Hepatitis C virus NS5A is able to competitively displace c-Myc from the Bin1 SH3 domain<i>in vitro</i>

Aladağ, Amine; Hoffmann, Silke; Stoldt, Matthias; Bösing, Christina; Willbold, Dieter; Schwarten, Melanie

doi:10.1002/psc.2618

Cited by 8 publications

(10 citation statements)

References 32 publications

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“…The exact function of amphiphysin in viral replication is unclear, but a role in formation or stabilization of the membranous replication structures by means of the BAR ((BIN/amphiphysin/Rvs) domain has been envisaged (9). Moreover, the interaction between HCV NS5A and amphiphysin can contribute to a favorable environment for productive viral replication by inhibiting Bin1-mediated apoptosis (27), possibly due to competitively blocking the binding of Myc to Bin1 (19).…”

Section: Discussionmentioning

confidence: 99%

“…We did not carry out ITC measurements for the HCV NS5A peptide, but based on the slow exchange kinetics observed in NMR titration and its highly similar amino acid composition with CHIKV and SFV, we presume that it also has a similar high amp-SH3 binding affinity. Indeed, a slowly dissociating complex in NMR-based titration experiments and a K d of 0.24 M for the subtype 1b peptide of HCV NS5A ( 347 -TKAPPIPPPRRKRTV 361 ) and amp-SH3 have recently been reported (19).…”

Section: C-terminal Tail Of Dynamin Is Disordered and Binds Amp-sh3 Withmentioning

confidence: 99%

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Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2

Tossavainen

Aitio

Hellman

et al. 2016

Journal of Biological Chemistry

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We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728 -1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (K d 24 nM). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this affinity. Thus, via optimization of such dynamic electrostatic forces, viral peptides have evolved a superior binding affinity for amphiphysin-2 SH3 compared with typical cellular ligands, such as dynamin, thereby enabling hijacking of amphiphysin-2 SH3-regulated host cell processes by these viruses. Moreover, our data show that the previously described consensus sequence PXRPXR for amphiphysin SH3 ligands is inaccurate and instead define it as an extended Class II binding motif PXXPXRpXR, where additional positive charges between the two constant arginine residues can give rise to extraordinary high SH3 binding affinity.

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Section: Discussionmentioning

confidence: 99%

Section: C-terminal Tail Of Dynamin Is Disordered and Binds Amp-sh3 Withmentioning

confidence: 99%

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2

Tossavainen

Aitio

Hellman

et al. 2016

Journal of Biological Chemistry

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“…This interaction is suspected to play a role in the development of hepatocellular carcinoma in HCV-infected liver cells. In particular, it has been shown that NS5A is able to competitively displace the oncogenic Myc protein from its complex formed with Bin1-SH3 (57). Previously, we have found (37,38) that NS5A displays a high-affinity binding site (PxxP region), as well as two low-affinity binding sites (transient helices H1b and H3) for Bin1-SH3.…”

Section: Ns5a-d2d3 Structural Changes Induced By Bin1-sh3 Bindingmentioning

confidence: 96%

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation

Solyom

Schwarten

et al. 2015

Biophysical Journal

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Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the IDP, and thus the distance over which the protein can catch binding partners via so-called fly-casting mechanisms. The modulation of long-range interactions also presents a convenient way of fine-tuning the protein's interaction network, by making binding sites more or less accessible. Here we studied, mainly by nuclear magnetic resonance spectroscopy, residual secondary structure and long-range interactions in nonstructural protein 5A (NS5A) from hepatitis C virus (HCV), a typical viral IDP with multiple functions during the viral life cycle. NS5A comprises an N-terminal folded domain, followed by a large (∼250-residue) disordered C-terminal part. Comparing nuclear magnetic resonance spectra of full-length NS5A with those of a protein construct composed of only the C-terminal residues 191-447 (NS5A-D2D3) allowed us to conclude that there is no significant interaction between the globular and disordered parts of NS5A. NS5A-D2D3, despite its overall high flexibility, shows a large extent of local residual (α-helical and β-turn) structure, as well as a network of electrostatic long-range interactions. Furthermore, we could demonstrate that these long-range interactions become modulated upon binding to the host protein Bin1, as well as after NS5A phosphorylation by CK2. As the charged peptide regions involved in these interactions are well conserved among the different HCV genotypes, these transient long-range interactions may be important for some of the functions of NS5A over the course of the HCV life cycle.

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“…Apparently, we are only at the beginning of understanding the complexity of CHIKV HVD interactions with SH3 domain-containing proteins in different cell types and hosts. Interestingly, similarly to NAP1L1, BIN1 was shown to interact with the disordered fragment of hepatitis C virus (HCV)-specific NS5A protein (62,(65)(66)(67). Alteration of this interaction has a negative effect on HCV replication.…”

Section: Fig 11mentioning

confidence: 99%

Multiple Host Factors Interact with the Hypervariable Domain of Chikungunya Virus nsP3 and Determine Viral Replication in Cell-Specific Mode

Meshram

Agback

Shiliaev

et al. 2018

J Virol

112

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Alphaviruses are widely distributed in both hemispheres and circulate between mosquitoes and amplifying vertebrate hosts. Geographically separated alphaviruses have adapted to replication in particular organisms. The accumulating data suggest that this adaptation is determined not only by changes in their glycoproteins, but also by the amino acid sequence of the hypervariable domain (HVD) of the alphavirus nsP3 protein. We performed a detailed investigation of chikungunya virus (CHIKV) nsP3 HVD interactions with host factors and their roles in viral replication in vertebrate and mosquito cells. The results demonstrate that CHIKV HVD is intrinsically disordered and binds several distinctive cellular proteins. These host factors include two members of the G3BP family and their mosquito homolog Rin, two members of the NAP1 family and several SH3 domain-containing proteins. Interaction with G3BP proteins or Rin is an absolute requirement for CHIKV replication, although it is insufficient to solely drive it in either vertebrate or mosquito cells. To achieve a detectable level of virus replication, HVD needs to bind members of at least one more protein family in addition to G3BPs. Interaction with NAP1L1 and NAP1L4 plays a more pro-viral role in vertebrate cells, while binding of SH3 domain-containing proteins to a proline-rich fragment of HVD is more critical for virus replication in the cells of mosquito origin. Modifications of binding sites in CHIKV HVD allow manipulation of the cell specificity of CHIKV replication. Similar changes may be introduced into HVDs of other alphaviruses to alter their replication in particular cells or tissues. Alphaviruses utilize a broad spectrum of cellular factors for efficient formation and function of replication complexes (RCs). Our data demonstrate for the first time that the hypervariable domain (HVD) of chikungunya virus nonstructural protein 3 (nsP3) is intrinsically disordered. It binds at least 3 families of cellular proteins, which play an indispensable role in viral RNA replication. The proteins of each family demonstrate functional redundancy. We provide a detailed map of the binding sites on CHIKV nsP3 HVD and show that mutations in these sites or the replacement of CHIKV HVD by heterologous HVD change cell specificity of viral replication. Such manipulations with alphavirus HVDs open an opportunity for development of new irreversibly attenuated vaccine candidates. To date, the disordered protein fragments have been identified in the nonstructural proteins of many other viruses. They may also interact with variety of cellular factors that determine critical aspects of virus-host interactions.

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Hepatitis C virus NS5A is able to competitively displace c-Myc from the Bin1 SH3 domainin vitro

Cited by 8 publications

References 32 publications

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation

Multiple Host Factors Interact with the Hypervariable Domain of Chikungunya Virus nsP3 and Determine Viral Replication in Cell-Specific Mode

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