Increasing Rate of Cleavage at Boundary between Non-structural Proteins 4B and 5A Inhibits Replication of Hepatitis C Virus

Herod, Morgan R.; Jones, Daniel M.; McLauchlan, John; McCormick, Christopher J.

doi:10.1074/jbc.m111.311407

Cited by 12 publications

(12 citation statements)

References 57 publications

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“…In HCV, polyprotein processing by NS3-4A is a tightly regulated process that occurs in a preferential order with rather protracted cleavage at the NS4B-5A site ( 33 – 37 ). Interestingly, mutations altering cleavage kinetics at this site are detrimental for HCV RNA replication, but the underlying mechanism has not been studied ( 38 ). Assuming that cleavage kinetics might play an important role in the biogenesis of the membranous HCV replication factory, we determined the impact of altered NS4B-5A cleavage kinetics on the formation of DMVs.…”

Section: Resultsmentioning

confidence: 99%

NS5A Domain 1 and Polyprotein Cleavage Kinetics Are Critical for Induction of Double-Membrane Vesicles Associated with Hepatitis C Virus Replication

Romero-Brey

Berger

Kallis

et al. 2015

mBio

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Induction of membrane rearrangements in the cytoplasm of infected cells is a hallmark of positive-strand RNA viruses. These altered membranes serve as scaffolds for the assembly of viral replication factories (RFs). We have recently shown that hepatitis C virus (HCV) infection induces endoplasmic reticulum-derived double-membrane vesicles (DMVs) representing the major constituent of the RF within the infected cell. RF formation requires the concerted action of nonstructural action of nonstructural protein (NS)3, -4A, protein (NS)3 -4A, -4B, -5A, and -5B. Although the sole expression of NS5A is sufficient to induce DMV formation, its efficiency is very low. In this study, we dissected the determinants within NS5A responsible for DMV formation and found that RNA-binding domain 1 (D1) and the amino-terminal membrane anchor are indispensable for this process. In contrast, deletion of NS5A D2 or D3 did not affect DMV formation but disrupted RNA replication and virus assembly, respectively. To identify cis- and trans-acting factors of DMV formation, we established a trans cleavage assay. We found that induction of DMVs requires full-length NS3, whereas a helicase-lacking mutant was unable to trigger DMV formation in spite of efficient polyprotein cleavage. Importantly, a mutation accelerating cleavage kinetics at the NS4B-5A site diminished DMV formation, while the insertion of an internal ribosome entry site mimicking constitutive cleavage at this boundary completely abolished this process. These results identify key determinants governing the biogenesis of the HCV RF with possible implications for our understanding of how RFs are formed in other positive-strand RNA viruses.

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Section: Resultsmentioning

confidence: 99%

NS5A Domain 1 and Polyprotein Cleavage Kinetics Are Critical for Induction of Double-Membrane Vesicles Associated with Hepatitis C Virus Replication

Romero-Brey

Berger

Kallis

et al. 2015

mBio

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“…Introduction of mutations at the cleavage boundaries within polyproteins is known to have effects on the rate of boundary cleavage [54, 68, 69]. For picornaviruses there is evidence from in vitro studies that the rate of processing can be largely influenced by the sequences at boundary junctions [38, 70, 71].…”

Section: Discussionmentioning

confidence: 99%

Genetic economy in picornaviruses: Foot-and-mouth disease virus replication exploits alternative precursor cleavage pathways

et al. 2017

Self Cite

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The RNA genomes of picornaviruses are translated into single polyproteins which are subsequently cleaved into structural and non-structural protein products. For genetic economy, proteins and processing intermediates have evolved to perform distinct functions. The picornavirus precursor protein, P3, is cleaved to produce membrane-associated 3A, primer peptide 3B, protease 3Cpro and polymerase 3Dpol. Uniquely, foot-and-mouth disease virus (FMDV) encodes three similar copies of 3B (3B1-3), thus providing a convenient natural system to explore the role(s) of 3B in the processing cascade. Using a replicon system, we confirmed by genetic deletion or functional inactivation that each copy of 3B appears to function independently to prime FMDV RNA replication. However, we also show that deletion of 3B3 prevents replication and that this could be reversed by introducing mutations at the C-terminus of 3B2 that restored the natural sequence at the 3B3-3C cleavage site. In vitro translation studies showed that precursors with 3B3 deleted were rapidly cleaved to produce 3CD but that no polymerase, 3Dpol, was detected. Complementation assays, using distinguishable replicons bearing different inactivating mutations, showed that replicons with mutations within 3Dpol could be recovered by 3Dpol derived from “helper” replicons (incorporating inactivation mutations in all three copies of 3B). However, complementation was not observed when the natural 3B-3C cleavage site was altered in the “helper” replicon, again suggesting that a processing abnormality at this position prevented the production of 3Dpol. When mutations affecting polyprotein processing were introduced into an infectious clone, viable viruses were recovered but these had acquired compensatory mutations in the 3B-3C cleavage site. These mutations were shown to restore the wild-type processing characteristics when analysed in an in vitro processing assay. Overall, this study demonstrates a dual functional role of the small primer peptide 3B3, further highlighting how picornaviruses increase genetic economy.

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“…Due to the lack of a tissue culture system for the human NoV, it remains to be determined whether alterations in processing order will affect human NoV replication. However, in hepatitis C virus (HCV), amino acid substitutions which enhanced cleavage at the NS4B/5A boundary were lethal, indicating that the precursor protein involved must exist for a finite amount of time (42). Future studies in our laboratory will be aimed at introducing altered polyprotein processing patterns into a cultivatable Calicivirus.…”

Section: Discussionmentioning

confidence: 99%

The P4-P2′ Amino Acids Surrounding Human Norovirus Polyprotein Cleavage Sites Define the Core Sequence Regulating Self-Processing Order

May

Viswanathan

et al. 2014

J Virol

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Noroviruses (NoV) are members of the family Caliciviridae. The human NoV open reading frame 1 (ORF1) encodes a 200-kDa polyprotein which is cleaved by the viral 20-kDa 3C-like protease (Pro, NS6) into 6 nonstructural proteins that are necessary for viral replication. The NoV ORF1 polyprotein is processed in a specific order, with "early" sites (NS1/2-3 and NS3-4) being cleaved rapidly and three "late" sites (NS4-5, NS5-6, and NS6-7) processed subsequently and less efficiently. Previously, we demonstrated that the NoV polyprotein processing order is directly correlated with the efficiency of the enzyme, which is regulated by the primary amino acid sequences surrounding ORF1 cleavage sites. Using fluorescence resonance energy transfer (FRET) peptides representing the NS2-3 and NS6-7 ORF1 cleavage sites, we now demonstrate that the amino acids spanning positions P4 to P2= (P4-P2=) surrounding each site comprise the core sequence controlling NoV protease enzyme efficiency. Furthermore, the NoV polyprotein self-processing order can be altered by interchanging this core sequence between NS2-3 and any of the three late sites in in vitro transcription-translation assays. We also demonstrate that the nature of the side chain at the P3 position for the NS1/2-3 (Nterm/NTPase) site confers significant influence on enzyme catalysis (k cat and k cat /K m ), a feature overlooked in previous structural studies. Molecular modeling provides possible explanations for the P3 interactions with NoV protease. IMPORTANCENoroviruses (NoV) are the prevailing cause of nonbacterial acute gastroenteritis worldwide and pose a significant financial burden on health care systems. Proteolytic processing of the viral nonstructural polyprotein is required for norovirus replication. Previously, the core sequence of amino acids surrounding the scissile bonds responsible for governing the relative processing order had not been determined. Using both FRET-based peptides and full-length NoV polyprotein, we have successfully demonstrated that the core sequences spanning positions P4-P2= surrounding the NS2-3, NS4-5, NS5-6, and NS6-7 cleavage sites contain all of the structural information necessary to control processing order. We also provide insight into a previously overlooked role for the NS2-3 P3 residue in enzyme efficiency. This article builds upon our previous studies on NoV protease enzymatic activities and polyprotein processing order. Our work provides significant additional insight into understanding viral polyprotein processing and has important implications for improving the design of inhibitors targeting the NoV protease.

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Increasing Rate of Cleavage at Boundary between Non-structural Proteins 4B and 5A Inhibits Replication of Hepatitis C Virus

Cited by 12 publications

References 57 publications

NS5A Domain 1 and Polyprotein Cleavage Kinetics Are Critical for Induction of Double-Membrane Vesicles Associated with Hepatitis C Virus Replication

NS5A Domain 1 and Polyprotein Cleavage Kinetics Are Critical for Induction of Double-Membrane Vesicles Associated with Hepatitis C Virus Replication

Genetic economy in picornaviruses: Foot-and-mouth disease virus replication exploits alternative precursor cleavage pathways

The P4-P2′ Amino Acids Surrounding Human Norovirus Polyprotein Cleavage Sites Define the Core Sequence Regulating Self-Processing Order

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