in hypotonic extracts of bleached ROS was also activated 2-4-fold by arrestin. Maximum activation of s-PLC by arrestin was observed at free Ca2+ of 80 nM. Arrestin activation of s-PLC was not affected by urea-treated and extensively washed ROS membranes, suggesting that rhodopsin was not required for the observed effect of arrestin on s-PLC. The results are indicative of a direct interaction of arrestin with s-PLC, resulting in the activation of the latter. Based on these results and the documented binding of arrestin to bleached and phosphorylated rhodopsin, a model for the light activation of PLC in ROS is proposed.

Activation of bovine rod outer segment phospholipase C by arrestin.

Phospholipase C (PLC) enzyme activity in rod outer segment (ROS) membranes bleached in the presence of ATP and GTP was assayed using exogenously added [3H]phosphatidylinositol 4,5-bisphosphate vesicles as substrate. The addition of the soluble ROS protein arrestin (also known as S-antigen or 48K protein) to ROS membranes activated PLC 2-3.4-fold. This activation was dose-dependent, and maximal activation was observed at an arrestin concentration of congruent to 110-220 nM. PLC activation by arrestin was dependent on ROS protein concentration and free Ca2+. Soluble PLC (s-PLC) enzyme activity present in hypotonic extracts of bleached ROS was also activated 2-4-fold by arrestin. Maximum activation of s-PLC by arrestin was observed at free Ca2+ of 80 nM. Arrestin activation of s-PLC was not affected by urea-treated and extensively washed ROS membranes, suggesting that rhodopsin was not required for the observed effect of arrestin on s-PLC. The results are indicative of a direct interaction of arrestin with s-PLC, resulting in the activation of the latter. Based on these results and the documented binding of arrestin to bleached and phosphorylated rhodopsin, a model for the light activation of PLC in ROS is proposed.

Rice hoja blanca virus genome characterization and expression in vitro.

No information exists on the organization and mechanisms of expression of the genome of rice hoja blanca virus (RHBV), a member of the tenuivirus group, but here we describe the first steps in its characterization. RHBV contains four ssRNA and three dsRNA species, the sizes of which were estimated by native and denaturing gel electrophoresis. Hybridization analyses using 32P-labelled riboprobes of viral and viral complementary polarities showed that unequal amounts of the two polarities of at least the smallest RNA are present in the virion, and indicated that the dsRNA species contain the same information as the ssRNA species of corresponding size. Total RHBV RNA directs the synthesis of two major proteins of 23K and 21K in vitro. RNA3 directs the synthesis of a 23K protein designated NS3, and RNA4 of a 21K protein designated NS4. The NS4 protein corresponds to the non-structural protein that accumulates in RHBV-infected rice tissue. The nuclecocapsid protein is not translated from either total RHBV RNA or any individual RHBV RNA in vitro.

Processing of hepatitis B virus surface antigen expressed by recombinant Oka varicella vaccine virus.

We have constructed a recombinant Oka varicella vaccine virus expressing hepatitis B virus (HBV) surface antigen (HBsAg). HBsAg was synthesized as 26K and 30K proteins in infected cells and secreted into the culture supernatant as 30K and 35K proteins. Inhibitors and glycosidase treatments, and pulse-chase labelling experiments, revealed the glycosylation process of HBsAg. The latter was synthesized as a non-glycosylated 26K protein and subjected to N-linked glycosylation to form a 30K protein with high mannose glycans. Three species of dimers composed of 26K and 30K subunits were then formed with disulphide bonds. Both subunits of the dimers were further subjected to O-linked glycosylation and conversion from high mannose glycans to complex glycans followed by sialylation. Three species of dimers composed of 30K and 35K subunits were secreted into the culture supernatant as HBsAg particles. HBsAg was synthesized, glycosylated with both N- and O-linked glycans, sialylated, and then secreted into the culture supernatant within 1 h. These modifications of HBsAg by glycans might stabilize its structure and enhance its immunogenicity as a live HBV vaccine.