scholarly journals Hepatitis C Virus Nonstructural Protein 5A (NS5A) Is an RNA-binding Protein

2005 ◽  
Vol 280 (43) ◽  
pp. 36417-36428 ◽  
Author(s):  
Luyun Huang ◽  
Jungwook Hwang ◽  
Suresh D. Sharma ◽  
Michele R. S. Hargittai ◽  
Yingfeng Chen ◽  
...  
Keyword(s):  
Hepatitis C ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Rna Binding Protein ◽  
Affinity Binding ◽  
Genome Replication ◽  
Subgenomic Replicon ◽  
High Affinity Binding ◽  
High Affinity ◽  
Nonstructural Protein 5A

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) has been shown to antagonize numerous cellular pathways, including the antiviral interferon-α response. However, the capacity of this protein to interact with the viral polymerase suggests a more direct role for NS5A in genome replication. In this study, we employed two bacterially expressed, soluble derivatives of NS5A to probe for novel functions of this protein. We find that NS5A has the capacity to bind to the 3′-ends of HCV plus and minus strand RNAs. The high affinity binding site for NS5A in the 3′-end of plus strand RNA maps to the polypyrimidine tract, an element known to be essential for genome replication and infectivity. NS5A has a preference for single-stranded RNA containing stretches of uridine or guanosine. Values for the equilibrium dissociation constants for high affinity binding sites were in the 10 nm range. Two-dimensional gel electrophoresis followed by Western blotting revealed the presence of unphosphorylated NS5A in Huh-7 cells stably expressing the subgenomic replicon. Moreover, RNA immunoprecipitation and NS5A pull-down experiments showed the capacity of replicon-derived NS5A to bind to synthetic RNA and the HCV genome, respectively. Deletion of all of the casein kinase II phosphorylation sites in NS5A supported stable replication of a subgenomic replicon in Huh-7. However, this derivative could not be labeled with inorganic phosphate, suggesting that extensive phosphorylation of NS5A is not required for the replication functions of NS5A. The discovery that NS5A is an RNA-binding protein defines a new functional target for development of agents to treat HCV infection and a new structural class of RNA-binding proteins.

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 Sequential S232/S235/S238 Phosphorylation of the Hepatitis C Virus Nonstructural Protein 5A

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Shih-Chin Hsu ◽  
Chia-Ni Tsai ◽  
Kuan-Ying Lee ◽  
Ting-Chun Pan ◽  
Chieh-Wen Lo ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Direct Evidence ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Low Complexity ◽  
Phosphorylation Cascade ◽  
Binding Groove ◽  
Domain I ◽  
Nonstructural Protein 5A

ABSTRACTThe hepatitis C virus (HCV) protein NS5A is a phosphorylated protein with crucial roles in viral replication and assembly. NS5A was thought to undergo sequential phosphorylation on a series of conserved serine residues; however, the phosphorylation cascade remained obscure. Using three phosphorylation-specific antibodies, we found that phosphorylation at S232, S235, and S238 occurred in parallel in HCV-infected Huh7.5.1 cells, suggestive of intramolecular sequential NS5A phosphorylation from S232 through S235 to S238 by casein kinase Iα (CKIα). In line with this, alanine mutation at S225, S229, or S232 reduced, whereas aspartate mutation at the same sites rescued, NS5A phosphorylation at S232, S235, and S238. In contrast, alanine or aspartate mutation at S235 or S238 had little or no effect on S232 or S235 phosphorylation. Consistent with an intramolecular sequential phosphorylation cascade, S232, S235, and S238 phosphorylation coexisted on one single NS5A molecule. Phosphorylation of NH2-terminal serine residues in one NS5A molecule did not rescue phosphorylation of COOH-terminal serine residues in another NS5A molecule. CKIα inhibition reduced NS5A phosphorylation at S232, S235, and S238. In summary, our results are indicative of a CKIα-mediated intramolecular, sequential phosphorylation cascade from S232 through S235 to S238 of the HCV NS5A protein. S225 and S229 also contribute substantially to the above sequential phosphorylation cascade of NS5A.IMPORTANCEThe nonstructural protein 5A (NS5A) of the hepatitis C virus was thought to undergo sequential intramolecular phosphorylation on a series of serine residues; however, direct evidence was missing. We offer the first direct evidence of a CKIα-mediated intramolecular sequential NS5A phosphorylation cascade from serine 232 through 235 to 238. This sequential phosphorylation cascade occurs in the disordered low-complexity sequence I region, which together with the domain I region forms an RNA-binding groove in an NS5A dimer. Sequential phosphorylation in the disordered region adds charge-charge repulsion to the RNA-binding groove and probably thereby regulates NS5A's RNA-binding ability and functions in viral RNA replication and assembly.

scholarly journals An AU-Rich Sequence Element (UUUN[A/U]U) Downstream of the Edited C in Apolipoprotein B mRNA Is a High-Affinity Binding Site for Apobec-1: Binding of Apobec-1 to This Motif in the 3′ Untranslated Region of c-myc Increases mRNA Stability

2000 ◽  
Vol 20 (6) ◽  
pp. 1982-1992 ◽  
Author(s):  
Shrikant Anant ◽  
Nicholas O. Davidson
Keyword(s):  
Binding Site ◽  
Apolipoprotein B ◽  
Half Life ◽  
Rna Binding ◽  
Cytidine Deaminase ◽  
Binding Activity ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site

ABSTRACT Apobec-1, the catalytic subunit of the mammalian apolipoprotein B (apoB) mRNA-editing enzyme, is a cytidine deaminase with RNA binding activity for AU-rich sequences. This RNA binding activity is required for Apobec-1 to mediate C-to-U RNA editing. Filter binding assays, using immobilized Apobec-1, demonstrate saturable binding to a 105-nt apoB RNA with a Kd of ∼435 nM. A series of AU-rich templates was used to identify a high-affinity (∼50 nM) binding site of consensus sequence UUUN[A/U]U, with multiple copies of this sequence constituting the high-affinity binding site. In order to determine whether this consensus site could be functionally demonstrated from within an apoB RNA, circular-permutation analysis was performed, revealing one major (UUUGAU) and one minor (UU) site located 3 and 16 nucleotides, respectively, downstream of the edited base. Secondary-structure predictions reveal a stem-loop flanking the edited base with Apobec-1 binding to the consensus site(s) at an open loop. A similar consensus (AUUUA) is present in the 3′ untranslated regions of several mRNAs, including that of c-myc, that are known to undergo rapid degradation. In this context, it is presumed that the consensus motif acts as a destabilizing element. As an independent test of the ability of Apobec-1 to bind to this sequence, F442A cells were transfected with Apobec-1 and the half-life of c-myc mRNA was determined following actinomycin D treatment. These studies demonstrated an increase in the half-life of c-myc mRNA from 90 to 240 min in control versus Apobec-1-expressing cells. Apobec-1 expression mutants, in which RNA binding activity is eliminated, failed to alter c-myc mRNA turnover. Taken together, the data establish a consensus binding site for Apobec-1 embedded in proximity to the edited base in apoB RNA. Binding to this site in other target RNAs raises the possibility that Apobec-1 may be involved in other aspects of RNA metabolism, independent of its role as an apoB RNA-specific cytidine deaminase.

scholarly journals DC-SIGN and L-SIGN Are High Affinity Binding Receptors for Hepatitis C Virus Glycoprotein E2

2003 ◽  
Vol 278 (22) ◽  
pp. 20358-20366 ◽  
Author(s):  
Pierre-Yves Lozach ◽  
Hugues Lortat-Jacob ◽  
Agnès De Lacroix De Lavalette ◽  
Isabelle Staropoli ◽  
Steven Foung ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Virus Glycoprotein ◽  
High Affinity ◽  
Glycoprotein E2

scholarly journals The hepatitis C viral nonstructural protein 5A stabilizes growth-regulatory human transcripts

2018 ◽  
Vol 46 (5) ◽  
pp. 2537-2547 ◽  
Author(s):  
Liang Guo ◽  
Suresh D Sharma ◽  
Jose D Debes ◽  
Daniel Beisang ◽  
Bernd Rattenbacher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatitis C ◽  
Chronic Infection ◽  
Nonstructural Protein ◽  
Host Gene Expression ◽  
Subgenomic Replicon ◽  
Mrna Stabilization ◽  
Infected Cells ◽  
Nonstructural Protein 5A

AbstractNumerous mammalian proto-oncogene and other growth-regulatory transcripts are upregulated in malignancy due to abnormal mRNA stabilization. In hepatoma cells expressing a hepatitis C virus (HCV) subgenomic replicon, we found that the viral nonstructural protein 5A (NS5A), a protein known to bind to viral RNA, also bound specifically to human cellular transcripts that encode regulators of cell growth and apoptosis, and this binding correlated with transcript stabilization. An important subset of human NS5A-target transcripts contained GU-rich elements, sequences known to destabilize mRNA. We found that NS5A bound to GU-rich elements in vitro and in cells. Mutation of the NS5A zinc finger abrogated its GU-rich element-binding and mRNA stabilizing activities. Overall, we identified a molecular mechanism whereby HCV manipulates host gene expression by stabilizing host transcripts in a manner that would promote growth and prevent death of virus-infected cells, allowing the virus to establish chronic infection and lead to the development of hepatocellular carcinoma.

scholarly journals Hepatitis C Virus Nonstructural Protein 5A: Biochemical Characterization of a Novel Structural Class of RNA-Binding Proteins

2010 ◽  
Vol 84 (24) ◽  
pp. 12480-12491 ◽  
Author(s):  
Jungwook Hwang ◽  
Luyun Huang ◽  
Daniel G. Cordek ◽  
Robert Vaughan ◽  
Shelley L. Reynolds ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Binding Activity ◽  
Cross Linking ◽  
Amino Terminal ◽  
Rna Complex ◽  
Domain I ◽  
Nonstructural Protein 5A

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) exhibits a preference for G/U-rich RNA in vitro. Biological analysis of the NS5A RNA-binding activity and its target sites in the genome will be facilitated by a description of the NS5A-RNA complex. We demonstrate that the C-4 carbonyl of the uracil base and, by inference, the C-6 carbonyl of the guanine base interact with NS5A. U-rich RNA of 5 to 6 nucleotides (nt) is sufficient for high-affinity binding to NS5A. The minimal RNA-binding domain of NS5A consists of residues 2005 to 2221 (referred to as domain I-plus). This region of the protein includes the amino-terminal domain I as well as the subsequent linker that separates domains I and II. This linker region is the site of adaptive mutations. U-rich RNA-binding activity is not observed for an NS5A derivative containing only residues 2194 to 2419 (domains II and III). Mass spectrometric analysis of an NS5A-poly(rU) complex identified domains I and II as sites for interaction with RNA. Dimerization of NS5A was demonstrated by glutaraldehyde cross-linking. This dimerization is likely mediated by domain I-plus, as dimers of this protein are trapped by cross-linking. Dimers of the domain II-III protein are not observed. The monomer-dimer equilibrium of NS5A shifts in favor of dimer when U-rich RNA is present but not when A-rich RNA is present, consistent with an NS5A dimer being the RNA-binding-competent form of the protein. These data provide a molecular perspective of the NS5A-RNA complex and suggest possible mechanisms for regulation of HCV and cellular gene expression.

scholarly journals Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes

2014 ◽  
Vol 89 (6) ◽  
pp. 3123-3135 ◽  
Author(s):  
Douglas Ross-Thriepland ◽  
Jamel Mankouri ◽  
Mark Harris
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Phosphorylation Site ◽  
Serine Phosphorylation ◽  
Genome Replication ◽  
Wild Type ◽  
Subgenomic Replicon ◽  
Replication Complex ◽  
Ns5a Protein

ABSTRACTThe hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is highly phosphorylated and involved in both virus genome replication and virion assembly. We and others have identified serine 225 in NS5A to be a phosphorylation site, but the function of this posttranslational modification in the virus life cycle remains obscure. Here we describe the phenotype of mutants with mutations at serine 225; this residue was mutated to either alanine (S225A; phosphoablatant) or aspartic acid (S225D; phosphomimetic) in the context of both the JFH-1 cell culture infectious virus and a corresponding subgenomic replicon. The S225A mutant exhibited a 10-fold reduction in genome replication, whereas the S225D mutant replicated like the wild type. By confocal microscopy, we show that, in the case of the S225A mutant, the replication phenotype correlated with an altered subcellular distribution of NS5A. This phenotype was shared by viruses with other mutations in the low-complexity sequence I (LCS I), namely, S229D, S232A, and S235D, but not by viruses with mutations that caused a comparable replication defect that mapped to domain II of NS5A (P315A, L321A). Together with other components of the genome replication complex (NS3, double-stranded RNA, and cellular lipids, including phosphatidylinositol 4-phosphate), the mutation in NS5A was restricted to a perinuclear region. This phenotype was not due to cell confluence or another environmental factor and could be partially transcomplemented by wild-type NS5A. We propose that serine phosphorylation within LCS I may regulate the assembly of an active genome replication complex.IMPORTANCEThe mechanisms by which hepatitis C virus replicates its RNA genome remain poorly characterized. We show here that phosphorylation of the viral nonstructural protein NS5A at serine residues is important for the efficient assembly of a complex that is able to replicate the viral genome. This research implicates cellular protein kinases in the control of virus replication and highlights the need to further understand the interplay between the virus and the host cell in order to develop potential avenues for future antiviral therapy.

scholarly journals Multivalency in the recognition and antagonism of a HIV TAR RNA–TAT assembly using an aminoglycoside benzimidazole scaffold

2016 ◽  
Vol 14 (6) ◽  
pp. 2052-2056 ◽  
Author(s):  
Sunil Kumar ◽  
Nihar Ranjan ◽  
Patrick Kellish ◽  
Changjun Gong ◽  
Derrick Watkins ◽  
...  
Keyword(s):  
Small Molecules ◽  
Rna Binding ◽  
Affinity Binding ◽  
Rna Recognition ◽  
High Affinity Binding ◽  
High Affinity ◽  
Tar Rna

Recognition of RNA by high-affinity binding small molecules is crucial for expanding existing approaches in RNA recognition, and for the development of novel RNA binding drugs.

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