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. 2016 Dec:499:259-266.
doi: 10.1016/j.virol.2016.09.022. Epub 2016 Oct 4.

Evasion of early innate immune response by 2'-O-methylation of dengue genomic RNA

David C Chang  1 , Long T Hoang  2 , Ahmad Nazri Mohamed Naim  2 , Hongping Dong  1 , Mark J Schreiber  1 , Martin L Hibberd  3 , Min Jie Alvin Tan  4 , Pei-Yong Shi  5
Affiliations

Evasion of early innate immune response by 2'-O-methylation of dengue genomic RNA

David C Chang et al. Virology. 2016 Dec.

Abstract

Dengue virus (DENV) is the most prevalent mosquito-borne virus pathogen in humans. There is currently no antiviral therapeutic or widely available vaccine against dengue infection. The DENV RNA genome is methylated on its 5' cap by its NS5 protein. DENV bearing a single E216A point mutation in NS5 loses 2'-O-methylation of its genome. While this mutant DENV is highly attenuated and immunogenic, the mechanism of this attenuation has not been elucidated. In this study, we find that replication of this mutant DENV is attenuated very early during infection. This early attenuation is not dependent on a functional type I interferon response and coincides with early activation of the innate immune response. Taken together, our data suggest that 2'-O-methylation of DENV genomic RNA is important for evasion of the host immune response during the very early stages of infection as the virus seeks to establish infection.

Keywords: Dengue virus; Innate immune response; Methyltransferase; NS5.

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Figures

Fig. 1.
Fig. 1
Absence of 2′-O-methylation is not lethal to viral replication but causes severe attenuation in interferon-competent cells. (A and B) BHK-21 cells electroporated with DENV RNA containing the 2′-O-MTase mutation display a slower progressive viral replication compared to those electroporated with WT DENV RNA, as demonstrated by immunofluorescence analysis of the DENV E protein. Replication of the 2′-O-MTase mutant DENV is significantly attenuated compared to WT DENV from day 1 post-infection, before catching up to statistically similar levels by day 5 post-infection. *Student’s t-test, p<0.01, between 2′-O-MTase mutant and WT DENV RNA. (C) A time course study reveals the replication kinetics of WT and 2′-O-MTase mutant DENV infection with the same MOI of A549 and Vero cells. Virus titers at each time point were collected and quantified by a plaque assay. Statistical significant differences are discussed in the text of the Results section.
Fig. 2.
Fig. 2
Lag in replication of 2′-O-MTase mutant RNA during the first 16 h of infection. DENV RNA copy number in A549 cells infected with wild type DENV increased exponentially from about 4 h post infection. In contrast, cells infected with 2′-O-MTase mutant DENV remained relatively stagnant in the first 16 h post infection before increasing, suggesting a severe attenuation in early replication. Data comes from 6 biological replicates *Student's t-test, p<0.01, between 2′-O-MTase mutant and WT .
Fig. 3.
Fig. 3
Innate immune and pro-inflammatory pathways are most significantly enriched canonical pathways after infection with the 2′-O-MTase mutant DENV compared to WT DENV. (A) Heat map depicting the expression pattern of the 1133 transcripts that are differentially expressed in 2′-O-MTase mutant DENV-infected samples in comparison with mock, heat-inactivated (HI) WT DENV- and WT DENV-infected samples. Transcripts with high expression levels are shown in red and those with low expression levels are in green. (B) Microarray analyses of differentially expressed genes between 12 and 24 h post infection with 2′-O-MTase mutant and wild-type DENV reveal that a significant enrichment in genes involved in the innate immune and type I interferon pathways. The p value was calculated by hypergeometric test with Benjamini-Hochberg (B-H) correction to investigate if a pathway was significantly up- or down-regulated. A p value of less than or equal to 0.05 indicates a pathway was less likely to be detected by chance. The ratio shows the proportion of genes in the gene list of interest that are involved in a certain pathway. (C) Many of the differentially expressed genes (2–fold difference, FDR <0.05) are involved in the innate immune and pro-inflammatory responses, as indicated by their classification in these canonical pathways.
Fig. 4
Fig. 4
The 2′-O-MTase mutant DENV induces a significant early innate immune response. (A) Heat map showing the normalized expression pattern of 48 genes that were selected for validation by qPCR. Genes with high expression levels were shown in red and those with low expression levels were in green. (B) Heat map showing the expression pattern of 43 of those genes as determined by qPCR. The expression levels were calculated using the delta-delta method. Genes with high expression levels were shown in red and those with low expression levels were in green. (C) The 2′-O-MTase mutant DENV induces a significant early innate immune response from as early as 2 h post-infection. In contrast, the wild-type DENV does not trigger an innate immune response until after 12 h post infection. The Loess method was used to fit the relative abundance of each group across each time point post-infection. It estimates the mean value non-parametrically for each data point of each data set (solid line) and the 95% confidence interval (CI) values of the mean (dashed lines). The time points at which the 95% CI lines of the 2′-O-MTase mutant DENV (green) and WT DENV (red) do not intersect denote instances of significant differences between them.
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