R7A mutation in N protein renders temperature sensitive phenotype of VSV by affecting its replication and transcription in vitro

Virus Genes ◽  
2019 ◽  
Vol 55 (4) ◽  
pp. 513-519 ◽  
Author(s):  
Longyun Chen ◽  
Yanyan Zhou ◽  
Min Zhao ◽  
Huimin Chen
Keyword(s):  
Temperature Sensitive ◽  
N Protein ◽  
Transcription In Vitro ◽  
Temperature Sensitive Phenotype

scholarly journals Yeast VSM1 Encodes a v-SNARE Binding Protein That May Act as a Negative Regulator of Constitutive Exocytosis

1999 ◽  
Vol 19 (6) ◽  
pp. 4480-4494 ◽  
Author(s):  
Vardit Lustgarten ◽  
Jeffrey E. Gerst
Keyword(s):  
Secretory Pathway ◽  
Snare Complex ◽  
Negative Regulator ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Interacting Protein ◽  
Cell Extracts ◽  
Temperature Sensitive Phenotype ◽  
Novel Protein

ABSTRACT We have screened for proteins that interact with v-SNAREs of the late secretory pathway in the yeast Saccharomyces cerevisiae. A novel protein, designated Vsm1, binds tightly to the Snc2 v-SNARE in the two-hybrid system and can be coimmunoprecipitated with Snc1 or Snc2 from solubilized yeast cell extracts. Disruption of the VSM1 gene results in an increase of proteins secreted into the medium but does not affect the processing or secretion of invertase. In contrast,VSM1 overexpression in cells which bear a temperature-sensitive mutation in the Sec9 t-SNARE (sec9-4cells) results in the accumulation of non-invertase-containing low-density secretory vesicles, inhibits cell growth and the secretion of proteins into the medium, and blocks rescue of the temperature-sensitive phenotype by SNC1 overexpression. Yet, VSM1 overexpression does not affect yeast bearing asec9-7 allele which, in contrast to sec9-4, encodes a t-SNARE protein capable of forming a stable SNARE complex in vitro at restrictive temperatures. On the basis of these results, we propose that Vsm1 is a novel v-SNARE-interacting protein that appears to act as negative regulator of constitutive exocytosis. Moreover, this regulation appears specific to one of two parallel exocytic paths which are operant in yeast cells.

scholarly journals Genetic and Cell Biological Characterization of the Vaccinia Virus A30 and G7 Phosphoproteins

2005 ◽  
Vol 79 (11) ◽  
pp. 7146-7161 ◽  
Author(s):  
Jason Mercer ◽  
Paula Traktman
Keyword(s):  
Vaccinia Virus ◽  
Temperature Sensitive ◽  
Phenotypic Analysis ◽  
Virion Morphogenesis ◽  
Release Assay ◽  
The Stability ◽  
Temperature Sensitive Phenotype

ABSTRACT The vaccinia virus proteins A30 and G7 are known to play essential roles in early morphogenesis, acting prior to the formation of immature virions. Their repression or inactivation results in the accumulation of large virosomes, detached membrane crescents, and empty immature virions. We have undertaken further study of these proteins to place them within the context of the F10 kinase, the A14 membrane protein, and the H5 phosphoprotein, which have been the focus of previous studies within our laboratory. Here we confirm that both A30 and G7 undergo F10 kinase-dependent phosphorylation in vivo and recapitulate that modification of A30 in vitro. Although the detached crescents observed upon loss of A30 or G7 echo those seen upon repression of A14, no interaction between A30/G7 and A14 could be detected. We did, however, determine that the A30 and G7 proteins are unstable during nonpermissive tsH5 infections, suggesting that the loss of A30/G7 is the underlying cause for the formation of lacy or curdled virosomes. We also determined that the temperature-sensitive phenotype of the Cts11 virus is due to mutations in two codons of the G7L gene. Phenotypic analysis of nonpermissive Cts11 infections indicated that these amino acid substitutions compromise G7 function without impairing the stability of either G7 or A30. Utilizing Cts11 in conjunction with a rifampin release assay, we determined that G7 acts at multiple stages of virion morphogenesis that can be distinguished both by ultrastructural analysis and by monitoring the phosphorylation status of several viral proteins that undergo F10-mediated phosphorylation.

scholarly journals Human and mouse Gpi1p homologues restore glycosylphosphatidylinositol membrane anchor biosynthesis in yeast mutants

1998 ◽  
Vol 334 (3) ◽  
pp. 609-616 ◽  
Author(s):  
Andreas TIEDE ◽  
Jörg SCHUBERT ◽  
Claudia NISCHAN ◽  
Irene JENSEN ◽  
Barbara WESTFALL ◽  
...  
Keyword(s):  
Surface Proteins ◽  
Synthetic Activity ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Temperature Dependent ◽  
Low Degree ◽  
Yeast Mutants ◽  
Human And Mouse ◽  
Temperature Sensitive Phenotype

Glycosylphosphatidylinositol (GPI) represents an important anchoring molecule for cell surface proteins. The first step in its synthesis is the transfer of N-acetylglucosamine (GlcNAc) from UDP to phosphatidylinositol (PI). The products of three mammalian genes, PIG-A, PIG-C and PIG-H, have previously been shown to be involved in the putative enzymic complex. Here we report the cloning of human and mouse cDNAs encoding a fourth participant in the GlcNAc transfer reaction which are homologues of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Gpi1 proteins. To provide evidence for their function, these proteins were expressed in GPI1-disrupted yeast strains. In Sacch. cerevisiae, where GPI1 disruption results in a temperature-sensitive phenotype and abolishes in vitro GlcNAc-PI synthesis, restoration of growth could be demonstrated in a temperature-dependent manner. In addition, in vitro GlcNAc-PI synthetic activity was again detectable. In Schiz. pombe, gpi1+ disruption is lethal. Using random spore analysis, we were able to show that the mammalian GPI1 homologues can rescue haploids harbouring the lethal gpi1+::his7+ allele. Our data demonstrate that the genes identified are indeed involved in the first step of GPI biosynthesis, and allow conclusions about a specific function for Gpi1p in stabilizing the enzymic complex. The finding that, despite a low degree of identity, the mammalian Gpi1 proteins are able to participate in the yeast GlcNAc-PI synthetic machinery as heterologous components further demonstrates that GPI biosynthesis has been highly conserved throughout evolution.

scholarly journals Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

2021 ◽  
Author(s):  
Li-Nan Wang ◽  
Xiang-Lei Peng ◽  
Min Xu ◽  
Yuan-Bo Zheng ◽  
Yue-Ying Jiao ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Gene Deletion ◽  
Human Respiratory Syncytial Virus ◽  
Temperature Sensitive ◽  
T7 Promoter ◽  
Vaccine Candidates ◽  
Rsv Infection ◽  
Syncytial Virus

AbstractHuman respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed  temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals Transcription signals and protein binding sites for sericin gene transcription in vitro

1989 ◽  
Vol 264 (31) ◽  
pp. 18707-18713 ◽  
Author(s):  
K Matsuno ◽  
C C Hui ◽  
S Takiya ◽  
T Suzuki ◽  
K Ueno ◽  
...  
Keyword(s):  
Protein Binding ◽  
Gene Transcription ◽  
Binding Sites ◽  
Protein Binding Sites ◽  
Transcription In Vitro ◽  
Transcription Signals
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