( A) PCIF1 KO HeLa cells reconstituted with PCIF1 or an empty vector were pretreated with vehicle (0.1% BSA) or 500 U ⋅ ml −1 of IFN-β for 5 h and infected with VSV at a MOI of 3. Solvents run in the first and second dimensions are marked.Įffect of IFN-β pretreatment of cells on viral infection. Total cellular RNA was extracted and, following poly(A) selection, hydrolyzed by the indicated nucleases into monophosphates that were resolved by 2D-TLC and detected by phosphorimaging (representative image n = 3). 293T cells were infected with VSV at a MOI of 3, cellular transcription was halted by adding 10 µg ⋅ ml −1 actinomycin D at 2.5 hpi, and viral RNA was labeled by metabolic incorporation of 100 ♜i ⋅ ml −1 phosphoric acid from 3 to 7 hpi. ( B) VSV contains m 6A m at the cap-proximal nucleotide. Sites of nuclease P1 and Cap-Clip pyrophosphatase cleavage are marked. VSV mRNAs contain the conserved 5′ gene start sequence AACAG, including the m 7GpppA m cap structures synthesized by the VSV polymerase, N6-methylation at the cap-proximal first nucleotide, and 2’O methylation at the second nucleotide made by the cell. ( A) VSV mRNA cap structures present in infected cells. VSV mRNAs contain a 5′ m 7Gpppm 6A m cap structure.
RNA modifications host–pathogen interactions innate immunity nonsegmented negative-sense RNA virus rhabdovirus. We further demonstrate that the mRNA cap structures of rabies and measles viruses are also modified by PCIF1 to m 7Gpppm 6A m This work identifies a function of PCIF1 and cap-proximal m 6A m in attenuation of the host response to VSV infection that likely extends to other viruses. Cells lacking PCIF1 or expressing a catalytically inactive PCIF1 exhibit an augmented inhibition of viral replication and gene expression following interferon-β treatment.
Functional assays revealed that the PCIF1-dependent modification of VSV mRNA cap structures is inert with regard to mRNA stability, translation, and viral infectivity but attenuates the antiviral effects of the treatment of cells with interferon-β. We employed cell-based and in vitro biochemical assays to demonstrate that PCIF1 efficiently modifies VSV mRNA cap structures to m 7Gpppm 6A m and define the substrate requirements for this modification.
Using vesicular stomatitis virus (VSV), we report that the host cell N6-adenosine messenger RNA (mRNA) cap methylase, phosphorylated C-terminal domain interacting factor 1 (PCIF1), attenuates the antiviral response. To overcome the suppressive action of interferons and their effectors, viruses have evolved diverse mechanisms. Interferons induce cell-intrinsic responses associated with resistance to viral infection.
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