4J1V

Functional and structural studies of MOBKL1B, a Salvador/Warts/Hippo tumor suppressor pathway, in HCV replication

Summary for 4J1V

• Entry DOI: 10.2210/pdb4j1v/pdb
• Descriptor: MOB kinase activator 1A, NS5A domain II peptide, ZINC ION, ... (4 entities in total)
• Functional Keywords: hcv ns5a binding protein, ns5a, peptide binding protein-viral protein complex, peptide binding protein/viral protein
• Biological source: Homo sapiens (human); More
• Cellular location: Core protein p21: Host endoplasmic reticulum membrane; Single-pass membrane protein (By similarity). Core protein p19: Virion (By similarity). Envelope glycoprotein E1: Virion membrane; Single-pass type I membrane protein (Potential). Envelope glycoprotein E2: Virion membrane; Single-pass type I membrane protein (Potential). p7: Host endoplasmic reticulum membrane; Multi-pass membrane protein. Protease NS2-3: Host endoplasmic reticulum membrane; Multi-pass membrane protein (Potential). Serine protease NS3: Host endoplasmic reticulum membrane; Peripheral membrane protein (Probable). Non-structural protein 4A: Host endoplasmic reticulum membrane; Single-pass type I membrane protein (Potential). Non-structural protein 4B: Host endoplasmic reticulum membrane; Multi-pass membrane protein (By similarity). Non-structural protein 5A: Host endoplasmic reticulum membrane; Peripheral membrane protein (By similarity). RNA-directed RNA polymerase: Host endoplasmic reticulum membrane; Single-pass type I membrane protein (Potential): Q99IB8
• Total number of polymer chains: 6
• Total formula weight: 52516.55

Authors

Chung, H.-Y.,Gu, M.,Rice, C.M. (deposition date: 2013-02-02, release date: 2014-08-06, Last modification date: 2024-02-28)

Primary citation

Chung, H.Y.,Gu, M.,Buehler, E.,MacDonald, M.R.,Rice, C.M.
Seed Sequence-Matched Controls Reveal Limitations of Small Interfering RNA Knockdown in Functional and Structural Studies of Hepatitis C Virus NS5A-MOBKL1B Interaction.
J.Virol., 88:11022-11033, 2014

PubMed Abstract: Hepatitis C virus (HCV) is a widespread human pathogen causing liver cirrhosis and cancer. Similar to the case for other viruses, HCV depends on host and viral factors to complete its life cycle. We used proteomic and yeast two-hybrid approaches to elucidate host factors involved in HCV nonstructural protein NS5A function and found that MOBKL1B interacts with NS5A. Initial experiments with small interfering RNA (siRNA) knockdown suggesting a role in HCV replication led us to examine the interaction using biochemical and structural approaches. As revealed by a cocrystal structure of a core MOBKL1B-NS5A peptide complex at 1.95 Å, NS5A binds to a hydrophobic patch on the MOBKL1B surface. Biosensor binding assays identified a highly conserved, 18-amino-acid binding site in domain II of NS5A, which encompasses residues implicated in cyclophilin A (CypA)-dependent HCV RNA replication. However, a CypA-independent HCV variant had reduced replication in MOBKL1B knockdown cells, even though its NS5A does not interact with MOBKL1B. These discordant results prompted more extensive studies of MOBKL1B gene knockdowns, which included additional siRNAs and specifically matched seed sequence siRNA controls. We found that reduced virus replication after treating cells with MOBKL1B siRNA was actually due to off-target inhibition, which indicated that the initial finding of virus replication dependence on the MOBKL1B-NS5A interaction was incorrect. Ultimately, using several approaches, we found no relationship of the MOBKL1B-NS5A interaction to virus replication. These findings collectively serve as a reminder to investigators and scientific reviewers of the pervasive impact of siRNA off-target effects on interpretation of biological data.

PubMed: 25031347
DOI: 10.1128/JVI.01582-14

Experimental method

X-RAY DIFFRACTION (1.95 Å)