Phosphoprotein mRNA Research Articles

Antiviral activity of anisomycin against spring viraemia of carp virus in epithelioma papulosum cyprini cells and zebrafish

Spring viraemia of carp (SVC) caused by spring viraemia of carp virus (SVCV) is an acute and highly lethal viral disease of cyprinid fish. However, effective therapy for SVC is still scarce until now. Here we evaluated the inhibition of anisomycin (Ani), a metabolite produced by Streptomyces griseolus, on the replication of SVCV in vitro and in vivo. Our results demonstrated that Ani could suppress SVCV replication with the maximum inhibitory rate > 95% in epithelioma papulosum cyprini (EPC) cells. And the half maximal inhibitory concentrations (IC50) of Ani on SVCV glycoprotein (G), nucleoprotein (N) and phosphoprotein mRNA expressions were 21.79, 13.13 and 12.24 nM, respectively. Besides, Ani decreased SVCV-induced cytopathic effects and nucleus damages. As expected, Ani also showed a strong anti-SVCV activity in vivo, as indicated by inhibiting viral gene expression and increasing the survival rate of zebrafish. Intraperitoneal injection of Ani increased the survival rate of zebrafish by 30% and markedly inhibited the expressions of G and N mRNA by > 60% in kidney and spleen at day 1 and day 4 post-infection. Results so far suggest that Ani as a powerful agent against SVCV can be applied to the control of SVC in aquaculture.

High-fat diet accelerates extreme obesity with hyperphagia in female heterozygous Mecp2-null mice.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutation of the methyl-CpG-binding protein 2 (MECP2) gene. Although RTT has been associated with obesity, the underlying mechanism has not yet been elucidated. In this study, female heterozygous Mecp2-null mice (Mecp2+/- mice), a model of RTT, were fed a normal chow diet or high-fat diet (HFD), and the changes in molecular signaling pathways were investigated. Specifically, we examined the expression of genes related to the hypothalamus and dopamine reward circuitry, which represent a central network of feeding behavior control. In particular, dopamine reward circuitry has been shown to regulate hedonic feeding behavior, and its disruption is associated with HFD-related changes in palatability. The Mecp2+/- mice that were fed the normal chow showed normal body weight and food consumption, whereas those fed the HFD showed extreme obesity with hyperphagia, an increase of body fat mass, glucose intolerance, and insulin resistance compared with wild-type mice fed the HFD (WT-HFD mice). The main cause of obesity in Mecp2+/--HFD mice was a remarkable increase in calorie intake, with no difference in oxygen consumption or locomotor activity. Agouti-related peptide mRNA and protein levels were increased, whereas proopiomelanocortin mRNA and protein levels were reduced in Mecp2+/--HFD mice with hyperleptinemia, which play an essential role in appetite and satiety in the hypothalamus. The conditioned place preference test revealed that Mecp2+/- mice preferred the HFD. Tyrosine hydroxylase and dopamine transporter mRNA levels in the ventral tegmental area, and dopamine receptor and dopamine- and cAMP-regulated phosphoprotein mRNA levels in the nucleus accumbens were significantly lower in Mecp2+/--HFD mice than those of WT-HFD mice. Thus, HFD feeding induced dysregulation of food intake in the hypothalamus and dopamine reward circuitry, and accelerated the development of extreme obesity associated with addiction-like eating behavior in Mecp2+/- mice.

Short interfering RNAs targeting a vampire-bat related rabies virus phosphoprotein mRNA.

The aim of this study was to assess the in vitro and in vivo effects of short-interfering RNAs (siRNAs) against rabies virus phosphoprotein (P) mRNA in a post-infection treatment for rabies as an extension of a previous report (Braz J Microbiol. 2013 Nov 15;44(3):879–82). To this end, rabies virus strain RABV-4005 (related to the Desmodus rotundus vampire bat) were used to inoculate BHK-21 cells and mice, and the transfection with each of the siRNAs was made with Lipofectamine-2000™. In vitro results showed that siRNA 360 was able to inhibit the replication of strain RABV-4005 with a 1 log decrease in virus titter and 5.16-fold reduction in P mRNA, 24 h post-inoculation when compared to non-treated cells. In vivo, siRNA 360 was able to induce partial protection, but with no significant difference when compared to non-treated mice. These results indicate that, despite the need for improvement for in vivo applications, P mRNA might be a target for an RNAi-based treatment for rabies.

A Novel Nuclear Trafficking Module Regulates the Nucleocytoplasmic Localization of the Rabies Virus Interferon Antagonist, P Protein

Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1-P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3-P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection.

Fbxw7-dependent Degradation of Notch Is Required for Control of “Stemness” and Neuronal-Glial Differentiation in Neural Stem Cells

Control of the growth and differentiation of neural stem cells is fundamental to brain development and is largely dependent on the Notch signaling pathway. The mechanism by which the activity of Notch is regulated during brain development has remained unclear, however. Fbxw7 (also known as Fbw7, SEL-10, hCdc4, or hAgo) is the F-box protein subunit of an Skp1-Cul1-F-box protein (SCF)-type ubiquitin ligase complex that plays a central role in the degradation of Notch family members. We now show that mice with brain-specific deletion of Fbxw7 (Nestin-Cre/Fbxw7(F/F) mice) die shortly after birth with morphological abnormalities of the brain and the absence of suckling behavior. The maintenance of neural stem cells was sustained in association with the accumulation of Notch1 and Notch3, as well as up-regulation of Notch target genes in the mutant mice. Astrogenesis was also enhanced in the mutant mice in vivo, and the differentiation of neural progenitor cells was skewed toward astrocytes rather than neurons in vitro, with the latter effect being reversed by treatment of the cells with a pharmacological inhibitor of the Notch signaling pathway. Our results thus implicate Fbxw7 as a key regulator of the maintenance and differentiation of neural stem cells in the brain.

Inhibition of Human Metapneumovirus Replication by Small Interfering RNA

Human metapneumovirus (hMPV) is a major respiratory viral pathogen in young children, elderly individuals and immunocompromised patients. Despite its major effects related to bronchiolitis, pneumonia and its potential role in recurrent wheezing episodes, there is still no commercial treatment or vaccine available against this paramyxovirus. We tested a therapeutic strategy for hMPV that was based on RNA interference. An hMPV genome-wide search for small interfering RNAs (siRNAs) by computational analysis revealed 200 potentially effective 21-mer siRNAs. Initial screening with a luciferase assay identified 57 siRNAs of interest. Further evaluation of their inhibitory potential against the four hMPV subgroups by quantitative real-time reverse transcriptase PCR and plaque immunoassay identified two highly potent siRNAs with 50% inhibitory concentration (IC50) values in the subnanomolar range. siRNA45 targets the nucleoprotein messenger RNA (mRNA) and had IC50 values <0.078 nM against representative strains from the four hMPV subgroups, whereas siRNA60, which targets the phosphoprotein mRNA, had IC50 values between 0.090-<0.078 nM against the same panel of hMPV strains. Longer25/27-mer siRNAs known as Dicer substrates designed from the top two siRNA candidates were also evaluated and were at least as effective as their corresponding 21-mer siRNAs. Interestingly, the presence of one or two nucleotide mismatches in the target mRNA sequence of some hMPV subgroups did not always affect hMPV inhibition in vitro. We successfully identified two highly efficient siRNAs against hMPV targeting essential components of the hMPV replication complex.

Paramyxovirus mRNA editing, the ‘rule of six’ and error catastrophe: a hypothesis

The order Mononegavirales includes three virus families that replicate in the cytoplasm: the Paramyxoviridae, composed of two subfamilies, the Paramyxovirinae and Pneumovirinae, the Rhabdoviridae and the Filoviridae. These viruses, also called non-segmented negative-strand RNA viruses (NNV), contain five to ten tandemly linked genes, which are separated by conserved junctional sequences that act as mRNA start and poly(A)/stop sites. For the NNV, downstream mRNA synthesis depends on termination of the upstream mRNA, and all NNV RNA-dependent RNA polymerases reiteratively copy ("stutter" on) a short run of template uridylates during transcription to polyadenylate and terminate their mRNAs. The RNA-dependent RNA polymerase of a subset of the NNV, all members of the Paramyxovirinae, also stutter in a very controlled fashion to edit their phosphoprotein gene mRNA, and Ebola virus, a filovirus, carries out a related process on its glycoprotein mRNA. Remarkably, all viruses that edit their phosphoprotein mRNA are also governed by the "rule of six", i.e. their genomes must be of polyhexameric length (6n+0) to replicate efficiently. Why these two seemingly unrelated processes are so tightly linked in the Paramyxovirinae has been an enigma. This paper will review what is presently known about these two processes that are unique to viruses of this subfamily, and will discuss whether this enigmatic linkage could be due to the phenomenon of RNA virus error catastrophe.

Translation initiation at alternate in-frame AUG codons in the rabies virus phosphoprotein mRNA is mediated by a ribosomal leaky scanning mechanism.

The phosphoprotein of rabies virus is a 297-amino-acid polypeptide encoded by the longest open reading frame of the P gene. Immunoprecipitation experiments using a monoclonal antiserum directed against the P protein detected the P protein and at least four additional shorter products in infected cells, cells transfected with a plasmid encoding the wild-type P protein, and purified virus (CVS strain). By means of deletion analyses, these proteins were shown to be translated from secondary downstream in-frame AUG initiation codons. Immunofluorescence experiments indicated that all these P products were found in the cytoplasm of transfected cells; however, the proteins initiated from the third, fourth, and fifth AUG codons were found mostly in the nucleus. Changes in the 5'-terminal region of the P mRNA (including site-specific mutations, deletions, and insertions) demonstrated that a leaky scanning mechanism is responsible for translation initiation of the P gene at several sites.

Effect of acidic ribosomal phosphoprotein mRNA 5'-untranslated region on gene expression and protein accumulation.

Constructions were made from genes encoding ribosomal acidic phosphoproteins YP1 beta (L44') and YP2 beta (L45) from Saccharomyces cerevisiae in which different parts of the 5'-untranslated regions were included. The constructs were inserted into centromeric plasmids under the control of the GAL1 promoter and expressed in yeast strains in which the genes coding for each acidic protein family, P1 and P2, had been disrupted. Deletions in the 5' region of the two genes have been found to oppositely affect their expression. Deletion of most of this region strongly stimulates the expression of YP2 beta (L45), increasing the translation efficiency of the mRNA, and generating a 6-fold excess of protein in the cell. A similar deletion in the rpYP1 beta gene represses the expression of the protein, reducing drastically the amount of the mRNA in the cell. The overexpression of rpYP2 beta affects the cell growth by inhibiting protein synthesis at the level of initiation. Reduction of the YP2 beta(L45) overproduction by growing in controlled concentrations of glucose abolishes the inhibitory effect. The excess protein, probably as a high molecular weight complex, apparently interferes with the joining of the 60 S subunit to the initiation complex generating the accumulation of polysome half-mers. In addition, the results indicate the existence of a regulatory mechanism by which each one of the two acidic proteins controls the expression of the other polypeptide. YP1 beta(L44') represses the expression of YP2 beta(L45), while this protein stimulates the expression of YP1 beta(L44').

Alternatively spliced pp52 mRNA in nonlymphoid stromal cells.

The 52-kDa phosphoprotein, also reported as lymphocyte-specific gene 1 and WP34, is transcribed as a 1.6-kb mRNA in B lymphocytes, B cell lines, and untransformed T cells. This gene encodes a cytoplasmic and plasma membrane-associated protein that is phosphorylated at a casein kinase II site and reportedly binds calcium. Based on these properties, it has been hypothesized that lymphoid form of the 52-kDa phosphoprotein protein may play a role in lymphocyte signal transduction. We show that alternatively spliced mRNA are expressed from this gene in nonlymphoid cell lines (myocytes, stromal cells, fibroblasts). These cell lines do not express the 1.6-kb lymphoid cell-specific transcript. Instead, mRNA of 2.0 and 2.8 kb are detected in varying abundance. A full-length 2.0-kb cDNA has been cloned and sequenced from the BMS2 stromal cell line by conventional screening and polymerase chain reaction-based methods. This cDNA clone, designated S37, has a single open reading frame encoding a 328 amino acid peptide. The nucleotide sequence of the S37 stromal cell cDNA is identical to that of the lymphocyte derived pp52 cDNA from the 3' poly(A) tail to the codon encoding the amino acid at residue 24. This region of the S37 cDNA clone encodes a protein that is identical to that encoded by the lymphoid pp52 cDNA and includes a casein kinase II phosphorylation site. However, the two clones differ in their 5' nucleotide sequence and their NH3 terminal amino acid sequence. This organization is consistent with alternative exon utilization. These results suggest that tissue-specific control mechanisms are used to generate different forms of lymphoid form of the 52-kDa phosphoprotein mRNA in lymphoid cells versus mesoderm-derived, nonlymphoid cell lineages.

Secreted phosphoprotein mrna is induced during multi‐stage carcinogenesis in mouse skin and correlates with the metastatic potential of murine fibroblasts

Secreted phosphoprotein I (SPP), also known as 2ar, osteopontin, 44-kDa bone phosphoprotein, bone sialoprotein I, and transformation-related phosphoprotein, is a 41.5-kDa glycosylated phosphoprotein secreted by many mammalian cell lines and expressed in a limited set of tissues. Using a cDNA probe, we found that SPP mRNA, which is barely detectable in normal mouse epidermis, was expressed at moderate-to-high levels in 2 of 3 epidermal papillomas and at consistently high levels in 7 of 7 squamous-cell carcinomas induced by an initiation-promotion regimen. This contrasts with the transient induction we had previously observed after a single application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). In a set of 5 independently isolated T24-H-ras-transfected mouse C3H 10T1/2 cell lines, the levels of SPP mRNA correlated well with ras mRNA levels and with both experimental and spontaneous metastatic ability. SPP mRNA expression was also elevated in a derivative of mouse LTA cells transfected with genomic DNA from B16F1 melanoma cells and selected for increased experimental metastatic ability in the chick embryo. This apparent association of SPP expression with invasion, progression and metastasis, along with the presence of a functional ArgGlyAsp (RGD) cell adhesion site in SPP (osteopontin), leads us to propose that SPP may act as an autocrine adhesion factor for tumor cells.

Stability of the nucleotide sequence of the phosphoprotein gene of measles virus during lytic infections

Three clones with cDNA inserts encoding large portions of the measles virus phosphoprotein mRNA were characterized and compared with a previously published sequence of the Edmonston strain of measles virus. The two cloned viruses were separated by more than 100 passages. Only one out of 1477 nucleotides differed in the two sequences reflecting a very low mutation rate of the phosphoprotein gene during dilute lytic passages. The discovery that a third reading frame in the phosphoprotein gene may code for a novel peptide chain in addition to the P and C peptides may explain some of the high stability of the gene. The new reading frame was accessed by a translational shift caused by insertion of one extra G at a particular site in one of three otherwise identical cDNA sequences. A discrepancy was also found between the presumably high error rate of viral RNA polymerases and the stability of nucleotides in which mutations would not lead to amino acid substitutions. A few errors in the previously published sequence were discovered and the corrections are presented.

Two species of the phosphoprotein mRNA with differently edited reading frames discovered in measles virus infected Vero cells. Brief report.

One cDNA clone representing the phosphoprotein mRNA sequence of the Edmonston strain of measles virus contained 4 G nucleotides at a particular position. Two other clones contained 3 G nucleotides at the same position. Otherwise the nucleotide sequence were identical. The mRNA with 3 G nucleotides codes for the 70 kD phosphoprotein. The mRNA with 4 G nucleotides may code for a putative new peptide with 231 aminoterminal amino acids in common with the P protein whereas the 68 carboxyterminal amino acids are different from any amino acid sequence of the phosphoprotein. Thus the consequence of the insertion of one additional G is a translational shift to a shorter open reading frame. There are several indications that the observation is of biological significance.

Characterization of the internal initiation of translation on the vesicular stomatitis virus phosphoprotein mRNA.

Internal initiation of translation on the vesicular stomatitis virus (VSV) phosphoprotein (P) mRNA leads to the synthesis of a second protein [Herman, R. C. (1986) J. Virol. 58, 797-804]. Characterization of this phenomenon shows that initiation at the 5'-proximal and internal AUG codons has different optima for mono- and divalent cations in the reticulocyte lysate. Whereas 5' initiation is stimulated by increasing concentration of K+ over the endogenous level, internal initiation is inhibited. Internal initiation is much less sensitive to the effects of the cap analogue 7mGpppG in both the reticulocyte lysate and the wheat-germ extract under conditions that reduce 5'-proximal initiation to only about 4-5% of the control level. These results imply that 5'-proximal and internal initiations are distinct biochemical processes.

Cloning and Expression of a Viral Phosphoprotein: Structure Suggests Vesicular Stomatitis Virus NS May Function by Mimicking an RNA Template

The phosphoprotein (NS) gene from the Indiana serotype of vesicular stomatitis virus (VSV; Mudd-Summers strain) was cloned and sequenced. The NS gene encodes a protein of 265 amino acids which was expressed from a simian virus 40 vector in COS cells. The post-translational modification characteristic of viral NS, the extensive phosphorylation of a cluster of serine and threonine residues, was also evident in recombinant NS protein. The NS gene displays a property common to the phosphoprotein genes of negative-strand RNA viruses: the phosphoprotein mRNA has a second open reading frame (ORF) which could encode a small (7500 mol. wt.) protein. Both measles virus and Sendai virus employ the second ORF of their phosphoprotein gene, and the resultant proteins have an amino acid composition similar to that predicted for the VSV ORF. Comparison of phosphoproteins from different VSV strains revealed two conserved domains that we propose are critical for the function of NS in transcription and replication.

Internal initiation of translation on the vesicular stomatitis virus phosphoprotein mRNA yields a second protein.

In vitro translation of a mixture of the vesicular stomatitis virus (VSV) polyadenylated mRNAs yielded a previously undetected protein with a molecular weight of approximately 7,000 (7K protein). Hybrid-arrested translation demonstrated that both the 7K protein and the VSV phosphoprotein (P protein) were encoded by the P protein message. Immunoprecipitation of the 7K protein with monoclonal antiserum directed against the P protein indicated that the two products were encoded in the same open reading frame. A protein of approximately the same size was immunoprecipitated from cytoplasmic extracts of VSV-infected cells by both the polyclonal and monoclonal antisera, and it is likely that it was a previously unrecognized viral gene product. Translational mapping of the P protein mRNA in vitro indicated that the 7K protein was encoded in the 3' one-third of the sequence. The synthesis of the 7K protein in vitro was unaffected by hybrid arrest conditions which blocked the 5' two-thirds of the mRNA and inhibited synthesis of the P protein. These results imply that the ribosomes bind and initiate translation internally on the P protein mRNA at a site located hundreds of nucleotides downstream from the capped 5' end.

Preliminary characterization of mouse dentine phosphoprotein mRNA: [formula omitted] and [formula omitted]. Univ. So. California, L.A., CA. 90089-0191

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Complete sequence analysis of cDNA clones encoding rat whey phosphoprotein: homology to a protease inhibitor.

Lactoprotein clones have been isolated from a rat mammary gland recombinant library of cDNA plasmids. Clones p-Wp 52 and p-Wp 47 were shown by hybrid selection, in vitro translation, and immunoprecipitation to represent a cloned DNA sequence encoding rat whey phosphoprotein. We report here the nucleotide sequence of the cDNA insert of p-Wp 52 and shows that it encodes the complete whey phosphoprotein sequence. The encoded sequence shows a high content of half-cystine, glutamic acid, aspartic acid, and serine but an absence of tyrosine. The half-cystines appear in unique arrangements and are repeated in two domains of the protein. The second domain has striking similarities with the second domain of the red sea turtle protease inhibitor. Clone p-Wp 52 has allowed the study of expression of whey phosphoprotein mRNA during functional differentiation of rat mammary gland and in mammary tumors. The whey phosphoprotein mRNA is detected during midpregnancy and lactation in the rat mammary gland but is barely detected in mammary tumors in which other milk protein mRNAs are expressed. The whey phosphoprotein gene in these tumors is hypermethylated, correlating with the reduced expression of this gene.