scholarly journals Revisiting the role of PML protein targeting and disruption of PML bodies in human cytomegalovirus infection

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Christina Paulus ◽  
Thomas Harwardt ◽  
Bernadette Walter ◽  
Andrea Marxreiter ◽  
Michael Nevels
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Critical Role ◽  
Wild Type ◽  
Early Protein ◽  
Type Protein ◽  
Wild Type Protein ◽  
Pml Bodies

Promyelocytic leukaemia (PML) bodies are nuclear organelles implicated in post-translational modification by small ubiquitin-like modifier (SUMO) proteins and in the antiviral host cell response to infection. The 72-kDa immediate-early protein 1 (IE1) is considered the principal antagonist of PML bodies encoded by the human cytomegalovirus, one of eight human herpesviruses. Previous work has suggested that the interaction between IE1 and PML proteins, the central organisers of PML bodies, and the subsequent disruption of these organelles serve a critical role in viral replication by counteracting intrinsic antiviral immunity and the induction of interferon (IFN)-stimulated genes. However, this picture has emerged largely from studying mutant IE1 proteins known or predicted to be globally misfolded und metabolically unstable. We systematically screened for stable IE1 mutants by clustered charge-to-alanine scanning. We identified a mutant protein (IE1cc172-176) selectively defective for PML interaction. Functional comparisons between the mutant and wild-type protein revealed that IE1 can undergo modification by mixed polymeric SUMO chains and that it targets PML and Sp100, the two main constituents of PML bodies, via distinct mechanisms. Unexpectedly, IE1cc172-176 supported viral replication almost as efficiently as wild-type IE1. Moreover, lower instead of higher (as expected) levels of tumor necrosis factor alpha, IFN-beta, IFN-lambda and IFN-stimulated gene expression were observed with the mutant compared to the wild-type protein and virus. These results suggest that the disruption of PML bodies is linked to induction rather than inhibition of antiviral gene expression. Our findings challenge current views regarding the role of PML bodies in viral infection.

scholarly journals A cis Repression Sequence Adjacent to the Transcription Start Site of the Human Cytomegalovirus US3 Gene Is Required To Down Regulate Gene Expression at Early and Late Times after Infection

1998 ◽  
Vol 72 (12) ◽  
pp. 9575-9584 ◽  
Author(s):  
Philip E. Lashmit ◽  
Mark F. Stinski ◽  
Eain A. Murphy ◽  
Grant C. Bullock
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Transcription Start Site ◽  
Human Cytomegalovirus ◽  
Maximum Level ◽  
Start Site ◽  
Wild Type ◽  
Transcription Start ◽  
Recombinant Viruses

ABSTRACT Human cytomegalovirus has two enhancer-containing immediate-early (IE) promoters with a cis repression sequence (CRS) positioned immediately upstream of the transcription start site, designated the major IE (MIE) promoter and the US3 promoter. The role of the CRS upstream of the US3 transcription start site in the context of the viral genome was determined by comparing the levels of transcription from these two enhancer-containing promoters in recombinant viruses with a wild-type or mutant CRS. Upstream of the CRS of the US3 promoter was either the endogenous enhancer (R2) or silencer (R1). The downstream US3 gene was replaced with the indicator gene chloramphenicol acetyltransferase (CAT). Infected permissive human fibroblast cells or nonpermissive, undifferentiated monocytic THP-1 cells were analyzed for expression from the US3 promoter containing either the wild-type or mutant CRS. With the wild-type CRS, the maximum level of transcription in permissive cells was detected within 4 to 6 h after infection and then declined. With the mutant CRS and the R2 enhancer upstream, expression from the US3 promoter continued to increase throughout the viral replication cycle to levels 20- to 40-fold higher than for the wild type. In nonpermissive or permissive monocytic THP-1 cells, expression from the US3 promoter was also significantly higher when the CRS was mutated. Less expression was obtained when only the R1 element was present, but expression was higher when the CRS was mutated. Thus, the CRS in the enhancer-containing US3 promoter appears to allow for a short burst of US3 gene expression followed by repression at early and late times after infection. Overexpression of US3 may be detrimental to viral replication, and its level of expression must be stringently controlled. The role of the CRS and the viral IE86 protein in controlling enhancer-containing promoters is discussed.

scholarly journals Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion

1998 ◽  
Vol 329 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Esther YÁÑEZ ◽  
A. Teresa CARMONA ◽  
Mercedes TIEMBLO ◽  
Antonio JIMÉNEZ ◽  
María FERNÁNDEZ-LOBATO
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catalytic Efficiency ◽  
Extracellular Enzyme ◽  
Site Directed Mutagenesis ◽  
Activity Levels ◽  
Wild Type ◽  
Schwanniomyces Occidentalis ◽  
Type Protein ◽  
Wild Type Protein

The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 α-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that α-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. α-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with endoglycosidase F. These findings indicate that Asn-229 is the single glycosylated residue in SWA2. Thermostability analysis of both purified wild-type (T50 = 50 °C, where T50 is the temperature resulting in 50% loss of activity) and mutant enzymes indicated that removal of carbohydrate from the 229 position results in a decrease of approx. 3 °C in the T50 of the enzyme. The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (Km) but decreases to 1/22 its apparent catalytic efficiency (kcat/Km). These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the Sw. occidentalis SWA2 α-amylase in both its wild-type and mutant forms.

scholarly journals Importance of the two tryptophan residues in the Streptomyces R61 exocellular dd-peptidase

1992 ◽  
Vol 282 (2) ◽  
pp. 361-367 ◽  
Author(s):  
C Bourguignon-Bellefroid ◽  
J M Wilkin ◽  
B Joris ◽  
R T Aplin ◽  
C Houssier ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Beta Lactams ◽  
Beta Lactamases ◽  
Type Protein ◽  
Wild Type Protein ◽  
Tryptophan Residues

Modification of the Streptomyces R61 DD-peptidase by N-bromosuccinimide resulted in a rapid loss of enzyme activity. In consequence, the role of the enzyme's two tryptophan residues was investigated by site-directed mutagenesis. Trp271 was replaced by Leu. The modification yielded a stable enzyme whose structural and catalytic properties were similar to those of the wild-type protein. Thus the Trp271 residue, though almost invariant among the beta-lactamases of classes A and C and the low-Mr penicillin-binding proteins, did not appear to be essential for enzyme activity. Mutations of the Trp233 into Leu and Ser strongly decreased the enzymic activity, the affinity for beta-lactams and the protein stability. Surprisingly, the benzylpenicilloyl-(W233L)enzyme deacylated at least 300-fold more quickly than the corresponding acyl-enzyme formed with the wild-type protein and gave rise to benzylpenicilloate instead of phenylacetylglycine. This mutant DD-peptidase thus behaved as a weak beta-lactamase.

scholarly journals SUMO-1 Modification of Human Cytomegalovirus IE1/IE72

2002 ◽  
Vol 76 (6) ◽  
pp. 2990-2996 ◽  
Author(s):  
Mary L. Spengler ◽  
Karen Kurapatwinski ◽  
Adrian R. Black ◽  
Jane Azizkhan-Clifford
Keyword(s):  
Gene Expression ◽  
Protein Stability ◽  
Western Blotting ◽  
Human Cytomegalovirus ◽  
Posttranslational Modifications ◽  
Nuclear Architecture ◽  
Wild Type ◽  
Cellular Processes ◽  
Early Protein ◽  
Cycle Regulation

ABSTRACT Human cytomegalovirus (HCMV) immediate-early protein IE1/IE72 is involved in undermining many cellular processes including cell cycle regulation, apoptosis, nuclear architecture, and gene expression. The multifunctional nature of IE72 suggests that posttranslational modifications may modulate its activities. IE72 is a phosphoprotein and has intrinsic kinase activity (S. Pajovic, E. L. Wong, A. R. Black, and J. C. Azizkhan, Mol. Cell. Biol. 17:6459-6464, 1997). We now demonstrate that IE72 is covalently conjugated to the small ubiquitin-like modifier (SUMO-1). SUMO-1 is an 11.5-kDa protein that is conjugated to multiple proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. A covalently modified protein migrating at ∼92 kDa, which is stabilized by a SUMO-1 hydrolase inhibitor, is revealed by Western blotting with anti-IE72 of lysates from cells infected with HCMV or cells expressing IE72. SUMO modification of IE72 was confirmed by immunoprecipitation with anti-IE72 and anti-SUMO-1 followed by Western blotting with anti-SUMO-1 and anti-IE72, respectively. Lysine 450 is within a sumoylation consensus site (I,V,L)KXE; changing lysine 450 to arginine by point mutation abolishes SUMO-1 modification of IE72. Inhibition of protein phosphatase 1 and 2A, which increases the phosphorylation of IE72, suppresses the formation of SUMO-1-IE72 conjugates. Both wild-type IE72 and IE72K450R localize to nuclear PML oncogenic domains and disrupt them. Studies of protein stability, transactivation, and complementation of IE72-deficient HCMV (CR208) have revealed no significant differences between wild-type IE72 and IE72K450R.

scholarly journals Reduced SCD1 activity alters markers of fatty acid reesterification, glyceroneogenesis, and lipolysis in murine white adipose tissue and 3T3-L1 adipocytes

2017 ◽  
Vol 313 (3) ◽  
pp. C295-C304 ◽  
Author(s):  
Steven M. Dragos ◽  
Karl F. Bergeron ◽  
Frédérik Desmarais ◽  
Katherine Suitor ◽  
David C. Wright ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Fatty Acid ◽  
White Adipose Tissue ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Critical Role ◽  
Wild Type ◽  
Important Regulator ◽  
Specific Regulation

White adipose tissue (WAT) has a critical role in lipid handling. Previous work demonstrated that SCD1 is an important regulator of WAT fatty acid (FA) composition; however, its influence on the various interconnected pathways influencing WAT lipid handling remains unclear. Our objective was to investigate the role of SCD1 on WAT lipid handling using Scd1 knockout (KO) mice and SCD1-inhibited 3T3-L1 adipocytes by measuring gene, protein, and metabolite markers related to FA reesterification, glyceroneogenesis, and lipolysis. Triacylglycerol (TAG) content was higher in inguinal WAT (iWAT) from KO mice compared with wild-type, but significantly lower in epididymal WAT (eWAT). The SCD1 desaturation index was decreased in both WAT depots in KO mice. FA reesterification, as measured with a NEFA:glycerol ratio, was reduced in both WAT depots in KO mice, as well as SCD1-inhibited 3T3-L1 adipocytes. Pck1, Atgl, and Hsl gene expression was reduced in both WAT depots of KO mice, while Pck2 and Pdk4 gene expression showed depot-specific regulation. Pck1, Atgl, and Hsl gene expression was reduced, and phosphoenolpyruvate carboxykinase protein content was ablated, in SCD1-inhibited adipocytes. Our data provide evidence that SCD1 has a broad impact on WAT lipid handling by altering TAG composition in a depot-specific manner, reducing FA reesterification, and regulating markers of lipolysis and glyceroneogenesis.

Role of tryptophan 135 of Chandipura virus phosphoprotein P in dimerization and complex formation with leader RNA: structural aspect using time resolved anisotropy and simulation

RSC Advances ◽  
2015 ◽  
Vol 5 (126) ◽  
pp. 104582-104593
Author(s):  
Manini Mukherjee ◽  
Aditya Sarkar ◽  
Arunava Roy ◽  
Pinki Saha Sardar ◽  
Ansuman Lahiri ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Structural Change ◽  
Complex Formation ◽  
Protein Concentration ◽  
Structural Aspect ◽  
Wild Type ◽  
Time Resolved ◽  
Type Protein ◽  
Wild Type Protein

The nanosecond and picosecond dynamics of wild type protein and its tryptophan mutants have been used to study structural change as a function of protein concentration and binding with leader RNA by time resolved anisotropy and molecular dynamics.

scholarly journals The Griffithsin Dimer Is Required for High-Potency Inhibition of HIV-1: Evidence for Manipulation of the Structure of gp120 as Part of the Griffithsin Dimer Mechanism

2013 ◽  
Vol 57 (8) ◽  
pp. 3976-3989 ◽  
Author(s):  
Jie Xue ◽  
Bart Hoorelbeke ◽  
Ioannis Kagiampakis ◽  
Borries Demeler ◽  
Jan Balzarini ◽  
...  
Keyword(s):  
Protein A ◽  
Carbohydrate Binding ◽  
Wild Type ◽  
Subtype C ◽  
Subtype B ◽  
Type Protein ◽  
Wild Type Protein ◽  
Protein Dimer ◽  
Anti Hiv

ABSTRACTGriffithsin (Grft) is a protein lectin derived from red algae that tightly binds the HIV envelope protein gp120 and effectively inhibits virus infection. This inhibition is due to the binding by Grft of high-mannose saccharides on the surface of gp120. Grft has been shown to be a tight dimer, but the role of the dimer in Grft's anti-HIV function has not been fully explored. To investigate the role of the Grft dimer in anti-HIV function, an obligate dimer of Grft was designed by expressing the protein with a peptide linker between the two subunits. This “Grft-linker-Grft” is a folded protein dimer, apparently nearly identical in structural properties to the wild-type protein. A “one-armed” obligate dimer was also designed (Grft-linker-Grft OneArm), with each of the three carbohydrate binding sites of one subunit mutated while the other subunit remained intact. While both constructed dimers retained the ability to bind gp120 and the viral surface, Grft-linker-Grft OneArm was 84- to 1,010-fold less able to inhibit HIV than wild-type Grft, while Grft-linker-Grft had near-wild-type antiviral potency. Furthermore, while the wild-type protein demonstrated the ability to alter the structure of gp120 by exposing the CD4 binding site, Grft-linker-Grft OneArm largely lost this ability. In experiments to investigate gp120 shedding, it was found that Grft has different effects on gp120 shedding for strains from subtype B and subtype C, and this might correlate with Grft function. Evidence is provided that the dimer form of Grft is critical to the function of this protein in HIV inhibition.

scholarly journals Expression and characterization of Pseudomonas aeruginosa cytochrome c-551 and two site-directed mutants: role of tryptophan 56 in the modulation of redox properties

1997 ◽  
Vol 322 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Francesca CUTRUZZOLÀ ◽  
Ilaria CIABATTI ◽  
Gabriella ROLLI ◽  
Sabrina FALCINELLI ◽  
Marzia ARESE ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Cytochrome C ◽  
Ph Dependence ◽  
Redox Properties ◽  
Expression Vectors ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Structural And Functional Properties

The gene coding for Pseudomonas aeruginosacytochrome c-551 was expressed in Pseudomonas putidaunder aerobic conditions, using two different expression vectors; the more efficient proved to be pNM185, induced by m-toluate. Mature holo-(cytochrome c-551) was produced in high yield by this expression system, and was purified to homogeneity. Comparison of the recombinant wild-type protein with that purified from Ps. aeruginosashowed no differences in structural and functional properties. Trp56, an internal residue in cytochrome c-551, is located at hydrogen-bonding distance from haem propionate-17, together with Arg47. Ionization of propionate-17 was related to the observed pH-dependence of redox potential. The role of Trp56 in determining the redox properties of Ps. aeruginosacytochrome c-551 was assessed by site-directed mutagenesis, by substitution with Tyr (W56Y) and Phe (W56F). The W56Y mutant is similar to the wild-type cytochrome. On the other hand, the W56F mutant, although similar to the wild-type protein in spectral properties and electron donation to azurin, is characterized by a weakening of the FeŐMet61 bond, as shown in the oxidized protein by the loss of the 695 nm band approx. 2 pH units below the wild-type. Moreover, in W56F, the midpoint potential and its pH-dependence are both different from the wild-type. These results are consistent with the hypothesis that hydrogen-bonding to haem propionate-17 is important in modulation of the redox properties of Ps. aeruginosacytochrome c-551.

scholarly journals Transcriptional Regulation of the Human Cytomegalovirus US11 Early Gene

1999 ◽  
Vol 73 (2) ◽  
pp. 863-870 ◽  
Author(s):  
Nha H. Chau ◽  
Cynthia D. Vanson ◽  
Julie A. Kerry
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Human Cytomegalovirus ◽  
Promoter Activity ◽  
Early Gene ◽  
Mrna Levels ◽  
Wild Type ◽  
Infected Cells ◽  
Wild Type Promoter

ABSTRACT The human cytomegalovirus (HCMV) US11 early gene encodes a protein involved in the down-regulation of major histocompatibility complex class I cell surface expression in HCMV-infected cells. Consequently, this gene is thought to play an important role in HCMV evasion of immune recognition. In this study, we examined the transcriptional regulation of US11 gene expression. Analysis of deletions within the US11 promoter suggests that two sequence elements are important for activation by the viral immediate-early (IE) proteins. Deletion of a CREB site located at −83 relative to the cap site resulted in a reduction in promoter activity to 50% of the wild-type level. Deletion of an additional ATF site immediately upstream of the TATA box resulted in abrogation of responsiveness to the IE proteins. To confirm the role of the CREB and ATF sites within the US11 promoter, mutagenesis of these two sites, both individually and in combination, was carried out. Results indicate that both the CREB element and the ATF site were required for full promoter activity, with the ATF site critical for US11 promoter activation. The loss of transcriptional activation correlated with a loss of cellular proteins binding to the mutated US11 promoter elements. In combination with the viral IE proteins, the HCMV tegument protein pp71 (UL82) was found to up-regulate the US11 promoter by six- to sevenfold in transient assays. These results suggest that pp71 may contribute to the activation of the US11 promoter at early times after infection. Up-regulation by pp71 required the presence of the CREB and ATF sites within the US11 promoter for full activation. The role of the ATF and CREB elements in regulating US11 gene expression during viral infection was then assessed. The US11 gene is not required for replication of HCMV in tissue culture. This property was exploited to generate US11 promoter mutants regulating expression of the endogenous US11 gene in the natural genomic context. We generated recombinant HCMV that contained the US11 promoter with mutations in either the CREB or ATF element or both regulating the expression of the endogenous US11 gene. Northern blot analysis of infected cell mRNA revealed that mutation of the CREB element reduced US11 mRNA expression to approximately 25% of that of the wild-type promoter, with identical kinetics of expression. Mutation of the ATF site alone reduced US11 mRNA levels to 6% of that of the wild-type promoter, with mRNA detectable only at 8 h after infection. Mutation of both the CREB and ATF elements in the US11 promoter reduced US11 gene expression to undetectable levels. These results demonstrate that the CREB and ATF sites cooperate to regulate the US11 promoter in HCMV-infected cells.

scholarly journals Transcriptome analysis of the role of autophagy in plant response to heat stress

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247783
Author(s):  
Yan Zhang ◽  
Haoxuan Min ◽  
Chengchen Shi ◽  
Gengshou Xia ◽  
Zhibing Lai
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Heat Tolerance ◽  
Stress Responses ◽  
Critical Role ◽  
Plant Response ◽  
Wild Type ◽  
Altered Expression ◽  
Regulated Gene Expression

Autophagy plays a critical role in plant heat tolerance in part by targeting heat-induced nonnative proteins for degradation. Autophagy also regulates metabolism, signaling and other processes and it is less understood how the broad function of autophagy affects plant heat stress responses. To address this issue, we performed transcriptome profiling of Arabidopsis wild-type and autophagy-deficient atg5 mutant in response to heat stress. A large number of differentially expressed genes (DEGs) were identified between wild-type and atg5 mutant even under normal conditions. These DEGs are involved not only in metabolism, hormone signaling, stress responses but also in regulation of nucleotide processing and DNA repair. Intriguingly, we found that heat treatment resulted in more robust changes in gene expression in wild-type than in the atg5 mutant plants. The dampening effect of autophagy deficiency on heat-regulated gene expression was associated with already altered expression of many heat-regulated DEGs prior to heat stress in the atg5 mutant. Altered expression of a large number of genes involved in metabolism and signaling in the autophagy mutant prior to heat stress may affect plant response to heat stress. Furthermore, autophagy played a positive role in the expression of defense- and stress-related genes during the early stage of heat stress responses but had little effect on heat-induced expression of heat shock genes. Taken together, these results indicate that the broad role of autophagy in metabolism, cellular homeostasis and other processes can also potentially affect plant heat stress responses and heat tolerance.

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