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Full length and protease domain activity of chikungunya virus nsP2 differ from other alphavirus nsP2 proteases in recognition of small peptide substrates.

ABSTRACT: Alphavirus nsP2 proteins are multifunctional and essential for viral replication. The protease role of nsP2 is critical for virus replication as only the virus protease activity is used for processing of the viral non-structural polypeptide. Chikungunya virus is an emerging disease problem that is becoming a world-wide health issue. We have generated purified recombinant chikungunya virus nsP2 proteins, both full length and a truncated protease domain from the C-terminus of the nsP2 protein. Enzyme characterization shows that the protease domain alone has different properties compared with the full length nsP2 protease. We also show chikungunya nsP2 protease possesses different substrate specificity to the canonical alphavirus nsP2 polyprotein cleavage specificity. Moreover, the chikungunya nsP2 also appears to differ from other alphavirus nsP2 in its distinctive ability to recognize small peptide substrates.

SUBMITTER: Saisawang C

PROVIDER: S-EPMC4445351 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Full length and protease domain activity of chikungunya virus nsP2 differ from other alphavirus nsP2 proteases in recognition of small peptide substrates.

Saisawang Chonticha C Sillapee Pornpan P Sinsirimongkol Kwanhathai K Ubol Sukathida S Smith Duncan R DR Ketterman Albert J AJ

Bioscience reports 20150422 3

Alphavirus nsP2 proteins are multifunctional and essential for viral replication. The protease role of nsP2 is critical for virus replication as only the virus protease activity is used for processing of the viral non-structural polypeptide. Chikungunya virus is an emerging disease problem that is becoming a world-wide health issue. We have generated purified recombinant chikungunya virus nsP2 proteins, both full length and a truncated protease domain from the C-terminus of the nsP2 protein. Enz ...[more]

PMID: 26182358

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Project description:Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for chikungunya fever. Nonstructural protein 2 (nsP2), a multifunctional protein essential for viral replication, has an N-terminal helicase region (nsP2h), which has both nucleotide triphosphatase and RNA triphosphatase activities, as well as a C-terminal cysteine protease region (nsP2p), which is responsible for nonstructural polyprotein processing. The two functional units are connected through a linker of 14 residues. Although crystal structures of the helicase and protease regions of CHIKV nsP2 have been solved separately, the conformational arrangement of the full-length nsP2 and the biological role of the linker remain elusive. Using the small-angle X-ray scattering (SAXS) method, we demonstrated that the full-length nsP2 is elongated and partially folded in solution. The reconstructed model of the structure of nsP2 contains a flexible interdomain linker, and there is no direct interaction between the two structured regions. To examine the function of the interdomain linker, we constructed and characterized a set of CHIKV mutants. The deletion of three or five amino acid residues in the linker region resulted in a modest defect in viral RNA replication and transcription but completely abolished viral infectivity. In contrast, increasing the flexibility of nsP2 by lengthening the interdomain linker increased both genomic RNA replication and viral infectivity. The enzymatic activities of the corresponding mutant proteins were largely unaffected. This work suggests that increasing the interdomain flexibility of nsP2 could facilitate the assembly of the replication complex (RC) with increased efficiency and promote virus production.IMPORTANCE CHIKV nsP2 plays multiple roles in viral RNA replication and virus-host interactions. The helicase and protease regions of nsP2 are connected through a short linker. Here, we determined that the conformation of full-length CHIKV nsP2 is elongated and that the protein is flexible in solution. We also highlight the importance of the flexibility of the interdomain of nsP2 on viral RNA synthesis and infectivity. CHIKV mutants harboring shortened linkers fail to produce infectious virus particles despite showing only relatively mild defects in genomic and subgenomic RNA synthesis. Mutations increasing the length of the interdomain linker have only mild and generally beneficial impacts on virus replication. Thus, our findings link interdomain flexibility with the regulation of viral RNA replication and infectivity of the viral genome.

Project description:Alphaviruses, such as Chikungunya virus, O’Nyong–Nyong virus, Ross River virus, have been widely known to cause fever, rash, and rheumatic diseases. In addition, several other alphaviruses, for instance Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, and Western equine encephalitis virus, potentially cause fatal encephalitis in humans. These diseases are considered as neglected tropical diseases for which there are no current antiviral therapies or vaccines available. The replication process in alphaviruses depends on four nonstructural proteins, NSP1–NSP4, which are produced as a single polyprotein. Therefore, the Alphavirus-mediated diseases in humans remain challenging among the virologists worldwide. Thus researchers are trying to find out proficient approaches, including the discovery of novel chemotherapeutic agents for the possible management and treatment of infected patients. Attempts were also made to identify an active compound against alphaviruses from natural sources. The genomes of various alphaviruses have already been revealed, and the function of proteins may be predicted by homology modeling, with the known proteins of closely related viruses. With the help of this information of protein modeling and subsequent virtual screening approach, the research teams will be able to identify few potential leads. The drug discovery against various alphaviruses is still in its early stages. Moreover, consolidating the available information and making it available for the scientific community are urgent requirements to expedite the research of potential drug discovery. The current chapter describes the techniques available to prevent Alphavirus infection and to treat Alphavirus-associated malignancies. In addition, we also discuss the recent outcomes in the fields of synthetic and natural medicinal chemistry research that were solely aimed to fight against Alphavirus infection. Thus the present chapter may also help and expedite the drug discovery and development of inhibitors against nonstructural proteins of various alphaviruses.

Dataset Information

Full length and protease domain activity of chikungunya virus nsP2 differ from other alphavirus nsP2 proteases in recognition of small peptide substrates.

Publications

Full length and protease domain activity of chikungunya virus nsP2 differ from other alphavirus nsP2 proteases in recognition of small peptide substrates.

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