Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2

Abstract

Chikungunya virus (CHIKV) (genus Alphavirus) has a positive-sense RNA genome. CHIKV nonstructural protein 2 (nsP2) proteolytically processes the viral nonstructural polyprotein, possesses nucleoside triphosphatase (NTPase), RNA triphosphatase, and RNA helicase activities, and induces cytopathic effects in vertebrate cells. Although alphaviral nsP2 mutations can result in a noncytotoxic phenotype, the effects of such mutations on nsP2 enzymatic activities are not well understood. In this study, we introduced a P718G (PG) mutation and selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a noncytotoxic phenotype in BHK-21 cells. Combinations of PG and either an E116K (EK) substitution or a GEEGS sequence insertion after residue T648 (5A) markedly reduced RNA synthesis; however, neither PG nor 5A prevented nsP2 nuclear translocation. Introducing PG into recombinant nsP2 inhibited proteolytic cleavage of nsP1/nsP2 and nsP3/nsP4 sites, reduced GTPase and RNA helicase activities, and abolished RNA stimulation of GTPase activity. 5A and EK modulated the effects of PG. However, only the RNA helicase activity of nsP2 was reduced by both of these mutations, suggesting that defects in this activity may be linked to a noncytotoxic phenotype. These results increase our understanding of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A and PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 and nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors and for screening inhibitors of CHIKV replication at lower biosafety levels. IMPORTANCECHIKV is a medically important pathogen that causes febrile illness and can cause chronic arthritis. No approved vaccines or antivirals are available for CHIKV. The attenuation of CHIKV is critical to the establishment of experimental systems that can be used to conduct virus replication studies at a lower biosafety level. We applied a functional selection approach to develop, for the first time, a noncytotoxic CHIKV replicon capable of persisting in human cell lines. We anticipate that this safe and efficient research tool will be valuable for screening CHIKV replication inhibitors and for identifying and analyzing host factors involved in viral replication. We also analyzed, from virological and protein biochemistry perspectives, the functional defects caused by mutations conferring noncytotoxic phenotypes; we found that all known enzymatic activities of CHIKV nsP2, as well as its RNAbinding capability, were compromised by these mutations, which led to a reduced capacity for replication. C hikungunya virus (CHIKV) is a mosquito-transmitted OldWorld alphavirus belonging to the Togaviridae family. CHIKV infection is characterized by acute illness associated with fever, skin rash, and arthralgia. Symptoms typically resolve within weeks, but frequently CHIKV in...

Differential Phosphatidylinositol-3-Kinase-Akt-mTOR Activation by Semliki Forest and Chikungunya Viruses Is Dependent on nsP3 and Connected to Replication Complex Internalization

“…The following antibodies were used according to the manufacturers' instructions: rabbit anti-phospho-Akt (S473) (catalog number 4060; Cell Signaling), rabbit anti-phospho-Akt (T308) (2965; Cell Signaling), rabbit anti-total Akt (4691; Cell Signaling), rabbit anti-phospho-4EBP1 (T37/46) (2855; Cell Signaling), rabbit antitotal 4EBP1 (9644; Cell Signaling), rabbit anti-phospho-S6 (5364; Cell Signaling), mouse anti-total S6 (2317; Cell Signaling), goat anti-actin (1616; Santa Cruz), and mouse anti-SFV-nsP2 (42) (a cocktail of 2H3, 2C7, and 3B5 at 1:1,000 each). Rabbit antisera directed against SFV-nsP3 (43), CHIKV-nsP3 (44), and CHIKV-nsP2 (45) were generated in the Merits lab and used at 1:5,000. All Western blot data are representative of at least three independent experiments.…”

Design and Validation of Novel Chikungunya Virus Protease Inhibitors

“…Cells were incubated at 37°C for 6 h, collected, and lysed, and total RNA was purified using TRIzol reagent (Thermo Scientific). Northern blotting was carried out as previously described (36). Briefly, equal amounts of total RNA samples (5 g) were denatured, separated by electrophoresis in a 1% agarose and 6.6% formaldehyde-containing denaturing gel, and transferred to a Hybond-Nϩ membrane (GE Healthcare).…”

“…Recombinant protein substrate contained the nsP2 cleavage site (P10 to P=5) from the nsP1/nsP2 junction, placed between enhanced green fluorescent protein (EGFP) and thioredoxin. The recombinant proteins were expressed and purified as described in detail earlier (16,36). Briefly, CHIKV nsP2 was expressed in Escherichia coli and the thioredoxin tag was removed by autocatalytic cleavage.…”

“…A protease inhibition assay using a recombinant protease substrate was carried out as described above except that the compounds were added to the reaction mixture at a final concentration of 1 mM; 10% DMSO was used as a solvent control. Products of protease reaction were resolved by 10% SDS-PAGE and detected using Coomassie blue staining as described earlier (35,36).…”

“…The protease activity of nsP2 is robust and can be easily analyzed (35,36). Such analysis represents the only option to provide direct proof regarding the true molecular target of the designed compounds, information regarding the exact mechanism of inhibition, and valuable input data for subsequent optimization of inhibitor structure.…”