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 Structural Properties of the C Terminus of Vesicular Stomatitis Virus N Protein Dictate N-RNA Complex Assembly, Encapsidation, and RNA Synthesis

2012 ◽  
Vol 86 (16) ◽  
pp. 8720-8729 ◽  
Author(s):  
Bianca S. Heinrich ◽  
Benjamin Morin ◽  
Amal A. Rahmeh ◽  
Sean P. J. Whelan
Keyword(s):  
Gene Expression ◽  
Vesicular Stomatitis Virus ◽  
Rna Synthesis ◽  
Critical Role ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
C Terminus ◽  
Vesicular Stomatitis ◽  
Rna Complex

The vesicular stomatitis virus (VSV) nucleoprotein (N) associates tightly with the viral genomic RNA. This N-RNA complex constitutes the template for the RNA-dependent RNA polymerase L, which engages the nucleocapsid via its phosphoprotein cofactor P. While N and P proteins play important roles in regulating viral gene expression, the molecular basis of this regulation remains incompletely understood. Here we show that mutations in the extreme C terminus of N cause defects in viral gene expression. To determine the underlying cause of such defects, we examined the effects of the mutations separately on encapsidation and RNA synthesis. Expression of N together with P inEscherichia coliresults predominantly in the formation of decameric N-RNA rings. In contrast, nucleocapsid complexes containing the substitution NY415Aor NK417Awere more loosely coiled, as revealed by electron microscopy (EM). In addition, the NEF419/420AAmutant was unable to encapsidate RNA. To further characterize these mutants, we engineered an infectious cDNA clone of VSV and employed N-RNA templates from those viruses to reconstitute RNA synthesisin vitro. The transcription assays revealed specific defects in polymerase utilization of the template that result in overall decreased RNA quantities, including reduced amounts of leader RNA. Passage of the recombinant viruses in cell culture led to the accumulation of compensatory second-site mutations in close proximity to the original mutations, underscoring the critical role of structural features within the C terminus in regulating N function.

scholarly journals Activation of TBK1 and IKKε Kinases by Vesicular Stomatitis Virus Infection and the Role of Viral Ribonucleoprotein in the Development of Interferon Antiviral Immunity

2004 ◽  
Vol 78 (19) ◽  
pp. 10636-10649 ◽  
Author(s):  
Benjamin R. tenOever ◽  
Sonia Sharma ◽  
Wen Zou ◽  
Qiang Sun ◽  
Nathalie Grandvaux ◽  
...  
Keyword(s):  
Virus Infection ◽  
Vesicular Stomatitis Virus ◽  
Transcriptional Activation ◽  
Early Recognition ◽  
A549 Cells ◽  
Viral Pathogen ◽  
Antiviral State ◽  
Vesicular Stomatitis ◽  
Biochemical Analyses

ABSTRACT Mounting an immune response to a viral pathogen involves the initial recognition of viral antigens through Toll-like receptor-dependent and -independent pathways and the subsequent triggering of signal transduction cascades. Among the many cellular kinases stimulated in response to virus infection, the noncanonical IKK-related kinases TBK1 and IKKε have been shown to phosphorylate and activate interferon regulatory factor 3 (IRF-3) and IRF-7, leading to the production of alpha/beta interferons and the development of a cellular antiviral state. In the present study, we examine the activation of TBK1 and IKKε kinases by vesicular stomatitis virus (VSV) infection in human lung epithelial A549 cells. We demonstrate that replication-competent VSV is required to induce activation of the IKK-related kinases and provide evidence that ribonucleoprotein (RNP) complex of VSV generated intracellularly during virus replication can activate TBK1 and IKKε activity. In TBK1-deficient cells, IRF-3 and IRF-7 activation is significantly reduced, although transcriptional upregulation of IKKε following treatment with VSV, double-stranded RNA, or RNP partially compensates for the loss of TBK1. Biochemical analyses with purified TBK1 and IKKε kinases in vitro demonstrate that the two kinases exhibit similar specificities with respect to IRF-3 and IRF-7 substrates and both kinases target serine residues that are important for full transcriptional activation of IRF-3 and IRF-7. These data suggest that intracellular RNP formation contributes to the early recognition of VSV infection, activates the catalytic activity of TBK1, and induces transcriptional upregulation of IKKε in epithelial cells. Induction of IKKε potentially functions as a component of the amplification mechanism involved in the establishment of the antiviral state.

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.

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

scholarly journals Matrix Protein Mutant of Vesicular Stomatitis Virus Stimulates Maturation of Myeloid Dendritic Cells

2006 ◽  
Vol 80 (5) ◽  
pp. 2194-2205 ◽  
Author(s):  
Maryam Ahmed ◽  
Kristina L. Brzoza ◽  
Elizabeth M. Hiltbold
Keyword(s):  
Gene Expression ◽  
Vesicular Stomatitis Virus ◽  
Host Gene ◽  
M Protein ◽  
Type I ◽  
Type I Ifn ◽  
Host Gene Expression ◽  
Tumor Immunosurveillance ◽  
Vesicular Stomatitis

ABSTRACT Matrix (M) protein mutants of vesicular stomatitis virus have recently been used as oncolytic viruses for tumor therapies and are being developed as vaccine vectors for heterologous antigens. Because dendritic cell (DC) maturation is an important correlate of tumor immunosurveillance and vaccine efficacy, we sought to determine the ability of a recombinant M protein mutant virus (rM51R-M virus) to mature DC in vitro. We have previously shown that rM51R-M virus is defective at inhibiting host gene expression in several cell lines compared to its recombinant wild-type counterpart, rwt virus. Therefore, rM51R-M virus allows the expression of genes involved in antiviral responses, such as the type I interferon (IFN) gene. Our results demonstrate that, in contrast to the rwt virus, rM51R-M virus induced the maturation of myeloid DC (mDC) populations, as indicated by an increase in the surface expression of CD40, CD80, and CD86 as well as the secretion of interleukin-12 (IL-12), IL-6, and type I IFN. In addition, mDC infected with rM51R-M virus effectively activated naïve T cells in vitro, whereas rwt virus-infected mDC were defective in antigen presentation. The inability of rwt virus to induce mDC maturation was correlated with the inhibition of host gene expression in rwt virus-infected cells. Our studies also indicated that the production of costimulatory molecules on mDC by rM51R-M virus was dependent on the type I IFN receptor, while maturation induced by this virus was largely independent of MyD88. These data indicate that rM51R-M virus effectively stimulates the maturation of mDC and has the potential to promote effective T-cell responses to vector-expressed antigens, activate DC at tumor sites during therapy, and aid in tumor immunosurveillance and destruction.

Sign in / Sign up

Export Citation Format

Share Document