scholarly journals Methylation of viral mRNA cap structures by PCIF1 attenuates the antiviral activity of interferon-β

2021 ◽  
Vol 118 (29) ◽  
pp. e2025769118
Author(s):  
Michael A. Tartell ◽  
Konstantinos Boulias ◽  
Gabriela Brunsting Hoffmann ◽  
Louis-Marie Bloyet ◽  
Eric Lieberman Greer ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Host Response ◽  
Interferon Β ◽  
Β Cells ◽  
Antiviral Effects ◽  
Biochemical Assays ◽  
Vesicular Stomatitis ◽  
Dependent Modification ◽  
Resistance To Viral Infection

Interferons induce cell-intrinsic responses associated with resistance to viral infection. To overcome the suppressive action of interferons and their effectors, viruses have evolved diverse mechanisms. 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. We employed cell-based and in vitro biochemical assays to demonstrate that PCIF1 efficiently modifies VSV mRNA cap structures to m7Gpppm6Am and define the substrate requirements for this modification. 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. Cells lacking PCIF1 or expressing a catalytically inactive PCIF1 exhibit an augmented inhibition of viral replication and gene expression following interferon-β treatment. We further demonstrate that the mRNA cap structures of rabies and measles viruses are also modified by PCIF1 to m7Gpppm6Am. This work identifies a function of PCIF1 and cap-proximal m6Am in attenuation of the host response to VSV infection that likely extends to other viruses.

scholarly journals Messenger RNA cap methylation by PCIF1 attenuates the interferon-β induced antiviral state

2020 ◽  
Author(s):  
Michael A. Tartell ◽  
Konstantinos Boulias ◽  
Gabriela Brunsting Hoffmann ◽  
Eric Lieberman Greer ◽  
Sean P. J. Whelan
Keyword(s):  
Gene Expression ◽  
Viral Infection ◽  
Vesicular Stomatitis Virus ◽  
Messenger Rna ◽  
Rna Stability ◽  
Innate Immune ◽  
Interferon Β ◽  
Antiviral State ◽  
Vesicular Stomatitis

AbstractInterferons induce cell intrinsic responses associated with resistance to viral infection. To overcome the suppressive action of interferons and their downstream effectors viruses have evolved diverse mechanisms. Working with vesicular stomatitis virus (VSV) we report a role for the host cell N6-adenosine mRNA cap-methylase, phosphorylated C-terminal domain interacting factor 1 (PCIF1), in attenuating the antiviral activity of interferon-β. Using cell based andin vitrobiochemical assays we demonstrate that PCIF1 efficiently modifies VSV mRNA cap structures to m7Gpppm6Am, and we identify thecis-acting elements required for this modification. Under basal conditions, N6-methylation of VSV mRNA cap structures is functionally inert with regard to mRNA stability, translation and viral infectivity. Induction of an antiviral state by treatment of cells with interferon-β prior to infection uncovered a functional role for PCIF1 in attenuation of the antiviral response. Cells lacking PCIF1 or expressing a catalytically inactive PCIF1, exhibit an augmented effect of interferon-β in the inhibition of viral replication and gene expression. This work identifies a function of PCIF1 and cap-proximal m6Amin attenuation of the host response to VSV infection that likely extends to other viruses.SignificanceThe cap structure present at the 5’ end of eukaryotic mRNAs regulates RNA stability, translation, and marks mRNA as self, thereby impeding recognition by the innate immune system. Cellular transcripts beginning with adenosine are additionally modified at the N6 position of the 2’-O methylated cap-proximal residue by the methyltransferase PCIF1 to m7Gpppm6Am. We define a function for this N6-adenosine methylation in attenuating the interferon-β mediated suppression of viral infection. Cells lacking PCIF1, or defective in its enzymatic activity, augment the cell intrinsic suppressive effect of interferon-β treatment on vesicular stomatitis virus gene expression. VSV mRNAs are efficiently methylated by PCIF1, suggesting this contributes to viral evasion of innate immune suppression.

scholarly journals Construction of a synthetic messenger RNA encoding a membrane protein.

1983 ◽  
Vol 96 (5) ◽  
pp. 1464-1469 ◽  
Author(s):  
J L Rubenstein ◽  
T G Chappell
Keyword(s):  
G Protein ◽  
Messenger Rna ◽  
In Vitro Transcription ◽  
Membrane Insertion ◽  
Wheat Germ Extract ◽  
E Coli ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Synthetic Mrna

We have synthesized microgram quantities of a functional eucaryotic mRNA by in vitro transcription. For this purpose, we constructed a plasmid in which the Escherichia coli lactose promoter was 5' to the vesicular stomatitis virus (VSV) G protein gene (Rose, J. K., and C. J. Gallione, 1981, J. Virol., 39:519-528). This DNA served as the template in an in vitro transcription reaction utilizing E. coli RNA polymerase. The RNA product was capped using the vaccinia guanylyltransferase. A typical preparation of the synthetic G mRNA was equivalent to the amount of G mRNA that can be isolated from approximately 10(8) VSV-infected cells. This synthetic mRNA was translated by a wheat germ extract in the presence of microsomes, producing a polypeptide that was indistinguishable from G protein in its size, antigenicity, degree of glycosylation, and its membrane insertion. This technique should aid in identifying features needed by proteins for insertion into membranes.

scholarly journals Nanoparticles of a different source induce different patterns of activation in key biochemical and cellular components of the host response

2009 ◽  
Vol 6 (41) ◽  
pp. 1213-1221 ◽  
Author(s):  
A.L. Guildford ◽  
T. Poletti ◽  
L.H. Osbourne ◽  
A. Di Cerbo ◽  
A.M. Gatti ◽  
...  
Keyword(s):  
Host Response ◽  
Mononuclear Cells ◽  
Inflammatory Cells ◽  
Clot Formation ◽  
Macrophage Phenotype ◽  
Industrial Processing ◽  
Biochemical Assays ◽  
Factor Α ◽  
Cellular Components

Nanoparticulate materials are produced by industrial processing or engineered for specific biomedical applications. In both cases, their contact with the human body may lead to adverse reactions. Most of the published papers so far have focused on the cytotoxic effects of nanoparticles (NPs). Instead, the present in vitro study investigates the effect of different types of NP on key components of the host response such as clot formation and the inflammatory cells. The different NPs were pre-conditioned with platelet-rich human plasma for 30 min and then incubated with the blood mononuclear cells for 20 hours. The potential of the different NPs to induce clot formation, platelet activation and monocyte/macrophage differentiation was assessed by morphological analysis, immunocytochemistry and biochemical assays. The data showed that nanoparticulate materials based on antimony, silver and nickel were capable of promoting the polymerization of fibrin and the aggregation and fragmentation of platelets, leading to a moderately activated monocyte phenotype. This process was more pronounced in the case of antimony- and silver-based NPs that share a similar size and round-shaped morphology. Conversely, NPs of cobalt, titanium and iron appeared to stimulate cells to acquire a macrophage phenotype able to secrete higher levels of tumour necrosis factor α, a pro-inflammatory cytokine. Therefore, the present study provides clear indications about the subtle and adverse effects that the invasion of these materials may produce in the cardiovascular system and in vital organs.

Studies on the in vivo and in vitro messenger RNA species of vesicular stomatitis virus

Virology ◽  
1975 ◽  
Vol 67 (2) ◽  
pp. 463-473 ◽  
Author(s):  
Sue A. Moyer ◽  
Marvin J. Grubman ◽  
Ellie Ehrenfeld ◽  
Amiya K. Banerjee
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Messenger Rna ◽  
Vesicular Stomatitis
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