Mutations conferring a noncytotoxic phenotype on Chikungunya virus replicons compromise enzymatic properties of nonstructural protein 2
Autor(es): Utt Age, Das Pratyush Kumar, Varjak Margus, Lulla Valeria, Lulla Aleksei, Merits Andres
Resumo: 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, - RNA helicase activities, - 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 - selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a noncytotoxic phenotype in BHK-21 cells. Combinations of PG - 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 - nsP3/nsP4 sites, reduced GTPase - RNA helicase activities, - abolished RNA stimulation of GTPase activity. 5A - 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 underst-ing of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A - PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 - nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors - for screening inhibitors of CHIKV replication at lower biosafety levels. CHIKV is a medically important pathogen that causes febrile illness - 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 - efficient research tool will be valuable for screening CHIKV replication inhibitors - for identifying - analyzing host factors involved in viral replication. We also analyzed, from virological - 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 RNA-binding capability, were compromised by these mutations, which led to a reduced capacity for replication.
Imprenta: Journal of Virology, v. 89, n. 6, p. 3145-3162, 2015
Identificador do objeto digital: 10.1128/JVI.03213-14
Descritores: Chikungunya virus - Biochemistry ; Chikungunya virus - Biosynthesis ; Chikungunya virus - Cell ; Chikungunya virus - Genome ; Chikungunya virus - Pathogenesis ; Chikungunya virus - Protein synthesis ; Chikungunya virus - Proteins ; Chikungunya virus - RNA ; Chikungunya Virus - Virus ; Chikungunya virus - Molecular screening ; Chikungunya virus - Vaccine
Data de publicação: 2015
