scholarly journals Specific Binding of Tombusvirus Replication Protein p33 to an Internal Replication Element in the Viral RNA Is Essential for Replication

2005 ◽  
Vol 79 (8) ◽  
pp. 4859-4869 ◽  
Author(s):  
Judit Pogany ◽  
K. Andrew White ◽  
Peter D. Nagy
Keyword(s):  
Rna Binding ◽  
Specific Binding ◽  
Specific Interaction ◽  
Genome Replication ◽  
Coding Region ◽  
Interaction Domain ◽  
Virus Family ◽  
Replicase Proteins

ABSTRACT The mechanism of template selection for genome replication in plus-strand RNA viruses is poorly understood. Using the prototypical tombusvirus, Tomato bushy stunt virus (TBSV), we show that recombinant p33 replicase protein binds specifically to an internal replication element (IRE) located within the p92 RNA-dependent RNA polymerase coding region of the viral genome. Specific binding of p33 to the IRE in vitro depends on the presence of a C · C mismatch within a conserved RNA helix. Interestingly, the absence of the p33:p33/p92 interaction domain in p33 prevented specific but allowed nonspecific RNA binding, suggesting that a multimeric form of this protein is involved in the IRE-specific interaction. Further support for the selectivity of p33 binding in vitro was provided by the inability of the replicase proteins of the closely related Turnip crinkle virus and distantly related Hepatitis C virus to specifically recognize the TBSV IRE. Importantly, there was also a strong correlation between p33:IRE complex formation in vitro and viral replication in vivo, where mutations in the IRE that disrupted selective p33 binding in vitro also abolished TBSV RNA replication both in plant and in Saccharomyces cerevisiae cells. Based on these findings and the other known properties of p33 and the IRE, it is proposed that the p33:IRE interaction provides a mechanism to selectively recruit viral RNAs into cognate viral replicase complexes. Since all genera in Tombusviridae encode comparable replicase proteins, these results may be relevant to other members of this large virus family.

Correlation between in vitro and in vivo Data of Radiolabeled Peptide for Tumor Targeting

2019 ◽  
Vol 19 (12) ◽  
pp. 950-960
Author(s):  
Soghra Farzipour ◽  
Seyed Jalal Hosseinimehr
Keyword(s):  
Tumor Targeting ◽  
Single Photon ◽  
Specific Binding ◽  
Specific Interaction ◽  
Photon Emission ◽  
Emission Computed Tomography ◽  
Mixed Effect ◽  
Radiolabeled Peptides

Tumor-targeting peptides have been generally developed for the overexpression of tumor specific receptors in cancer cells. The use of specific radiolabeled peptide allows tumor visualization by single photon emission computed tomography (SPECT) and positron emission tomography (PET) tools. The high affinity and specific binding of radiolabeled peptide are focusing on tumoral receptors. The character of the peptide itself, in particular, its complex molecular structure and behaviors influence on its specific interaction with receptors which are overexpressed in tumor. This review summarizes various strategies which are applied for the expansion of radiolabeled peptides for tumor targeting based on in vitro and in vivo specific tumor data and then their data were compared to find any correlation between these experiments. With a careful look at previous studies, it can be found that in vitro unblock-block ratio was unable to correlate the tumor to muscle ratio and the success of radiolabeled peptide for in vivo tumor targeting. The introduction of modifiers’ approaches, nature of peptides, and type of chelators and co-ligands have mixed effect on the in vitro and in vivo specificity of radiolabeled peptides.

scholarly journals Genetic and Biochemical Studies of Polioviruscis-Acting Replication Element cre in Relation to VPg Uridylylation

2000 ◽  
Vol 74 (22) ◽  
pp. 10371-10380 ◽  
Author(s):  
Elizabeth Rieder ◽  
Aniko V. Paul ◽  
Dong Wook Kim ◽  
Jacques H. van Boom ◽  
Eckard Wimmer
Keyword(s):  
Genome Replication ◽  
Coding Region ◽  
Stem Loop ◽  
Genetic Changes ◽  
Poliovirus Type ◽  
Type 3 ◽  
Type Sequence

ABSTRACT In addition to highly conserved stem-loop structures located in the 5′- and 3′-nontranslated regions, genome replication of picornaviruses requires cis-acting RNA elements located in the coding region (termed cre) (K. L. McKnight and S. M. Lemon, J. Virol. 70:1941–1952, 1996; P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560–11565, 1999; I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590–4600, 2000). cre elements appear to be essential for minus-strand RNA synthesis by an as-yet-unknown mechanism. We have discovered that the cre element of poliovirus (mapping to the 2C coding region of poliovirus type 1; nucleotides 4444 to 4505 in 2C), which is homologous to thecre element of poliovirus type 3, is preferentially used as a template for the in vitro uridylylation of VPg catalyzed by 3Dpol in a reaction that is greatly stimulated by 3CDpro (A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74:10359–10370, 2000). Here we report a direct correlation between mutations that eliminate, or severely reduce, the in vitro VPg-uridylylation reaction and produce replication phenotypes in vivo. None of the genetic changes significantly influenced translation or polyprotein processing. A substitution mapping to the first A (A4472C) of a conservedAAACA sequence in the loop of PV-cre(2C) eliminated the ability of the cre RNA to serve as template for VPg uridylylation and abolished RNA infectivity. Mutagenesis of the second A (A4473C; AAACA) severely reduced the yield of VPgpUpU and RNA infectivity was restored only after reversion to the wild-type sequence. The effect of substitution of the third A (A4474G; AAACA) was less severe but reduced both VPg uridylylation and virus yield. Disruption of base pairing within the upper stem region of PV-cre(2C) also affected uridylylation of VPg. Virus derived from transcripts containing mutations in the stem was either viable or quasi-infectious.

scholarly journals All Three Domains of the Hepatitis C Virus Nonstructural NS5A Protein Contribute to RNA Binding

2010 ◽  
Vol 84 (18) ◽  
pp. 9267-9277 ◽  
Author(s):  
Toshana L. Foster ◽  
Tamara Belyaeva ◽  
Nicola J. Stonehouse ◽  
Arwen R. Pearson ◽  
Mark Harris
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Binding Capacity ◽  
Specific Interaction ◽  
Genome Replication ◽  
Mobility Shift ◽  
Positive Strand Rna

ABSTRACT The hepatitis C virus (HCV) nonstructural protein NS5A is critical for viral genome replication and is thought to interact directly with both the RNA-dependent RNA polymerase, NS5B, and viral RNA. NS5A consists of three domains which have, as yet, undefined roles in viral replication and assembly. In order to define the regions that mediate the interaction with RNA, specifically the HCV 3′ untranslated region (UTR) positive-strand RNA, constructs of different domain combinations were cloned, bacterially expressed, and purified to homogeneity. Each of these purified proteins was probed for its ability to interact with the 3′ UTR RNA using filter binding and gel electrophoretic mobility shift assays, revealing differences in their RNA binding efficiencies and affinities. A specific interaction between domains I and II of NS5A and the 3′ UTR RNA was identified, suggesting that these are the RNA binding domains of NS5A. Domain III showed low in vitro RNA binding capacity. Filter binding and competition analyses identified differences between NS5A and NS5B in their specificities for defined regions of the 3′ UTR. The preference of NS5A, in contrast to NS5B, for the polypyrimidine tract highlights an aspect of 3′ UTR RNA recognition by NS5A which may play a role in the control or enhancement of HCV genome replication.

scholarly journals Fitness barriers limit reversion of a proofreading-deficient coronavirus

2019 ◽  
Author(s):  
Kevin W. Graepel ◽  
Maria L. Agostini ◽  
Xiaotao Lu ◽  
Nicole R. Sexton ◽  
Mark R. Denison
Keyword(s):  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Genome Replication ◽  
Nucleotide Substitutions ◽  
Experimental Conditions ◽  
Competitive Fitness ◽  
Adaptive Mutations ◽  
Replicase Proteins

ABSTRACTThe 3′-to-5′ exoribonuclease in coronavirus (CoV) nonstructural protein 14 (nsp14-ExoN) mediates RNA proofreading during genome replication. ExoN catalytic residues are arranged in three motifs: I (DE), II (E), III (D). Alanine substitution of the motif I residues (AA-E-D, four nucleotide substitutions) in murine hepatitis virus (MHV) and SARS-CoV yields viable mutants with impaired replication and fitness, increased mutation rates, and attenuated virulencein vivo. Despite these impairments, MHV- and SARS-CoV ExoN motif I AA mutants (ExoN-AA) have not reverted at motif I in diversein vitroandin vivoenvironments, suggesting that profound fitness barriers prevent motif I reversion. To test this hypothesis, we engineered MHV-ExoN-AA with 1, 2 or 3 nucleotide mutations along genetic pathways to AA-to-DE reversion. We show that engineered intermediate revertants were viable but had no increased replication or competitive fitness compared to MHV-ExoN-AA. In contrast, a low passage (P10) MHV-ExoN-AA showed increased replication and competitive fitness without reversion of ExoN-AA. Finally, engineered reversion of ExoN-AA to ExoN-DE in the presence of ExoN-AA passage-adaptive mutations resulted in significant fitness loss. These results demonstrate that while reversion is possible, at least one alternative adaptive pathway is more rapidly advantageous than intermediate revertants and may alter the genetic background to render reversion detrimental to fitness. Our results provide an evolutionary rationale for lack of ExoN-AA reversion, illuminate potential multi-protein replicase interactions and coevolution, and support future studies aimed at stabilizing attenuated CoV ExoN-AA mutants.IMPORTANCECoronaviruses encode an exoribonuclease (ExoN) that is important for viral replication, fitness, and virulence, yet coronaviruses with a defective ExoN (ExoN-AA) have not reverted under diverse experimental conditions. In this study, we identify multiple impediments to MHV-ExoN-AA reversion. We show that ExoN-AA reversion is possible but evolutionarily unfavorable. Instead, compensatory mutations outside of ExoN-AA motif I are more accessible and beneficial than partial reversion. We also show that coevolution between replicase proteins over long-term passage partially compensates for ExoN-AA motif I but renders the virus inhospitable to a reverted ExoN. Our results reveal the evolutionary basis for the genetic stability of ExoN-inactivating mutations, illuminate complex functional and evolutionary relationships between coronavirus replicase proteins, and identify potential mechanisms for stabilization of ExoN-AA coronavirus mutants.

scholarly journals A Nucleolin-Binding 3′ Untranslated Region Element Stabilizes β-Globin mRNA In Vivo

2006 ◽  
Vol 26 (6) ◽  
pp. 2419-2429 ◽  
Author(s):  
Yong Jiang ◽  
Xiang-Sheng Xu ◽  
J. Eric Russell
Keyword(s):  
Mrna Stability ◽  
Untranslated Region ◽  
Rna Binding ◽  
Critical Role ◽  
Specific Interaction ◽  
Globin Mrna ◽  
Intact Cells ◽  
Normal Expression

ABSTRACT The normal expression of human β globin is critically dependent upon the constitutively high stability of its encoding mRNA. Unlike with α-globin mRNA, the specific cis-acting determinants and trans-acting factors that participate in stabilizing β-globin mRNA are poorly described. The current work uses a linker-scanning strategy to identify a previously unknown determinant of mRNA stability within the β-globin 3′ untranslated region (3′UTR). The new determinant is positioned on an mRNA half-stem opposite a pyrimidine-rich sequence targeted by αCP/hnRNP-E, a factor that plays a critical role in stabilizing human α-globin mRNA. Mutations within the new determinant destabilize β-globin mRNA in intact cells while also ablating its 3′UTR-specific interaction with the polyfunctional RNA-binding factor nucleolin. We speculate that 3′UTR-bound nucleolin enhances mRNA stability by optimizing αCP access to its functional binding site. This model is favored by in vitro evidence that αCP binding is enhanced both by cis-acting stem-destabilizing mutations and by the trans-acting effects of supplemental nucleolin. These studies suggest a mechanism for β-globin mRNA stability that is related to, but distinct from, the mechanism that stabilizes human α-globin mRNA.

scholarly journals A 4-Base-Pair Core-Enclosing Helix in Telomerase RNA Is Essential for Activity and for Binding to the Telomerase Reverse Transcriptase Catalytic Protein Subunit

2020 ◽  
Vol 40 (24) ◽  
Author(s):  
Melissa A. Mefford ◽  
Evan P. Hass ◽  
David C. Zappulla
Keyword(s):  
Reverse Transcriptase ◽  
Rna Binding ◽  
Telomerase Reverse Transcriptase ◽  
Genome Replication ◽  
Telomerase Rna ◽  
Protein Subunit ◽  
Binding Interaction ◽  
Catalytic Core

ABSTRACT The telomerase ribonucleoprotein (RNP) counters the chromosome end replication problem, completing genome replication to prevent cellular senescence in yeast, humans, and most other eukaryotes. The telomerase RNP core enzyme is composed of a dedicated RNA subunit and a reverse transcriptase (telomerase reverse transcriptase [TERT]). Although the majority of the 1,157-nucleotide (nt) Saccharomyces cerevisiae telomerase RNA, TLC1, is rapidly evolving, the central catalytic core is largely conserved, containing the template, template-boundary helix, pseudoknot, and core-enclosing helix (CEH). Here, we show that 4 bp of core-enclosing helix is required for telomerase to be active in vitro and to maintain yeast telomeres in vivo, whereas the ΔCEH and 1- and 2-bp alleles do not support telomerase function. Using the CRISPR/nuclease-deactivated Cas9 (dCas9)-based CARRY (CRISPR-assisted RNA–RNA-binding protein [RBP] yeast) two-hybrid assay to assess binding of our CEH mutant RNAs to TERT, we find that the 4-bp CEH RNA binds to TERT but the shorter-CEH constructs do not, consistent with the telomerase activity and in vivo complementation results. Thus, the CEH is essential in yeast telomerase RNA because it is needed to bind TERT to form the core RNP enzyme. Although the 8 nt that form this 4-bp stem at the base of the CEH are nearly invariant among Saccharomyces species, our results with sequence-randomized and truncated-CEH helices suggest that this binding interaction with TERT is dictated more by secondary than by primary structure. In summary, we have mapped an essential binding site in telomerase RNA for TERT that is crucial to form the catalytic core of this biomedically important RNP enzyme.

scholarly journals Multimerization of human cytomegalovirus regulatory protein UL69 via a domain that is conserved within its herpesvirus homologues

2007 ◽  
Vol 88 (2) ◽  
pp. 405-410 ◽  
Author(s):  
Peter Lischka ◽  
Marco Thomas ◽  
Zsolt Toth ◽  
Regina Mueller ◽  
Thomas Stamminger
Keyword(s):  
Human Cytomegalovirus ◽  
Nuclear Export ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Transcriptional Level ◽  
Homologous Proteins ◽  
Interaction Domain ◽  
Self Association

The UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that has counterparts in all herpesviruses. Some of these proteins have been shown to function primarily at the post-transcriptional level in promoting nuclear export of viral transcripts. Consistently, this group has reported recently that pUL69 is an RNA-binding, nucleocytoplasmic shuttling protein that facilitates the cytoplasmic accumulation of unspliced mRNA via its interaction with the cellular mRNA export factor UAP56. Evidence has been presented to suggest that some of the pUL69 homologues self-interact and function in vivo as multimers. Herein, the possibility of pUL69 self-association was examined and it has been demonstrated that pUL69 can interact with itself in vitro and in vivo in order to form high-molecular-mass complexes. The self-interaction domain within pUL69 was mapped to a central domain of this viral protein that is conserved within the homologous proteins of other herpesviruses, suggesting that multimerization is a conserved feature of this protein family.

scholarly journals Fitness Barriers Limit Reversion of a Proofreading-Deficient Coronavirus

2019 ◽  
Vol 93 (20) ◽  
Author(s):  
Kevin W. Graepel ◽  
Maria L. Agostini ◽  
Xiaotao Lu ◽  
Nicole R. Sexton ◽  
Mark R. Denison
Keyword(s):  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Genome Replication ◽  
Nucleotide Substitutions ◽  
Experimental Conditions ◽  
Competitive Fitness ◽  
Adaptive Mutations ◽  
Replicase Proteins

ABSTRACT The 3′-to-5′ exoribonuclease in coronavirus (CoV) nonstructural protein 14 (nsp14-ExoN) mediates RNA proofreading during genome replication. ExoN catalytic residues are arranged in three motifs: I (DE), II (E), and III (D). Alanine replacement of the motif I residues (AA-E-D; four nucleotide substitutions) in murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV yields viable mutants with impaired replication and fitness, increased mutation rates, and attenuated virulence in vivo. Despite these impairments, MHV- and SARS-CoV ExoN motif I AA mutants (ExoN-AA) have not reverted at motif I in diverse in vitro and in vivo environments, suggesting that profound fitness barriers prevent motif I reversion. To test this hypothesis, we engineered MHV-ExoN-AA with 1, 2, or 3 nucleotide mutations along genetic pathways to AA-to-DE reversion. We show that engineered intermediate revertants were viable but had no increased replication or competitive fitness compared to that of MHV-ExoN-AA. In contrast, a low-passage-number (passage 10 [P10]) MHV-ExoN-AA showed increased replication and competitive fitness without reversion of ExoN-AA. Finally, engineered reversion of ExoN-AA to ExoN-DE in the presence of ExoN-AA passage-adaptive mutations resulted in significant fitness loss. These results demonstrate that while reversion is possible, at least one alternative adaptive pathway is more rapidly advantageous than intermediate revertants and may alter the genetic background to render reversion detrimental to fitness. Our results provide an evolutionary rationale for lack of ExoN-AA reversion, illuminate potential multiprotein replicase interactions and coevolution, and support future studies aimed at stabilizing attenuated CoV ExoN-AA mutants. IMPORTANCE Coronaviruses encode an exoribonuclease (ExoN) that is important for viral replication, fitness, and virulence, yet coronaviruses with a defective ExoN (ExoN-AA) have not reverted under diverse experimental conditions. In this study, we identify multiple impediments to MHV-ExoN-AA reversion. We show that ExoN-AA reversion is possible but evolutionarily unfavorable. Instead, compensatory mutations outside ExoN-AA motif I are more accessible and beneficial than partial reversion. We also show that coevolution between replicase proteins over long-term passage partially compensates for ExoN-AA motif I but renders the virus inhospitable to a reverted ExoN. Our results reveal the evolutionary basis for the genetic stability of ExoN-inactivating mutations, illuminate complex functional and evolutionary relationships between coronavirus replicase proteins, and identify potential mechanisms for stabilization of ExoN-AA coronavirus mutants.

scholarly journals Delineation of the Protein Module That Anchors HMGN Proteins to Nucleosomes in the Chromatin of Living Cells

2008 ◽  
Vol 28 (9) ◽  
pp. 2872-2883 ◽  
Author(s):  
Tetsuya Ueda ◽  
Frédéric Catez ◽  
Gabi Gerlitz ◽  
Michael Bustin
Keyword(s):  
Dna Sequences ◽  
Specific Binding ◽  
Specific Interaction ◽  
Living Cells ◽  
Mobility Shift ◽  
Generic Structure ◽  
Conserved Sequence ◽  
Nucleosome Core

ABSTRACT Numerous nuclear proteins bind to chromatin by targeting unique DNA sequences or specific histone modifications. In contrast, HMGN proteins recognize the generic structure of the 147-bp nucleosome core particle. HMGNs alter the structure and activity of chromatin by binding to nucleosomes; however, the determinants of the specific interaction of HMGNs with chromatin are not known. Here we use systematic mutagenesis, quantitative fluorescence recovery after photobleaching, fluorescence imaging, and mobility shift assays to identify the determinants important for the specific binding of these proteins to both the chromatin of living cells and to purified nucleosomes. We find that several regions of the protein affect the affinity of HMGNs to chromatin; however, the conserved sequence RRSARLSA, is the sole determinant of the specific interaction of HMGNs with nucleosomes. Within this sequence, each of the 4 amino acids in the R-S-RL motif are the only residues absolutely essential for anchoring HMGN protein to nucleosomes, both in vivo and in vitro. Our studies identify a new chromatin-binding module that specifically recognizes nucleosome cores independently of DNA sequence or histone tail modifications.

scholarly journals PDZ binding motif of HTLV-1 Tax promotes virus-mediated T-cell proliferation in vitro and persistence in vivo

Blood ◽  
2006 ◽  
Vol 107 (5) ◽  
pp. 1980-1988 ◽  
Author(s):  
Li Xie ◽  
Brenda Yamamoto ◽  
Abdelali Haoudi ◽  
O. John Semmes ◽  
Patrick L. Green
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
Specific Binding ◽  
Cellular Transformation ◽  
T Cell Proliferation ◽  
Binding Motif ◽  
Interaction Domain ◽  
Pdz Proteins

HTLV-1 cellular transformation and disease induction is dependent on expression of the viral Tax oncoprotein. PDZ is a modular protein interaction domain used in organizing signaling complexes in eukaryotic cells through recognition of a specific binding motif in partner proteins. Tax-1, but not Tax-2, contains a PDZ-binding domain motif (PBM) that promotes the interaction with several cellular PDZ proteins. Herein, we investigate the contribution of the Tax-1 PBM in HTLV-induced proliferation and immortalization of primary T cells in vitro and viral survival in an infectious rabbit animal model. We generated several HTLV-1 and HTLV-2 Tax viral mutants, including HTLV-1ΔPBM, HTLV-2+C22(+PBM), and HTLV-2+ C18(ΔPBM). All Tax mutants maintained the ability to significantly activate the CREB/ATF or NFκB signaling pathways. Microtiter proliferation assays revealed that the Tax-1 PBM significantly increases both HTLV-1– and HTLV-2–induced primary T-cell proliferation. In addition, Tax-1 PBM was responsible for the micronuclei induction activity of Tax-1 relative to that of Tax-2. Viral infection and persistence were severely attenuated in rabbits inoculated with HTLV-1ΔPBM. Our results provide the first direct evidence suggesting that PBM-mediated associations between Tax-1 and cellular proteins play a key role in HTLV-induced cell proliferation and genetic instability in vitro and facilitate viral persistence in vivo.

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