Endomembrane targeting of human OAS1 p46 augments antiviral activity
- Department of Immunology, School of Medicine, University of Washington, United States
- Center for Innate Immunity and Immune Disease, University of Washington, United States
- Benaroya Research Institute at Virginia Mason, United States
- Roslin Institute, University of Edinburgh, United Kingdom
- Department of Molecular Genetics and Microbiology, School of Medicine, University of New Mexico, United States
- Department of Microbial Infection and Immunity, College of Medicine, The Ohio State University, United States
- Department of Microbiology, School of Medicine, University of Washington, United States
- Department of Microbiology and Immunology, University of Texas Medical Center, United States
- Department of Basic Sciences, College of Osteopathic Medicine, Touro University Nevada, United States
- Department of Infectious Disease, Virginia Mason Medical Center, United States
- Department of Medicine, Section of Infectious Diseases, University of Washington, United States
- Cancer Virology Program, University of Pittsburgh Cancer Institute, University of Pittsburgh, United States
- Center for Immunity and Immunotherapies, Seattle Children's Research Institute, United States
- MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, United Kingdom
Figures
Figure 1 with 2 supplements
The p46 isoform of OAS1 is targeted to the endomembrane system.
(A) Differential C-terminal splicing of OAS1 creates isoform diversity. (B) Immunoblot analysis of OAS1 isoform expression across cell lines treated with 1000 U/mL rIFNβ for 24 hr (n=3). (C) …
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Figure 1—source data 1
Uncropped gels for the associated panels in Figure 1 and Figure 1—figure suppleent 2.
- https://cdn.elifesciences.org/articles/71047/elife-71047-fig1-data1-v2.zip
Figure 1—figure supplement 1
Alignment and sequence analysis of OAS1.
(A) Sequence alignment of human OAS1 isoforms. (B) Distribution of allelic frequency of rs10774671 (A/G) in the human population (http://popgen.uchicago.edu/ggv/) (Marcus and Novembre, 2017). (C) …
Figure 1—figure supplement 2
Biochemical and confocal analysis of human OAS1 isoforms.
(A) Human OAS1 isoforms with last 10 amino acids. (B) Immunoblot analysis of OAS1 expression in A549 and PH5CH8 cells following treatment with rIFNβ (1000 U/mL) or Sendai virus (100 HAU/mL) for the …
Figure 2 with 1 supplement
OAS1 isoforms are differentially antiviral.
(A) Immunoblot analysis of p42 and p46 in OAS1 KO 293 T cells at 24 hr post transfection. (B) Quantification of EMCV 5′UTR by RT-qPCR at 24 hr post-infection with EMCV (MOI=0.001) in OAS1 KO 293 T …
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Figure 2—source data 1
Uncropped gels for the associated panels in Figure 2.
- https://cdn.elifesciences.org/articles/71047/elife-71047-fig2-data1-v2.zip
Figure 2—figure supplement 1
Antiviral activity of human OAS1 isoforms.
Figure 3 with 1 supplement
OAS1 isoforms require catalytic and RNase L activity.
(A) Expression of OAS1 p42 and p46 along with their corresponding catalytic mutants (250 ng) at 24 hr post transfection in OAS1 KO 293 T cells. (B) Quantification of EMCV 5′UTR by RT-qPCR in OAS1 KO …
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Figure 3—source data 1
Uncropped gels for the associated panels in Figure 3 and Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/71047/elife-71047-fig3-data1-v2.zip
Figure 3—figure supplement 1
RNase L-dependent activity of OAS1 isoforms.
(A) Expression of OAS1 p42 and p46 along with their corresponding catalytic mutants at 24 hr post transfection in OAS1 KO 293 T cells. (B) Quantification of EMCV 5′UTR by RT-qPCR from OAS1 KO 293 T …
Figure 4 with 1 supplement
Endomembrane targeting of OAS1 p46 through the CaaX motif enhances access to viral RNA.
(A) Confocal micrographs from mock or EMCV infected (MOI=0.001, 12 hr) OAS1 KO Huh7 cells ectopically expressing p42 or p46 and stained with anti-OAS1 (green) and anti-dsRNA (magenta) antibodies and …
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Figure 4—source data 1
Uncropped gels for the associated panels in Figure 4 and Figure 4—figure suppleent 1.
- https://cdn.elifesciences.org/articles/71047/elife-71047-fig4-data1-v2.zip
Figure 4—figure supplement 1
Role of CaaX motif in OAS1 localization.
(A) Immunoblot of RNase L expression in untreated cell lines. (B) Representative confocal micrographs of OAS1 KO Huh7 cells expressing OAS1 p46 and OAS1 common +CTIL stained with anti-OAS1 (green) …
Figure 5 with 1 supplement
Combined effects of CaaX motif, C-terminus length and oligomerization domain confer differential antiviral activity of OAS1 isoforms.
(A) C-termini of OAS1 p46 alanine substitution mutants 1–9. (B) Immunoblot of control EV, p46, p46ATIL, and p46 alanine substitution mutants 1–9 in OAS1 KO 293 T cells at 24 hr post transfection. (C)…
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Figure 5—source data 1
Uncropped gels for the associated panels in Figure 5 and Figure 5—figure suppleent 1.
- https://cdn.elifesciences.org/articles/71047/elife-71047-fig5-data1-v2.zip
Figure 5—figure supplement 1
Enhanced antiviral activity of OAS1 p46 is mediated by p46-specific coding region.
(A) Quantification of EMCV 5´UTR copies by RT-qPCR from OAS1 KO 293 T cells expressing control EV, p46, p46ATIL, and p46 alanine substitution mutants 1–9 at 24 hr post EMCV infection (MOI=0.001). (B)…
Figure 6 with 1 supplement
OAS1 p46 has broad antiviral activity against viruses that use the endomembrane system for replication.
(A) WNV Texas titers (percent titer normalized to EV) 48 hr post WNV infection (MOI=0.001) taken from OAS1 KO 293 T cells transfected with control EV, p42, and p46. (B) Representative confocal …
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Figure 6—source data 1
Excel file with the summary statistics for the association analysis for Figure 6G.
- https://cdn.elifesciences.org/articles/71047/elife-71047-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Endomembrane targeting of OAS1 p46 confers its enhanced antiviral activity.
(A) Representative confocal micrographs from mock or WNV Texas (MOI=1, 24 hr) infected OAS1 KO 293 T cells expressing p42 or p46 and stained with DAPI (blue) and anti-OAS1 (green), Golgin-97 (red), …
Figure 7
Schematic depicting how endomembrane targeting of OAS1 p46 primes antiviral activity against positive-strand RNA viruses.
A splice-acceptor SNP (rs10774671) controls production of the p42/p46 OAS1 isoforms. Isoform-specific prenylation localizes p46 to the Golgi apparatus, while OAS1 p42 is cytosolic. During …
Author response image 1
Primary human fibroblasts do not induce OAS1 expression upon infection with EMCV.
Primary human fibroblasts of (A/A) and (A/G) genotype at rs10774671 were infected with EMCV (MOI=0.01) or treated with 25 U/mL rIFNβ for 24h. OAS1 expression was assessed by Western blot.
Author response image 2
Additional files
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Source data 1
Cumulative PDF file of all the source data in the manuscript.
- https://cdn.elifesciences.org/articles/71047/elife-71047-data1-v2.pdf
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Supplementary file 1
Cells used in this study.
- https://cdn.elifesciences.org/articles/71047/elife-71047-supp1-v2.docx
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Supplementary file 2
Antibodies used in this study.
- https://cdn.elifesciences.org/articles/71047/elife-71047-supp2-v2.docx
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Supplementary file 3
Nucleic acids used in this study.
- https://cdn.elifesciences.org/articles/71047/elife-71047-supp3-v2.docx
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Supplementary file 4
Viruses used in this study.
- https://cdn.elifesciences.org/articles/71047/elife-71047-supp4-v2.docx
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Supplementary file 5
Clinical and demographic information for COVID-19 and matched healthy control cohort.
- https://cdn.elifesciences.org/articles/71047/elife-71047-supp5-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/71047/elife-71047-transrepform-v2.docx
