scholarly journals Cytomegalovirus Basic Phosphoprotein (pUL32) Binds to Capsids In Vitro through Its Amino One-Third

2001 ◽  
Vol 75 (15) ◽  
pp. 6865-6873 ◽  
Author(s):  
Michael K. Baxter ◽  
Wade Gibson
Keyword(s):  
Molecular Structure ◽  
Binding Sites ◽  
Sequence Conservation ◽  
Binding Region ◽  
Present Evidence ◽  
Tegument Proteins ◽  
Infected Cells ◽  
Human Cmv

ABSTRACT The cytomegalovirus (CMV) basic phosphoprotein (BPP) is a component of the tegument. It remains with the nucleocapsid fraction under conditions that remove most other tegument proteins from the virion, suggesting a direct and perhaps tight interaction with the capsid. As a step toward localizing this protein within the molecular structure of the virion and understanding its function during infection, we have investigated the BPP-capsid interaction. In this report we present evidence that the BPP interacts selectively, through its amino one-third, with CMV capsids. Radiolabeled simian CMV (SCMV) BPP, synthesized in vitro, bound to SCMV B-capsids, and C-capsids to a lesser extent, following incubation with either isolated capsids or lysates of infected cells. Human CMV (HCMV) BPP (pUL32) also bound to SCMV capsids, and SCMV BPP likewise bound to HCMV capsids, indicating that the sequence(s) involved is conserved between the two proteins. Analysis of SCMV BPP truncation mutants localized the capsid-binding region to the amino one-third of the molecule—the portion of BPP showing the greatest sequence conservation between the SCMV and HCMV homologs. This general approach may have utility in studying the interactions of other proteins with conformation-dependent binding sites.

scholarly journals Multiple capsid protein binding sites mediate selective packaging of the alphavirus genomic RNA

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Rebecca S. Brown ◽  
Dimitrios G. Anastasakis ◽  
Markus Hafner ◽  
Margaret Kielian
Keyword(s):  
Capsid Protein ◽  
Binding Sites ◽  
Semliki Forest Virus ◽  
Virus Assembly ◽  
Genomic Rna ◽  
Packaging Signal ◽  
Genome Packaging ◽  
Genome Recognition ◽  
Infected Cells

Abstract The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp’s top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly.

scholarly journals Mutational Scanning and Affinity Cleavage Analysis of UhpA-Binding Sites in the Escherichia coli uhpT Promoter

2002 ◽  
Vol 184 (10) ◽  
pp. 2682-2691 ◽  
Author(s):  
Igor N. Olekhnovich ◽  
Robert J. Kadner
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Promoter Activity ◽  
Consensus Sequence ◽  
Response Regulators ◽  
Binding Region ◽  
Nucleotide Substitutions ◽  
Iron Chelate ◽  
Affinity Cleavage

ABSTRACT UhpA, a member of the NarL family of response regulators, activates transcription of the Escherichia coli uhpT gene for the sugar phosphate transporter UhpT in response to extracellular glucose-6-phosphate. UhpA binds with different affinities to adjacent regions in the uhpT promoter, termed the strong-binding (S) region from −80 to −50 and the weak-binding (W) region from −50 to −32. Transcription activation by UhpA is stimulated by the catabolite gene activator protein (CAP)-cyclic AMP complex and depends on the C-terminal domains of the RNA polymerase RpoA and RpoD subunits. Because single-base substitutions in the UhpA-binding region had little effect on promoter activity, nucleotide substitutions in successive 4-bp blocks throughout this region were examined for their effects on promoter activation and UhpA binding. Changes in three of four blocks within the W region substantially impaired the ability of UhpA to bind to this region, to drive expression of a uhpT-lacZ reporter, and to support UhpA-dependent in vitro transcription. These W region variant promoters were strongly stimulated by CAP. Changes in several parts of the S region impaired UhpA binding to both the S and W regions and decreased promoter activity in vivo and in vitro. Thus, binding of UhpA to the W region is crucial for UhpA-dependent activation and depends on occupancy of the S region. None of these substitutions eliminated promoter function. The orientation of UhpA-binding sites was assessed by the affinity cleavage method. The iron chelate FeBABE [iron (S)-1-(p-bromoacetamidobenzyl) EDTA] was covalently attached to engineered cysteine residues near the DNA-binding region in UhpA. Hydroxyl radicals generated by the iron chelate attached at position 187 resulted in DNA strand cleavages in two clusters of sites located in the middle of the S and W regions. These results are consistent with the binding of two dimers of UhpA. Each dimer binds to an inverted repeat of monomer-binding sites with the consensus sequence CCTGRR, where R is A or G, and each is separated by 6 bp. It is likely that members of the NarL family bind to dyad targets, in contrast to the binding of OmpR family response regulators to direct-repeat targets.

scholarly journals gadd153/Chop10, a potential target gene of the transcriptional repressor ATF3.

1997 ◽  
Vol 17 (11) ◽  
pp. 6700-6707 ◽  
Author(s):  
C D Wolfgang ◽  
B P Chen ◽  
J L Martindale ◽  
N J Holbrook ◽  
T Hai
Keyword(s):  
Binding Sites ◽  
Target Gene ◽  
Mrna Level ◽  
Transcriptional Repressor ◽  
Transient Transfection ◽  
Negative Regulation ◽  
Present Evidence ◽  
Ccl4 Treatment

Recently, we demonstrated that the function of ATF3, a stress-inducible transcriptional repressor, is negatively regulated by a bZip protein, gadd153/Chop10. In this report, we present evidence that ATF3 can repress the expression of its own inhibitor, gadd153/Chop10. First, ATF3 represses a chloramphenicol acetyltransferase reporter gene driven by the gadd153/Chop10 promoter when assayed by a transfection assay in vivo and a transcription assay in vitro. Second, the gadd153/Chop10 promoter contains two functionally important binding sites for ATF3: an AP-1 site and a C/EBP-ATF composite site, a previously unidentified binding site for ATF3. The absence of either site reduces the ability of ATF3 to repress the promoter. Third, overexpression of ATF3 by transient transfection results in a reduction of the endogenous gadd153/Chop10 mRNA level. Fourth, as described previously, ATF3 is induced in the liver upon CCl4 treatment. Intriguingly, we show in this report that gadd153/Chop10 mRNA is not present in areas where ATF3 is induced. Taken together, these results strongly suggest that ATF3 represses the expression of gadd153/Chop10. The mutual negative regulation between ATF3 and gadd153/Chop10 is discussed.

scholarly journals Corepression of the P1 Addiction Operon by Phd and Doc

1998 ◽  
Vol 180 (23) ◽  
pp. 6342-6351 ◽  
Author(s):  
Roy Magnuson ◽  
Michael B. Yarmolinsky
Keyword(s):  
Fusion Protein ◽  
Binding Sites ◽  
Wild Type ◽  
Dna Complexes ◽  
Present Evidence ◽  
Cooperative Interactions ◽  
Regulatory Properties

ABSTRACT The P1 plasmid addiction operon encodes Doc, a toxin that kills plasmid-free segregants, and Phd, an unstable antidote that neutralizes the toxin. Additionally, these products repress transcription of the operon. The antidote binds to two adjacent sites in the promoter. Here we present evidence concerning the regulatory role of the toxin, which we studied with the aid of a mutation,docH66Y. The DocH66Y protein retained the regulatory properties of the wild-type protein, but not its toxicity. In vivo, DocH66Y enhanced repression by Phd but failed to affect repression in the absence of Phd, suggesting that DocH66Y contacts Phd. In vitro, a MalE-DocH66Y fusion protein was found to bind Phd. Binding of toxin to antidote may be the physical basis for the neutralization of toxin. DocH66Y failed to bind DNA in vitro yet enhanced the affinity, cooperativity, and specificity with which Phd bound the operator. Although DocH66Y enhanced the binding of Phd to two adjacent Phd-binding sites, DocH66Y had relatively little effect on the binding of Phd to a single Phd-binding site, indicating that DocH66Y mediates cooperative interactions between adjacent Phd-binding sites. Several electrophoretically distinct protein-DNA complexes were observed with different amounts of DocH66Y relative to Phd. Maximal repression and specificity of DNA binding were observed with subsaturating amounts of DocH66Y relative to Phd. Analogous antidote-toxin pairs appear to have similar autoregulatory circuits. Autoregulation, by dampening fluctuations in the levels of toxin and antidote, may prevent the inappropriate activation of the toxin.

scholarly journals Three Regions within Acta Promote Arp2/3 Complex-Mediated Actin Nucleation and Listeria monocytogenes Motility

2000 ◽  
Vol 150 (3) ◽  
pp. 527-538 ◽  
Author(s):  
Justin Skoble ◽  
Daniel A. Portnoy ◽  
Matthew D. Welch
Keyword(s):  
Listeria Monocytogenes ◽  
Actin Polymerization ◽  
Sequence Similarity ◽  
Binding Region ◽  
Actin Nucleation ◽  
Homology Sequence ◽  
Infected Cells ◽  
Wasp Family Proteins ◽  
In Cells

The Listeria monocytogenes ActA protein induces actin-based motility by enhancing the actin nucleating activity of the host Arp2/3 complex. Using systematic truncation analysis, we identified a 136-residue NH2-terminal fragment that was fully active in stimulating nucleation in vitro. Further deletion analysis demonstrated that this fragment contains three regions, which are important for nucleation and share functional and/or limited sequence similarity with host WASP family proteins: an acidic stretch, an actin monomer–binding region, and a cofilin homology sequence. To determine the contribution of each region to actin-based motility, we compared the biochemical activities of ActA derivatives with the phenotypes of corresponding mutant bacteria in cells. The acidic stretch functions to increase the efficiency of actin nucleation, the rate and frequency of motility, and the effectiveness of cell–cell spread. The monomer-binding region is required for actin nucleation in vitro, but not for actin polymerization or motility in infected cells, suggesting that redundant mechanisms may exist to recruit monomer in host cytosol. The cofilin homology sequence is critical for stimulating actin nucleation with the Arp2/3 complex in vitro, and is essential for actin polymerization and motility in cells. These data demonstrate that each region contributes to actin-based motility, and that the cofilin homology sequence plays a principal role in activation of the Arp2/3 complex, and is an essential determinant of L. monocytogenes pathogenesis.

scholarly journals Multiple Capsid Protein Binding Sites Mediate Selective Packaging of the Alphavirus Genomic RNA

2020 ◽  
Author(s):  
Rebecca S. Brown ◽  
Dimitrios G. Anastasakis ◽  
Markus Hafner ◽  
Margaret Kielian
Keyword(s):  
Capsid Protein ◽  
Binding Sites ◽  
Semliki Forest Virus ◽  
Virus Assembly ◽  
Genomic Rna ◽  
Packaging Signal ◽  
Genome Packaging ◽  
Genome Recognition ◽  
Infected Cells

ABSTRACTThe alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, we comprehensively defined binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrated Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments showed that PS was not required for production of infectious SFV or Chikungunya virus. Instead, we identified multiple novel Cp binding sites that were enriched on gRNA-specific regions and promoted infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrated that budding caused discrete changes in Cp-gRNA interactions. Notably, Cp’s top binding site was maintained throughout virus assembly, and specifically bound and assembled with Cp into core-like particles in vitro. Together our data suggest a new model for selective alphavirus genome recognition and assembly.

scholarly journals Enhancing safety of cytomegalovirus-based vaccine vectors by engaging host intrinsic immunity

2019 ◽  
Vol 11 (501) ◽  
pp. eaaw2603 ◽  
Author(s):  
Emily E. Marshall ◽  
Daniel Malouli ◽  
Scott G. Hansen ◽  
Roxanne M. Gilbride ◽  
Colette M. Hughes ◽  
...  
Keyword(s):  
T Cell ◽  
Close Contact ◽  
Effector Memory ◽  
Growth Defect ◽  
Intrinsic Immunity ◽  
Pathogenic Potential ◽  
Tegument Proteins ◽  
Human Cmv

Rhesus cytomegalovirus (RhCMV)–based vaccines maintain effector memory T cell responses (TEM) that protect ~50% of rhesus monkeys (RMs) challenged with simian immunodeficiency virus (SIV). Because human CMV (HCMV) causes disease in immunodeficient subjects, clinical translation will depend upon attenuation strategies that reduce pathogenic potential without sacrificing CMV’s unique immunological properties. We demonstrate that “intrinsic” immunity can be used to attenuate strain 68-1 RhCMV vectors without impairment of immunogenicity. The tegument proteins pp71 and UL35 encoded by UL82 and UL35 of HCMV counteract cell-intrinsic restriction via degradation of host transcriptional repressors. When the corresponding RhCMV genes, Rh110 and Rh59, were deleted from 68-1 RhCMV (ΔRh110 and ΔRh59), we observed only a modest growth defect in vitro, but in vivo, these modified vectors manifested little to no amplification at the injection site and dissemination to distant sites, in contrast to parental 68-1 RhCMV. ΔRh110 was not shed at any time after infection and was not transmitted to naïve hosts either by close contact (mother to infant) or by leukocyte transfusion. In contrast, ΔRh59 was both shed and transmitted by leukocyte transfusion, indicating less effective attenuation than pp71 deletion. The T cell immunogenicity of ΔRh110 was essentially identical to 68-1 RhCMV with respect to magnitude, TEM phenotype, epitope targeting, and durability. Thus, pp71 deletion preserves CMV vector immunogenicity while stringently limiting vector spread, making pp71 deletion an attractive attenuation strategy for HCMV vectors.

A Panel of Artificial Antigen Presenting Cells (AAPC) Permits Immediate Sensitization and Rapid Generation of Virus-Specific T-Cells of Desired HLA Restriction for Adoptive Immunotherapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3268-3268
Author(s):  
Aisha N. Hasan ◽  
Wouter J. Kollen ◽  
Deepa Trivedi ◽  
A. Selvakumar ◽  
Michel Sadelain ◽  
...  
Keyword(s):  
T Cells ◽  
Adoptive Immunotherapy ◽  
Effector Memory ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Hla Restriction ◽  
Infected Cells ◽  
Rapid Generation ◽  
Human Cmv

Abstract Adoptive transfer of donor-derived virus- specific T-cells can treat or prevent severe infections in allogeneic hematopoietic stem cell transplant recipients, but maybe ineffective in HLA-disparate recipients if the T-cells are restricted by unshared alleles. We previously described an AAPC consisting of mouse 3T3 fibroblasts transduced to express human B7.1, LFA-3, ICAM-1, β2-microglobulin and the alpha chain of HLA A*0201 (Papanicolaou et al. Blood2003;102:2498). To test the potential of AAPC to present CMV pp65 epitopes and elicit virus-specific T-cells restricted by other, less common alleles, we generated a panel of AAPC, each expressing one of a series of HLA class-I alleles, including HLA A*0201, A*0301, A*2402, B*0702, B*0801 and C*0401. We have subsequently tested the AAPC expressing HLA A*0201, A*2402 and B*0702 for their capacity to sensitize human T-cells when loaded with overlapping 15-mer peptides spanning the CMV pp65 sequence or transduced to express the CMV pp65 protein. T-cells from groups of up to 4 CMV seropositive donors, each expressing one of these HLA alleles, when sensitized with either peptide loaded or transduced AAPC expanded 15–38 fold in vitro over 21 days. The T-cells generated were predominantly CD8+ T-cells containing central memory and effector memory subsets. As shown in table.1, T-cells elicited by each AAPC exhibited a high level of HLA-restricted, CMV-specific cytotoxic activity despite the variability in the percentage of HLA-tetramer positive and IFNγ producing T-cells. Responses against unmodified autologous or allogeneic targets was <5%. Similarly, over 2% of the T-cells in the cultures generated IFNγ in response to secondary stimulation with autologous PBMC loaded with the peptide pool, versus 0.1–0.3% in controls. In comparative assays, the yields of specific CTL generated in response to AAPC transduced to express CMV pp65 were higher than those generated in response to pool-loaded AAPC in each of the 9 donors tested. Strikingly, staining by HLA-tetramers demonstrated that sensitization by mouse derived, CMV pp65 transduced AAPC generates high proportions of T cells (7–51%) recognizing epitopes known to be immunogenic when presented by that human HLA allele on CMV infected cells i.e. NLVPMAVTV by HLA A*0201, QYDPVAALF by A*2402 and RPHERNGFTV by B*0702. These data indicate that the human HLA expressing 3T3 cells process CMV pp65 protein and transport and present epitopes which are similar or identical to those presented on human, CMV-infected cells. Thus, a replenishable panel of 3T3 based AAPC, each expressing a single HLA allele, can be used for immediate sensitization and efficient expansion of human CMV-specific T-cells. Use of this panel permits rapid generation of virus-specific T-cells of desired HLA restriction for adoptive immunotherapy and maybe of particular value for treatment of patients receiving grafts from HLA-disparate donors. Table 1. Analysis of CTL Generated with AAPC (Transduced with CMV pp65 or Peptide Pulsed) Donors Fold T-cell Expansion % CD8 CD8 % Tetramer [+] CD8 % IFNγ 51-Cr Cytotoxicity All values represent average +/− SEM A*0201 Donors N=4 28 +/− 3 84% +/− 3 51% +/− 0.1 2.6% +/− 0.03 54% +/− 0.1 A*2402 Donors N=4 15 +/− 2 62% +/− 5 15% +/− 0.2 2.43% +/− 0.003 34% +/− 0.03 B*0702 Donors N=1 38 85% 7% 2% 53%

scholarly journals Tau proteins: the molecular structure and mode of binding on microtubules.

1988 ◽  
Vol 107 (4) ◽  
pp. 1449-1459 ◽  
Author(s):  
N Hirokawa ◽  
Y Shiomura ◽  
S Okabe
Keyword(s):  
Molecular Structure ◽  
Binding Sites ◽  
Competitive Binding ◽  
Tau Proteins ◽  
Molar Ratio ◽  
Cross Link ◽  
Binding Studies ◽  
Related Proteins ◽  
Better Than

Tau is a family of closely related proteins (55,000-62,000 mol wt) which are contained in the nerve cells and copolymerize with tubulin to induce the formation of microtubules in vitro. All information so far has indicated that tau is closely apposed to the microtubule lattice, and there was no indication of domains projecting from the microtubule polymer lattice. We have studied the molecular structure of the tau factor and its mode of binding on microtubules using the quick-freeze, deep-etch method (QF.DE) and low angle rotary shadowing technique. Phosphocellulose column-purified tubulin from porcine brain was polymerized with tau and the centrifuged pellets were processed by QF.DE. We observed periodic armlike elements (18.7 +/- 4.8 nm long) projecting from the microtubule surface. Most of the projections appeared to cross-link adjacent microtubules. We measured the longitudinal periodicity of tau projections on the microtubules and found it to match the 6-dimer pattern better than the 12-dimer pattern. The stoichiometry of tau versus tubulin in preparations of tau saturated microtubules was 1:approximately 5.0 (molar ratio). Tau molecules adsorbed on mica took on rodlike forms (56.1 +/- 14.1 nm long). Although both tau and MAP1 are contained in axons, competitive binding studies demonstrated that the binding sites of tau and MAP1A on the microtubule surfaces are most distinct, although they may partially overlap.

scholarly journals Identification of Sperm-Binding Sites in the N-Terminal Domain of Bovine Egg Coat Glycoprotein ZP4

2022 ◽  
Vol 23 (2) ◽  
pp. 762
Author(s):  
Kamila Dilimulati ◽  
Misaki Orita ◽  
Yoshiki Yonahara ◽  
Fabiana Lica Imai ◽  
Naoto Yonezawa
Keyword(s):  
Binding Sites ◽  
Binding Activity ◽  
Binding Region ◽  
Terminal Domain ◽  
Sperm Binding ◽  
Bovine Sperm ◽  
Selective Interaction ◽  
Egg Coat

The species-selective interaction between sperm and egg at the beginning of mammalian fertilisation is partly mediated by a transparent envelope called the zona pellucida (ZP). The ZP is composed of three or four glycoproteins (ZP1–ZP4). The functions of the three proteins present in mice (ZP1–ZP3) have been extensively studied. However, the biological role of ZP4, which was found in all other mammals studied so far, has remained largely unknown. Previously, by developing a solid support assay system, we showed that ZP4 exhibits sperm-binding activity in bovines and the N-terminal domain of bovine ZP4 (bZP4 ZP-N1 domain) is a sperm-binding region. Here, we show that bovine sperm bind to the bZP4 ZP-N1 domain in a species-selective manner and that N-glycosylation is not required for sperm-binding activity. Moreover, we identified three sites involved in sperm binding (site I: from Gln-41 to Pro-46, site II: from Leu-65 to Ser-68 and site III: from Thr-108 to Ile-123) in the bZP4 ZP-N1 domain using chimeric bovine/porcine and bovine/human ZP4 recombinant proteins. These results provide in vitro experimental evidence for the role of the bZP4 ZP-N1 domain in mediating sperm binding to the ZP.

Sign in / Sign up

Export Citation Format

Share Document