Positive-sense RNA viruses reveal the complexity and dynamics of the cellular and viral epitranscriptomes during infection

Abstract

More than 140 post-transcriptional modifications (PTMs) are known to decorate cellular RNAs, but their incidence, identity and significance in viral RNA are still largely unknown. We have developed an agnostic analytical approach to comprehensively survey PTMs on viral and cellular RNAs. Specifically, we used mass spectrometry to analyze PTMs on total RNA isolated from cells infected with Zika virus, Dengue virus, hepatitis C virus (HCV), poliovirus and human immunodeficiency virus type 1. All five RNA viruses significantly altered global PTM landscapes. Examination of PTM profiles of individual viral genomes isolated by affinity capture revealed a plethora of PTMs on viral RNAs, which far exceeds the handful of well-characterized modifications. Direct comparison of viral epitranscriptomes identified common and virus-specific PTMs. In particular, specific dimethylcytosine modifications were only present in total RNA from virus-infected cells, and in intracellular HCV RNA, and viral RNA from Zika and HCV virions. Moreover, dimethylcytosine abundance during viral infection was modulated by the cellular DEAD-box RNA helicase DDX6. By opening the Pandora's box on viral PTMs, this report presents numerous questions and hypotheses on PTM function and strongly supports PTMs as a new tier of regulation by which RNA viruses subvert the host and evade cellular surveillance systems.

Figure 1.

Venn diagram showing unique and overlapping PTM profiles of total RNA extracts obtained from mock- and virus-infected Huh7 cells. Cultured cells were mock-infected with medium or infected with ZIKV, DENV, HCV and PV, and harvested at specific time points post-infection (see ‘Materials and Methods’ section). Because HIV-1 does not replicate in Huh7 cells, no corresponding data are included in this diagram. Instead, data obtained from HIV-1 grown in 293T cells are provided in Supplementary Table S4 and Figure S5. For the sake of simplicity, the diagram was based exclusively on the presence or absence of each PTM in the sample, whereas comprehensive quantitative data, which may help appreciate any up- and downregulation, are provided separately in Supplementary Table S2. Full PTM names are listed in Supplementary Table S3.

Figure 2.

Venn diagram generated from PTM profiles of total RNA extracts from mock-infected, ZIKV-infected, arsenite-treated and poly(I:C)-transfected Huh7 cells (see ‘Materials and Methods’ section for details). The diagram was based exclusively on the presence or absence of each PTM in the samples, whereas comprehensive quantitative data, which may help appreciate any up- and downregulation, are provided separately in Supplementary Table S10. Full PTM names are listed in Supplementary Table S3.

Figure 3.

Venn diagrams generated from PTM profiles of viral RNAs isolated from whole cell lysates of infected cells (top) and virions harvested from the culture media (bottom, see ‘Materials and Methods’ section for details). Only the ss(+) RNA viruses grown in Huh7 cells are included in the diagrams to preserve the comparison consistency. Data obtained from isolated HIV-1 in cell lysate and virions are provided separately in Supplementary Table S8. These diagrams were based exclusively on the presence/absence of each PTM, while comprehensive data displaying abundance variations are reported separately in Supplementary Table S9. Full PTM names are listed in Supplementary Table S3.

Figure 4.

DDX6 modulates the presence of m⁵Cm and m⁴₄C during viral infection. (A) The abundance of m⁵Cm and m⁴₄C are presented above each western and northern blot. These levels show changes in m⁵Cm and m⁴₄C in viral infected cells without arsenite treatment. (B) Western blots show expression of viral protein (ZIKV and DENV capsid, HCV core and PV 3D proteins), DDX6, Flag MS2 and GAPDH in Huh7 cells transfected with control (siGL2) and DDX6-specific (siDDX6) siRNAs, and 3xFlag-Bacterial Alkaline Phosphatase (BAP) or 3xFlag-DDX6 resistant to siRNA targeting (DDX6Δsi). (*) indicates detection of 3xFlag- DDX6Δsi by using the anti-DDX6 antibody. (C) Northern blots show abundance of viral genomic RNA and actin mRNA. (D) Quantification of viral RNA on northern blot. Viral RNA was normalized to actin mRNA, and a percentage relative to infected Huh7 cells transfected with siGL2 is shown. The quantitative data were obtained from at least three independent experiments, and SD of mean is shown. (E) Titers of virions released into the extracellular media. The viral titers of ZIKV and PV were calculated by plaque assay (PFU/ml), and DENV and HCV by TCID₅₀ assay (TCID₅₀/ml). Titers are from at least three independent experiments, and SD of mean is shown. (F) The abundance of m⁵Cm and m⁴₄C in uninfected cells transfected with the respective siRNAs and 3xFlag plasmids, and treated with 1 mM sodium arsenite for 30 min at 37°C. The color gradient provides their abundances relative to the combined abundances of the canonical ribonucleotides, calculated as described in ‘Materials and Methods’ section. Any color change along the gradient carries a P < 0.05 statistical significance. Full PTM names are listed in Supplementary Table S3.