Viral Polymerase-Helicase Complexes Regulate Replication Fidelity To Overcome Intracellular Nucleotide Depletion

Abstract

To date, the majority of work on RNA virus replication fidelity has focused on the viral RNA polymerase, while the potential role of other viral replicase proteins in this process is poorly understood. Previous studies used resistance to broad-spectrum RNA mutagens, such as ribavirin, to identify polymerases with increased fidelity that avoid misincorporation of such base analogues. We identified a novel variant in the alphavirus viral helicase/protease, nonstructural protein 2 (nsP2) that operates in concert with the viral polymerase nsP4 to further alter replication complex fidelity, a functional linkage that was conserved among the alphavirus genus. Purified chikungunya virus nsP2 presented delayed helicase activity of the high-fidelity enzyme, and yet purified replication complexes manifested stronger RNA polymerization kinetics. Because mutagenic nucleoside analogs such as ribavirin also affect intracellular nucleotide pools, we addressed the link between nucleotide depletion and replication fidelity by using purine and pyrimidine biosynthesis inhibitors. High-fidelity viruses were more resistant to these conditions, and viral growth could be rescued by the addition of exogenous nucleosides, suggesting that mutagenesis by base analogues requires nucleotide pool depletion. This study describes a novel function for nsP2, highlighting the role of other components of the replication complex in regulating viral replication fidelity, and suggests that viruses can alter their replication complex fidelity to overcome intracellular nucleotide-depleting conditions.

Importance: Previous studies using the RNA mutagen ribavirin to select for drug-resistant variants have highlighted the essential role of the viral RNA-dependent RNA polymerase in regulating replication fidelity. However, the role of other viral replicase components in replication fidelity has not been studied in detail. We identified here an RNA mutagen-resistant variant of the nsP2 helicase/protease that conferred increased fidelity and yet could not operate in the same manner as high-fidelity polymerases. We show that the alphavirus helicase is a key component of the fidelity-regulating machinery. Our data show that the RNA mutagenic activity of compounds such as ribavirin is coupled to and potentiated by nucleotide depletion and that RNA viruses can fine-tune their replication fidelity when faced with an intracellular environment depleted of nucleotides.

FIG 1

Chikungunya virus nsP2 regulates virus fidelity. (A) Mutation frequencies of wild-type (WT) and nsP2 (G641D) viruses in mammalian cells. (B) Virus titers of wild-type and high-fidelity variants grown in HeLa cells in the presence of the RNA mutagens ribavirin (200 μM) and 5-fluorouracil (40 μg/ml). Mean values ± the standard error of the mean (SEM) are shown (n = 3; *, P < 0.05; **, P < 0.01; **, P < 0.001 [two-way ANOVA with Bonferroni posttest]). (C) Mutation frequencies of wild type (WT) and high-fidelity variants grown in the absence (black bars) or presence (white bars) of 200 μM ribavirin. (D) Mutation frequencies by molecular cloning of wild type, high-fidelity nsp2 G641D, mutator nsp4 C483G, and the nsp2 G641D-nsp4 C483G variants (*, P = 0.05; **, P = 0.0098 [Mann-Whitney U two-tailed test]). (E) Mutation frequencies by deep sequencing as in panel D (***, P < 0.0001 [mean values ± the SEM, Mann-Whitney U two-tailed test]).

FIG 2

Regulation of virus fidelity by nsP2 is conserved among alphaviruses. (A) Amino acid alignment of the alphavirus nsp2 region. Shading indicates CHIKV position 641 and analogous residues in other alphaviruses (ONNV, O'nyong nyong virus; RRV, Ross River virus; SFV, Semliki Forest virus; VEEV, Venezuelan equine encephalitis virus). (B) Mutation frequency of Chikungunya virus wild type (WT 641G), high-fidelity 641D and 641E, and Sindbis virus wild-type 650E and low-fidelity 650G. (C) SINV wild-type and low-fidelity E650G grown in the absence (black bars) or presence (white bars) of 200 μM ribavirin. At all concentrations tested, the low-fidelity variant exhibited significant sensitivity (mean values ± the SEM, n = 3, two-tailed Student t test [*, P < 0.05; **, P < 0.01]).

FIG 3

Chikungunya virus nsP2 high-fidelity variant does not alter viral growth or polyprotein processing. (A and B) One-step viral growth kinetics measured by infectious virion production determined by TCID₅₀ (A) and extracellular viral genome copies determined by qRT-PCR (B) (mean values ± the SEM, n = 3). (C and D) BHK-21 cells were infected at an MOI of 10 with wild type and nsP2 G641D variant. At 3 h postinfection, the cells were starved in methionine-cysteine-free medium and then pulsed with 50 μCi of [³⁵S]methionine-cysteine mixture for 15 min. The cells were collected immediately after the pulse, after 15 min, or after a 45-min chase. Synthesized nonstructural polyproteins, their processing intermediates, and mature nsPs were immunoprecipitated using either CHIKV anti-nsP1 (C) and anti-nsP2 (D) antibodies. The percent cleaved represents the fraction of fully cleaved nsP1 (C) and nsP2 (D) compared to the nsP123 polyprotein. The data for one of two reproducible independent experiments are shown.

FIG 4

Purified CHIKV nsP2 G641D increases protease activity in vitro. (A) SDS-PAGE gel of purified wild-type and nsP2 G641D proteins. (B) Circular dichroism spectra of purified WT, an unrelated nsP2 mutant as a control, and nsP2 G641D. (C) Coomassie blue-stained protein gel showing the ability of purified nsP2 to cleave the nsP2/3 cleavage sites using an in vitro protease assay. Substrates were incubated for 6 min or 1 h with purified proteins. UT, no enzyme control. (D) Schematic of FRET-based protease assay. (E) Quantification of FRET-based protease assay using wild-type nsP2, nsP2 G641D, and nsP2 5A-PG as a negative control (mean values ± the SEM, n = 2).

FIG 5

Purified CHIKV nsP2 G641D has reduced helicase activity and increased NTPase activity in vitro. (A) One-hour endpoint helicase assay. Purified proteins were incubated with dsRNA subtract for 1 h in the presence or absence of ATP and Mg²⁺. nsP2 5A-PG was used as a negative control (lanes 6 and 7). (B) Quantification of the data in panel A. (C) Kinetic analysis of wild-type nsP2 and G641D helicase activity. Purified proteins and dsRNA substrates were incubated at 30°C, and aliquots were removed at the indicated time points. (D) Quantification of kinetic analysis in panel C. (E) NTPase activity of wild-type nsP2 (white bars) and nsP2 5A-PG (black bars) as a negative control. (F) NTPase activity of wild-type nsP2 (white bars) and nsP2 G641D (black bars). Mean values ± the SEM are shown (n = 5, Student t test [*, P < 0.05]).

FIG 6

CHIKV high-fidelity replication complexes have increased in vitro RNA polymerase activity and are less sensitive to nucleotide depletion. (A) Immunoblot showing subcellular localizations of viral proteins (n = 2). T, total extract; S, soluble fraction; P, pellet. (B) Autoradiogram of time course of in vitro replication for CHIKV wild type, nsp4 C483Y, nsp2 G641D, and DM. (C) Quantification subgenomic RNA synthesis from in vitro replication assay in panel B (mean values ± the SEM, n = 3). (D) In vitro replication assay in the presence of 10, 100, and 1,000 μM NTP. Membranes were incubated for 3 h at 37°C. (E) Quantification of subgenomic synthesis in nucleotide depleted conditions in panel D (Student t test, n = 3 [*, P < 0.05; **, P < 0.01]).

FIG 7

CHIKV high-fidelity variants are resistant to nucleotide biosynthesis inhibitors and can be rescued with exogenous nucleotides. (A) Viruses were grown in the presence of 200 μM ribavirin and either left untreated (no exogenous nucleosides) or complemented with 10 or 100 μg of guanosine/ml or 20 μg of uracil/ml. (B) Viruses were grown in the presence of mycophenolic acid and either left untreated (no exogenous nucleosides) or complemented with 30 μg of guanosine/ml or 20 μg of uracil/ml. (C) Viruses were grown in the presence of brequinar and either left untreated (no exogenous nucleosides) or complemented with 20 μg of uracil/ml or 30 μg of guanosine/ml. For all panels, mean values ± the SEM are shown (n = 3; *, P < 0.05 [two-way analysis of variance with Bonferroni posttest]).