A quantitative infection assay for human type I, II, and III interferon antiviral activities

Voigt, Emily A.; İnankur, Bahar; Baltes, Ashley; Yin, John

doi:10.1186/1743-422x-10-224

Cited by 20 publications

(13 citation statements)

References 45 publications

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“…Further, depletion of CMTR1 resulted in increased viral spread as compared to control cells, in which viral replication was limited to discrete foci ( Figure 2C ). To determine directly whether CMTR1 contributed to viral restriction by type I IFN, we measured the ability of type I IFN to restrict infection by another IFN-sensitive virus, vesicular stomatitis virus (VSV), a negative-sense RNA virus which also encodes its own Cap 1 2’O methyltransferase (37-39), following CMTR1 depletion. In IFN-β treated Huh7 cells, CMTR1 depletion resulted in an increase in the percentage of VSV-infected cells as compared to control cells.…”

Section: Resultsmentioning

confidence: 99%

The mRNA cap 2’O methyltransferase CMTR1 regulates the expression of certain interferon-stimulated genes

Williams

Gokhale

Snider

et al. 2020

Preprint

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AbstractType I interferons (IFN) initiate an antiviral state through a signal transduction cascade that leads to the induction of hundreds of IFN-stimulated genes (ISGs) to restrict viral infection. Recently, RNA modifications on both host and viral RNAs have been described as regulators of infection. However, the impact of host mRNA cap modifications on the IFN response and how this regulates viral infection is unknown. Here, we reveal that CMTR1, an ISG that catalyzes 2’O methylation of the first transcribed nucleotide in cellular mRNA (Cap 1), promotes the protein expression of specific ISGs that contribute to the antiviral response. Depletion of CMTR1 reduces the IFN-induced protein levels of ISG15, MX1, and IFITM1, without affecting their transcript abundance. However, CMTR1 depletion does not significantly affect the IFN-induced protein or transcript abundance of IFIT1 and IFIT3. Importantly, knockdown of IFIT1, which acts with IFIT3 to inhibit the translation of RNAs lacking Cap 1 2’O methylation, restores protein expression of ISG15, MX1, and IFITM1 in cells depleted of CMTR1. Finally, we found that CMTR1 plays a role in restricting RNA virus replication, likely by ensuring the expression of specific antiviral ISGs. Taken together, these data reveal that CMTR1 is required to establish an antiviral state by ensuring the protein expression of a subset of ISGs during the type I IFN response.ImportanceInduction of an efficient type I IFN response is important to control viral infection. We show that the host 2’O methyltransferase CMTR1 facilitates the protein expression of ISGs in human cells by preventing IFIT1 from inhibiting the translation of these mRNAs lacking cap 2’O methylation. Thus, CMTR1 promotes the IFN-mediated antiviral response.

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Section: Resultsmentioning

confidence: 99%

The mRNA cap 2’O methyltransferase CMTR1 regulates the expression of certain interferon-stimulated genes

Williams

Gokhale

Snider

et al. 2020

Preprint

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“…Upon thawing, active virus in the supernate samples was inactivated by exposure to 7000 J/m 2 UVC irradiation with rocking over 20 min. Samples were then assayed for antiviral activity using published methods ( Voigt et al, 2013 ). Briefly, samples were diluted serially 1:2, incubated for 24 h over A549 human lung epithelial cells (American Type Culture Collection strain #CCL-185) responsive to human antivirals, and challenged with a DsRed2-VSV reporter virus.…”

Section: Methodsmentioning

confidence: 99%

Genome rearrangement affects RNA virus adaptability on prostate cancer cells

et al. 2015

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Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wild type gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense non-segmented RNA viruses (order Mononegavirales) have specific genome architecture: 3′ UTR – core protein genes – envelope protein genes – RNA-dependent RNA-polymerase gene – 5′ UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV) variants with the nucleocapsid (N) gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N gene translocation toward the 5′ end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC-3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function), especially on PC-3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture) adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective environments for a given gene order.

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“…Fluorescent reporter virus strains incorporating DsRed2 or DsRed-Express fluorescent genes into the fifth genomic position of VSV-lndiana were created previously using reverse genetics techniques (Swick et al, 2013;Voigt et al, 2013). The M51R reporter DsRed-Express virus strain used in this work was originally created by introducing the methionineto-arginine substitution in the 51 st position of the M protein via oligonucleotide-directed mutagenesis using the same viral genomic backbone as the other viral strains (Swick et al, 2013).…”

Section: Virus Strainsmentioning

confidence: 99%

“…Supernates collected from one-step virus infections were thawed and irradiated with 7000 J/m 2 of UVC irradiation over 20 minutes to inactivate infectious virus. The supernates were then assayed for paracrine antiviral signaling molecules as previously described (Voigt et al, 2013). Briefly, serial 1:2 dilutions were incubated over A549 cells in 96-well plates for 24 hours, and the antiviral state of the cells was then challenged by infection with VSV-DsRed2 virus at MOI 5.…”

Section: Antiviral Secretion Assaymentioning

confidence: 99%

Rapid induction and persistence of paracrine-induced cellular antiviral states arrest viral infection spread in A549 cells

2016

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The virus/host interaction is a complex interplay between pro- and anti-viral factors that ultimately determines the spread or halt of virus infections in tissues. This interplay develops over multiple rounds of infection. The purpose of this study was to determine how cellular-level processes combine to impact the spatial spread of infection. We measured the kinetics of virus replication (VSV), antiviral paracrine signal upregulation and secretion, spatial spread of virus and paracrine antiviral signaling, and inhibition of virus production in antiviral-exposed A549 human lung epithelial cells. We found that initially infected cells released antiviral signals 4-to-7 hours following production of virus. However, the subsequent rapid dissemination of signal and fast induction of a robust and persistent antiviral state ultimately led to a suppression of infection spread. This work shows how cellular responses to infection and activation of antiviral responses can integrate to ultimately control infection spread across host cell populations.

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A quantitative infection assay for human type I, II, and III interferon antiviral activities

Cited by 20 publications

References 45 publications

The mRNA cap 2’O methyltransferase CMTR1 regulates the expression of certain interferon-stimulated genes

The mRNA cap 2’O methyltransferase CMTR1 regulates the expression of certain interferon-stimulated genes

Genome rearrangement affects RNA virus adaptability on prostate cancer cells

Rapid induction and persistence of paracrine-induced cellular antiviral states arrest viral infection spread in A549 cells

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