4x2v | pdb_00004x2v
Crystal structure of the Murine Norovirus NS6 protease (inactive C139A mutant) with a C-terminal extension to include residue P1 prime of NS7
Structural highlights
FunctionPOLG_MNV1 Induces the proliferation of the host smooth ER membranes forming long tubular structures (By similarity). These remodeled membranes probably form the viral factories that contain the replication complex (By similarity). May play a role in viral replication by interacting with host VAPA, a vesicle-associated membrane protein that plays a role in SNARE-mediated vesicle fusion. This interaction may target replication complex to intracellular membranes (Probable).[UniProtKB:P54634][1] Promotes intestinal tropism and persistent fecal shedding in strain CR6 (PubMed:23077309, PubMed:31130511, PubMed:31329638). This function requires Glu-94 and is present in persistant strains (PubMed:23077309).[2] [3] [4] Displays NTPase activity, but probably no helicase activity (PubMed:30265237). Displays RNA chaperone-like activity and destabilizes dsRNA (PubMed:30265237). Induces the formation of convoluted membranes derived from the host ER (By similarity). These remodeled membranes probably form the viral factories that contain the replication complex (By similarity). Initiates host cell death by targeting the mitochondrial outer membrane, leading to the permeabilization of mitochondria, programmed host cell death and viral egress (PubMed:36991121). Externalization of host cardiolipin seems to be involved in the process (PubMed:36991121). Probably plays a role in preventing the assembly of host stress granules (PubMed:31905230).[UniProtKB:P54634][5] [6] [7] Probable key protein responsible for the formation of membrane alterations by the virus (By similarity). Induces the formation of convoluted membranes derived from the host ER (By similarity). These remodeled membranes probably form the viral factories that contain the replication complex (By similarity). May play a role in targeting replication complex to intracellular membranes.[UniProtKB:P54634] Viral genome-linked protein is covalently linked to the 5'-end of the positive-strand, negative-strand genomic RNAs and subgenomic RNA (By similarity). Acts as a genome-linked replication primer (By similarity). May recruit ribosome to viral RNA thereby promoting viral proteins translation (By similarity). Interacts with host translation initiation complex to allow the translation of viral proteins (PubMed:16835235, PubMed:24928504). Induces the formation of aggregates of RNA-directed RNA polymerase in the presence of RNA (PubMed:30038601). Through its interaction with the viral RNA-directed RNA polymerase, plays a crucial role in enhancing the polymerase activity (PubMed:30038601).[UniProtKB:P27409][8] [9] [10] Processes the polyprotein. 3CLpro-RdRp is first released by autocleavage, then all other proteins are cleaved (PubMed:26363064). May cleave host polyadenylate-binding protein thereby inhibiting cellular translation. Does not cleave host G3BP1 (PubMed:27147742).[11] [12] Replicates genomic and antigenomic RNA by recognizing replications specific signals. Transcribes also a subgenomic mRNA by initiating RNA synthesis internally on antigenomic RNA. This sgRNA codes for structural proteins. Catalyzes the covalent attachment VPg with viral RNAs (By similarity).[UniProtKB:Q86119] Publication Abstract from PubMedNoroviruses are positive-sense single-stranded RNA viruses. They encode an NS6 protease that cleaves a viral polyprotein at specific sites to produce mature viral proteins. In an earlier study we obtained crystals of murine norovirus (MNV) NS6 protease in which crystal contacts were mediated by specific insertion of the C-terminus of one protein (which contains residues P5-P1 of the NS6-7 cleavage junction) into the peptide binding site of an adjacent molecule, forming an adventitious protease-product complex. We sought to reproduce this crystal form to investigate protease-substrate complexes by extending the C-terminus of NS6 construct to include residues on the C-terminal (P') side of the cleavage junction. We report the crystallization and crystal structure determination of inactive mutants of murine norovirus NS6 protease with C-terminal extensions of one, two and four residues from the N-terminus of the adjacent NS7 protein (NS6 1', NS6 2', NS6 4'). We also determined the structure of a chimeric extended NS6 protease in which the P4-P4' sequence of the NS6-7 cleavage site was replaced with the corresponding sequence from the NS2-3 cleavage junction (NS6 4' 2|3).The constructs NS6 1' and NS6 2' yielded crystals that diffracted anisotropically. We found that, although the uncorrected data could be phased by molecular replacement, refinement of the structures stalled unless the data were ellipsoidally truncated and corrected with anisotropic B-factors. These corrections significantly improved phasing by molecular replacement and subsequent refinement.The refined structures of all four extended NS6 proteases are very similar in structure to the mature MNV NS6-and in one case reveal additional details of a surface loop. Although the packing arrangement observed showed some similarities to those observed in the adventitious protease-product crystals reported previously, in no case were specific protease-substrate interactions observed. Structure determination of Murine Norovirus NS6 proteases with C-terminal extensions designed to probe protease-substrate interactions.,Fernandes H, Leen EN, Cromwell H Jr, Pfeil MP, Curry S PeerJ. 2015 Feb 26;3:e798. doi: 10.7717/peerj.798. eCollection 2015. PMID:25755927[13] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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