Oxidative stress influences positive strand RNA virus genome synthesis and capping

Abstract

Flaviviruses are 5' capped positive-stranded RNA viruses that replicate their genomes within endoplasmic reticulum-derived vesicles. Flaviviruses are well known to induce oxidative stress late in infection but it is unknown if oxidative stress plays a positive role in the viral RNA replication cycle. We therefore examined how oxidation affects flavivirus RNA replication. We found that antioxidant treatment reduced virus production, reduced the viral positive-to-negative strand RNA ratio, and resulted in the accumulation of uncapped positive-sense viral RNAs. Treatment of the NS5 RNA capping enzyme in vitro with oxidizing agents enhanced guanylyltransferase activity, indicating that the guanylyltransferase function of the flavivirus NS5 RNA capping enzyme is activated by oxidative conditions. Antioxidant treatment also reduced alphavirus RNA replication and protein expression while enhancing nsP1 capping activity. These findings suggest that RNA viruses may utilize oxidative stress induced during infection to help temporally control genome RNA capping and genome replication.

Keywords: Flavivirus; Guanylyltransferase; Oxidation; RNA capping.

Fig. 1

Antioxidants reduce flavivirus RNA replication. (A) The antioxidant BHA blocks Kunjin virus (KUNV) induced oxidative stress. BHK cells were treated or infected as shown for 24 h, then incubated with the fluorescent oxidation sensor CM-H₂DCFDA (green) and imaged. (B) BHA reduces KUNV replication. BHK cells were infected with KUNV (MOI=0.01) and treated with the indicated concentration of BHA. Supernatants were collected at 48 h, RNA extracted, and qRT-PCR analysis performed to determine PFU equivalents. 100 PFU/ml (2 logs) is the limit of detection in these assays. C_q values were converted to PFU equivalents using a standard curve. A pairwise t test with Bonferroni correction showed that 300 and 400 =M BHA treatments were significantly different than DMSO with p<0.01. (n=3) (C) BHA reduces KUNV replication in different cell species. Huh7, BHK, or C6/36 cells were infected with KUNV (MOI=0.01) at mock treated or treated with 200 μM BHA for 72 h. Viral particles were quantified by KUNV plaque assay. Two-way ANOVA demonstrated a significant reduction in viral titer upon treatment with BHA with p<0.0001 in each cell type. (n=3) (D) Antiviral effect of BHA on DENV-2 replicon replication. BHK cells containing a stable DENV-2 replicon expressing Renilla luciferase were treated with the indicated concentration of BHA. Renilla luciferase and CellTiter-Glo signals were measured at 72 h post BHA addition, and the signal was reported as a percentage of the untreated sample. (n=3).

Fig. 2

BHA reduces positive strand RNA synthesis and capping. (A) Treatment of cells with BHA alters the ratio of positive to negative strand RNAs in infected cells. BHK cells were infected with KUNV (MOI=0.1) and treated with 200 μM BHA for 48 h. Total RNA was extracted and strand specific qRT-PCR was performed. The ratio of average positive and negative strand C_q values for each sample are shown and average C_q values are in the text. An unpaired t test with equal variance yielded p=0.0129, demonstrating a significant reduction in positive strand KUNV RNA in BHA as compared to DMSO. (n=3) (B) Treatment with BHA increased the abundance of uncapped viral RNA. BHK cells were infected with KUNV as above and treated with DMSO or BHA and total RNA extracted at 48 h post infection. Capped and uncapped RNAs were fractionated by immunoprecipitation from total RNA samples, and KUNV and cellular GAPDH RNAs were detected from the uncapped fraction by qRT-PCR. An unpaired t test with equal variance yielded p=0.048 for uncapped KUNV RNA but insignificant differences for GAPDH, demonstrating a significant increase in uncapped KUNV RNA but not GAPDH RNA with BHA treatment. (n=3).

Fig. 3

Oxidizing agents increase capping enzyme guanylation activity. (A) DENV-2 NS5 capping enzyme guanylation is enhanced with oxidizing agents. Recombinant DENV-2 capping enzyme was incubated with 1 μM GTP-ATTO 680 and the indicated concentration of H₂O₂ or diamide for 4 h at 37 °C, then resolved on a 12% polyacrylamide gel. Protein guanylation activity was measured by ATTO-680 signal tracking with protein. CIP was used as a negative control in these experiments. Representative gels are shown. (n=3) (B) Oxidizing and reducing agents do not significantly affect DENV-2 capping enzyme GTP binding. 500 nM DENV-2 capping enzyme was incubated with 10 nM GTP-Bodipy, then the indicated concentrations of TCEP or H₂O₂ were added and the reaction was incubated at 22 °C for 1 h. Fluorescence polarization signal was detected and plotted against concentration of H₂O₂ or TCEP. (n=3).

Fig. 4

Oxidation induces NS5 dimerization via disulfide bond formation. (A) Oxidation induces NS5 dimerization. Recombinant DENV-2 capping enzyme was incubated with 1 μM GTP-ATTO 680 and the indicated concentration of H₂O₂ or diamide for 4 h at 37 °C. Samples were boiled in Laemmli buffer with or without 2.5% β-mercaptoethanol and resolved on 12% polyacrylamide gels. Protein guanylation activity was measured by GMP-ATTO 680 signal tracking with protein. A representative gel is shown. (B) The NS5 guanylation reaction is concentration dependent. 30 pMol of dengue NS5 capping enzyme was incubated with 1 μM GTP-ATTO 680 in 100 μl, 30 μl, and 10 μl volumes for 4 h at 37 °C. At the end of the incubation sample volumes were all increased to 100 μl, Laemmli buffer added, and proteins boiled and resolved on 12% SDS-PAGE gels. GMP-ATTO 680 guanylation signals for each concentration were normalized for protein loading and the 3 μM dengue NS5 guanylation signal was set to 1. One-way ANOVA analysis yielded a significant different between the relative guanylation signal at 0.3 and 1 μM with p=0.0041 as compared to 3 μM concentration. (n=3) (C) Mutation of cysteine residues alters NS5 oxidative activation. 3 μM of mutant DENV-2 NS5 capping enzyme proteins were incubated with 1 μM GTP-ATTO 680 in the presence or absence of 1 mM H₂O₂ for 4 h at 37 °C. Reactions were resolved on 12% SDS-PAGE gels and GMP-ATTO 680 signal detected. A representative gel is shown. (n=3).

Fig. 5

Met219 influences oxidative activation of NS5 guanylyltransferase function. (A) Oxidation of DENV-2 capping enzyme increases proportion of methionine sulfoxide and sulfone species. Wild-type DENV-2 capping enzyme was mock treated or treated with 1 mM H₂O₂ for 1 h at 37 °C and resolved on SDS-PAGE gel. Proteins were trypsin digested, extracted from gels, and subjected to Orbitrap mass spectrometry. The proportion of Met219 sulfoxide and sulfone species for each sample was determined and compared to unmodified Met219. A representative experiment showing the proportion of Met219 sulfoxide and sulfone species for each sample compared to unmodified Met219 is shown. (B) Mutation of oxidation sensitive residues alters response to oxidizing agents. 3 μM of wild-type or mutant NS5 proteins were incubated with 1 mM H₂O₂ for 4 h, then resolved on SDS-PAGE gel and GMP-ATTO 680 detected. Guanylation signal was normalized to Commassie blue staining. A one-way ANOVA yielded significant difference with p=0.00693 and a pairwise t test with a Bonferroni comparison yielded significant difference between WT M219E with p=0.00347. (n=3) (C) Mutants mimicking Met219 methionine sulfoxide and sulfone have altered thermal stability. 30 μM of wild-type or mutant NS5 proteins were denatured in the presence of Krypton Infrared Protein stain, and melting temperature was determined by maximum d(RFU)/dT value. A one-way ANOVA yielded significant difference between samples with p=0.026 and a pairwise t test with a bonferroni comparison yielded significant difference between WT and each mutant with p<0.001. (n=3) (D) Oxidation mimicking mutations of NS5 Met219 block replicon replication. Mutant West Nile virus replicon plasmids that express firefly luciferase as a function of viral positive strand RNA abundance were transfected into BHK cells, and luciferase activity was determined 24 h later on a Victor X5 platereader. A pairwise t test with Bonferroni correction yielded a significant difference for each mutant compared to the wild type with p<0.001 in each case. Additionally, each mutant was significantly different than M219Q with p<0.001. (n=3).

Fig. 6

Alphavirus replication and nsP1 guanylyltransferase activity is sensitive to oxidation. (A) Antioxidant treatment reduces alphavirus replication. BHK cells treated with the indicated concentrations of BHA were infected with firefly luciferase expressing Sindbis virus for 24 h. Firefly luciferase activity was detected in infected cell lysates on a Victor X5 platereader as a measure of viral propagation (n=3). (B) Oxidants and antioxidants agents alter Sindbis virus mediated mCherry expression. BHK cells were treated with DMSO, 200 μM BHA, or 200 μM H₂O₂ and infected with mCherry expressing Sindbis virus. At 24 h post infection fluorescent images were collected. (C) Alphavirus nsP1 guanylyltransferase is activated by oxidant treatment. 3 μM recombinant VEEV nsP1 protein was incubated with 1 μM GTP-ATTO 680 in the presence of the indicated concentration of diamide for 4 h at 37 °C. Samples were boiled in Laemmli buffer with β-mercaptoethanol and resolved on 12% polyacrylamide gels. A representative gel is shown. (n=3).