Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0038362 (stomatitis)
 8,852 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Encapsidation of presynthesized and nascent (synthesized de novo) vesicular stomatitis virus (VSV) leader RNA in vitro by the nucleocapsid protein (N) and the role of the phosphoprotein (P, previously known as NS) in this process were examined. Presynthesized VSV leader RNAs were derived from the SP6 transcription vectors containing both (+) and (-) leader genes while the nascent RNA was derived from transcription of viral ribonucleoprotein (RNP) complex. The N and the P proteins were made by transcription from SP6 vectors containing the genes, followed by translation of the mRNAs in rabbit reticulocyte lysate. Here, we demonstrate that the N protein alone encapsidated presynthesized VSV leader RNA; however, prior formation of N-P complex totally abolished the encapsidation property of N. On the other hand, encapsidation of nascent RNA by the N protein was stimulated by the N-P complex. These results suggest that encapsidation by the N protein of presynthesized and nascent VSV RNA are separate biochemical processes which can be distinguished by the differential role of the phosphoprotein P in the two reactions.
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PMID:Role of the phosphoprotein (P) in the encapsidation of presynthesized and de novo synthesized vesicular stomatitis virus RNA by the nucleocapsid protein (N) in vitro. 131 45

Protein secretion is blocked in Xenopus oocytes arrested at second meiotic metaphase. In this report, we show that secretion becomes blocked coincident with germinal vesicle breakdown (GVBD). Transport through the metaphase-arrested oocyte's secretory pathway continues unimpeded until proteins reach the trans-Golgi. These conclusions are drawn from experiments using exogenous prolactin and vesicular stomatitis virus G protein (VSV G) encoded by SP6 transcripts and endogenous glycosaminoglycan (GAG) chains initiated on beta-D-4-methylumbelliferyl-xyloside. From the initiation of maturation with progesterone until GVBD, secretion of prolactin synthesized before the start of maturation is comparable to secretion in immature oocytes, but after GVBD secretion of prolactin declines approximately 63% in the first hour. Not all steps in the secretory pathway are blocked when oocytes mature. Since VSV G protein acquires resistance to endo H digestion with equal efficiency in immature oocytes (arrested in first meiotic prophase) and matured oocytes (arrested in second meiotic metaphase), we conclude that transport of this protein from the ER to the Golgi is not inhibited at meiotic metaphase. Using [35S]sulfate to label xyloside-initiated GAG chains we find that transport of GAG chains from the trans-Golgi to the cell surface is 15-fold lower in matured oocytes than in immature oocytes. Examination of the size of GAG chains by SDS-PAGE and HPLC indicates that matured oocytes produce GAG chains significantly larger than GAG chains from immature oocytes. This increase in size suggests that GAG chains from matured oocytes have a longer residence time in the trans-Golgi than GAG chains from immature oocytes. Hence, part of the block to secretion in metaphase-arrested oocytes could be an inhibition of vesicle budding from the trans-Golgi.
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PMID:The secretory pathway is blocked between the trans-Golgi and the plasma membrane during meiotic maturation in Xenopus oocytes. 239 Sep 97

The interaction of the nucleocapsid protein N and the phosphoprotein NS of vesicular stomatitis virus (VSV) was studied, free of other viral proteins, by transcription from SP6 vectors, followed by translation in a rabbit reticulocyte lysate. N-NS complex formation depended strongly on cotranslation of the two proteins; when N and NS were mixed following separate translation of each, very little complex formation occurred. Conditions were found under which at least six N-NS complexes were separated from each other by electrophoresis in a nondenaturing gel system, and the following findings were made. (i) These complexes fell into two groups; complexes 1 through 5 all had a stoichiometry of two molecules of N to one molecule of NS, whereas N-NS complex 6 had an equimolar ratio of the two proteins. (ii) N-NS complexes 1 through 5 predominated at lower concentrations of NS relative to N, but N-NS complex 6 was the major or sole product when NS was equimolar to or in excess of N. (iii) The two sets of complexes were formed by two distinct types of interactions of NS with N. The formation of N-NS complexes 1 through 5 was abolished by the removal of as few as 11 amino acid residues from the basic, highly conserved carboxy-terminal domain of NS, which is essential for the binding of NS to the N-RNA template of VSV. In contrast, formation of complex 6 was unaffected by removal of as many as 62 of the carboxy-terminal amino acids of NS, a region encompassing both the terminal basic domain and an adjacent domain which is required for VSV RNA polymerase function. The significance of these observations for the mechanism of VSV genome replication is discussed.
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PMID:Resolution of multiple complexes of phosphoprotein NS with nucleocapsid protein N of vesicular stomatitis virus. 283 92

The interactions between the nucleocapsid protein N and either RNA or the phosphoprotein NS of vesicular stomatitis virus (VSV) were studied by the transcription of N and NS mRNAs from SP6 vectors, followed by translation in a rabbit reticulocyte lysate. Nascent N protein bound tightly to added labeled RNA, as well as to endogenous RNA in the reticulocyte lysate. This binding was demonstrated by three independent techniques. First, labeled N protein and labeled RNA migrated identically as a series of sharp, closely spaced bands in a nondenaturing gel system. Second, translated N protein behaved as a stable ribonucleoprotein complex in CsCl gradients and sedimented to the same density as the authentic N-RNA template of VSV. Third, translated N protein protected a series of labeled RNA fragments from digestion by RNase A. None of the three RNA-binding criteria was satisfied by either translated NS protein or two deletion mutants of N protein or by other components of the reticulocyte lysate. The evidence suggests that the observed binding of RNA by nascent N was not RNA sequence specific, in contrast to the encapsidation process during VSV replication. Moreover, the prior formation of N-NS complexes totally abolished the observed binding of RNA by N. Thus, we propose that NS may be responsible for conferring the sequence specificity of the RNA binding that occurs during VSV genome replication.
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PMID:Complex formation with vesicular stomatitis virus phosphoprotein NS prevents binding of nucleocapsid protein N to nonspecific RNA. 283 93

A full-length cDNA clone of the mRNA encoding the phosphoprotein (NS) of the Indiana serotype of vesicular stomatitis virus was inserted into the SP6 transcription vector. By in vitro transcription of the inserted gene followed by translation of the mRNA in a rabbit reticulocyte lysate, NS protein was synthesized. The biological activity of the protein was demonstrated by RNA synthesis in vitro by reconstitution with L protein and N-RNA template purified from virions. Using oligonucleotide-directed RNase H cleavage of the full-length NS mRNA, a series of deleted RNAs were made which gave rise to corresponding size classes of truncated NS protein after translation in vitro. The N-RNA template binding site was located at the C-terminal domain (21 amino acids) of the NS protein and the L-protein binding site was present within 14 amino acids spanning the NH2-terminal side of the N-RNA binding site. These results are similar to that obtained with the NS protein of the New Jersey serotype of VSV, indicating conservation of the functional domains within the VSV serotypes.
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PMID:The functional domains of the phosphoprotein (NS) of vesicular stomatitis virus (Indiana serotype). 284 48

The phosphoprotein (NS) of vesicular stomatitis virus is an indispensable subunit of the virion-associated RNA polymerase (L). NS consists of a highly acidic NH2-terminal domain and a basic COOH-terminal domain. Unlike the latter, the amino acid sequences of the NH2-terminal regions are highly dissimilar among different viral serotypes, although they share structural similarities. We have cloned an NS gene into the SP6 transcription vector and replaced the 5'-terminal 80% by a full-length gene for beta-tubulin, which contains an acidic COOH-terminal domain. Here we present evidence that the chimeric tubulin-NS protein is biologically active and that the acidic region in tubulin directly affects the transcription reaction. These observations indicate that NS probably functions as an activator protein in which the acidic domain stimulates transcription of the viral genes by interacting with the RNA polymerase as observed for eukaryotic cellular transcription activators.
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PMID:NH2-terminal acidic region of the phosphoprotein of vesicular stomatitis virus can be functionally replaced by tubulin. 284 50

A full-length cDNA copy of the phosphoprotein (NS) mRNA of vesicular stomatitis virus (New Jersey serotype) was inserted into pGEM4 vector downstream of the promoter for bacteriophage SP6 RNA polymerase. Transcription of the cDNA in vitro resulted in the synthesis of NS mRNA, which was subsequently translated into NS protein in a cell-free rabbit reticulocyte system. The biological activity of the expressed NS protein was demonstrated by in vitro synthesis of mRNA by transcription-reconstitution with purified viral L protein and N-RNA template. Deletion mapping of the NS gene defined a specific domain between amino acid residues 213 and 247, which was essential for in vitro transcription. Removal of the COOH-terminal 21 amino acids, on the other hand, did not have a significant effect on transcription. This domain appears to be involved in efficient binding of NS protein to the N protein-RNA template.
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PMID:Identification of a domain within the phosphoprotein of vesicular stomatitis virus that is essential for transcription in vitro. 302 53

The homologous and heterologous interactions between the nucleocapsid protein N and the phosphoprotein P of New Jersey and Indiana serotypes of vesicular stomatitis virus were studied. SP6 derived N and P mRNAs were cotranslated in rabbit reticulocyte lysate and the complexes formed thereof were analyzed by 7.5% nondenaturing polyacrylamide gel electrophoresis. P protein of VSV(NJ) has two binding sites for homologous N protein: One located within the C-terminal 11 amino acids (within domain III) is responsible for the formation of five specific complexes while the other site, which spans the acidic domain I, is necessary for the formation of the sixth complex only. In contrast, P(IND) does not form the sixth complex when interacted with homologous N protein. Interestingly, P(NJ) forms only complexes 1 to 5 when it interacts with N(IND). The above results suggest that the complex 6 formation or domain I interacting site is NJ-serotype specific. Two chimeric P proteins were made using heterologous domains I and II/III of the P proteins of both serotypes. The soluble interaction of the chimeric proteins with the N protein supported the observed serotype specific interactions. The chimeric P proteins bound with equal efficiency with N-RNA template of both serotypes. These results strongly suggest that the acidic domain I of the P protein differentially interacts with homologous and heterologous N proteins. The biological significance of these findings is discussed.
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PMID:Acidic domain of the phosphoprotein (P) of vesicular stomatitis virus differentially interacts with homologous and heterologous nucleocapsid protein (N). 822 May 88

The encapsidation of vesicular stomatitis virus (VSV) genome RNA, a prerequisite step to the replication process by the nucleocapsid protein (N) was studied by its ability to package VSV leader RNA in vitro in a RNase-resistant form. The VSV leader RNA was derived from the SP6 transcription vector while the N protein was made in rabbit reticulocyte lysate. The in vitro encapsidation was carried out by translating N mRNA in the presence of 32P-labeled presynthesized leader RNA. The RNA encapsidation property of the N protein was completely abrogated when the C-terminal five amino acids (VEFDK-COOH) were deleted. Systematic mutational analyses within the C-terminal five amino acid regions reveal that the RNA encapsidation activity was lost in all mutants except K --> A and K --> R, indicating that C-terminal five amino acids, in particular the lysine residue play critical role in genome RNA encapsidation. To correlate the in vitro encapsidation abilities of these mutant N proteins with genome RNA replication, we have used a full-length cDNA clone of VSV genome RNA to rescue infectious virions from cells expressing L, P, and wt or mutant N proteins and measured the recovery of plaque forming units. The results indicate that the N mutants that are defective in in vitro encapsidation of leader RNA do not support replication, establishing the requirement of C-terminal five amino acids of the N protein in viral replication.
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PMID:Carboxy-terminal five amino acids of the nucleocapsid protein of vesicular stomatitis virus are required for encapsidation and replication of genome RNA. 1036 6