<i>Apol9</i> expression in mouse tissues and in L929 cells.

<p><i>Apol9</i> transcripts were quantified by RT-qPCR in mouse tissues and cells. A. <i>Apol9a</i> and <i>Apol9b</i> cDNA copies (mean and SD) detected per 10⁶ β-actin cDNA copies, in tissues of naive C57BL/6 mice (n = 3). The column indicated “+ IFN-α” gives the mean upregulation (x-fold) of <i>Apol9</i> expression observed in the presence of systemic IFN-α. This corresponds to the ratio between the number of <i>Apol9</i> cDNA copies detected in tissues of FVB/N mice electro-injected in the tibialis muscle with plasmid pcDNA3-IFN-α6T, which expresses IFN-α that circulates in the blood stream (n = 6) and that of control mice that were electro-injected with an empty pcDNA3 plasmid. N.D. = not determined. B. <i>Apol9b</i> expression in the central nervous system of FVB/N mice that were mock-infected (n = 3) or infected intracerebrally with 10⁶ PFU of the DA1 strain of TMEV (n = 5) for the indicated time. *: indicates a significant difference with the mock-infected mice at 7dpi. C. Time course of <i>Oasl2</i>, <i>Apol9a</i> and <i>Apol9b expression</i> in L929 cells treated with 5 units/ml of IFN-β (n = 3).</p

The murine ApoL family.

<p>A. Phylogenetic tree of the murine <i>Apol</i> gene family, generated by the Gene Orthology/Paralogy prediction method available on the Ensemble server (<a href="http://www.ensembl.org/" target="_blank">http://www.ensembl.org</a>). Duplication nodes are indicated by black squares. Numbers represent the duplication confidence score. B. Organization of the mouse <i>Apol</i> genes cluster. Underlined regions (<i>Apol7b/9a</i> and <i>Apol7e/9b</i>) share 98% of nucleotide sequence identity and likely arose from a duplication event. C. Alignment of the human ApoL6 BH3 domain with the hypothetical BH3 domain of murine ApoL proteins. Two amino acids, <i>L</i> and <i>D</i>, shown in bold letters, are functionally conserved in all the BH3 domains identified. D. Structural organization of ApoL9 proteins. The putative BH3 domain (residues 81 to 89) is shown in dark gray. The white area (residues 128 to 144) corresponds to a potential transmembrane (TM) domain.</p

Mouse infections with RNA viruses.

a<p>1 day post-infection was reported to correspond to the peak of LDV replication and of IFN expression in vivo.</p>b<p>Mice infected with highly neurovirulent viruses were sampled when signs of encephalitis were prominent (generally less than 24 hours before death).</p>c<p>The DA1 strain of TMEV produces a transient encephalitis lasting about 1 or 2 weeks. In mice with the H-2 b haplotype, the virus is then rapidly cleared by the cytolytic T lymphocyte response <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000017#ppat.1000017-Brahic1" target="_blank">[40]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000017#ppat.1000017-Roos1" target="_blank">[41]</a>. Mice were sampled at 5 days post-infection, a time-point representative of the acute phase of infection.</p>d<p>Preliminary RT-PCR experiments failed to reveal a clear difference in IFN expression and viral load between 129/Sv mice infected i.p. for 2 days and for 7 days. Only mice with amounts of MHV-A59 detectable by conventional RT-PCR were taken into account.</p>e<p>These samples from TMEV (GDVII strain) and LACVdelNSs infected brains were from a previous work <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000017#ppat.1000017-Delhaye1" target="_blank">[33]</a>.</p

Activity of FLAG-tagged and untagged mouse IFNs.

<p>Histograms show the log₂ of antiviral activities detected in the supernatant of Neuro2A cells collected 24h after transfection of the pcDNA3 derivatives expressing the indicated FLAG-tagged (+) or untagged (-) IFNs or after transfection of the corresponding empty vectors (pcDNA3). Antiviral activities are presented relative to those of culture medium. NS: non significant (Mann–Whitney <i>U</i> test).</p

The Interferon-Inducible Mouse Apolipoprotein L9 and Prohibitins Cooperate to Restrict Theiler’s Virus Replication

<div><p><i>Apolipoprotein L9b</i> (<i>Apol9b</i>) is an interferon-stimulated gene (ISG) that has antiviral activity and is weakly expressed in primary mouse neurons as compared to other cell types. Here, we show that both <i>Apol9</i> isoforms (<i>Apol9b</i> and <i>Apol9a</i>) inhibit replication of Theiler’s murine encephalomyelitis virus (TMEV) but not replication of vesicular stomatitis virus (VSV), Murid herpesvirus-4 (MuHV-4), or infection by a lentiviral vector. <i>Apol9</i> genes are strongly expressed in mouse liver and, to a lesser extent, in pancreas, adipose tissue and intestine. Their expression is increased by type I interferon and viral infection. In contrast to genuine apolipoproteins that are involved in lipid transport, ApoL9 has an intracytoplasmic localization and does not seem to be secreted. The cytoplasmic localization of ApoL9 is in line with the observation that ApoL9 inhibits the replication step of TMEV infection. In contrast to human ApoL6, ApoL9 did not sensitize cells to apoptosis, in spite of the presence of a conserved putative BH3 domain, required for antiviral activity. ApoL9a and b isoforms interact with cellular prohibitin 1 (Phb1) and prohibitin 2 (Phb2) and this interaction might contribute to ApoL9 antiviral activity. Knocking down <i>Phb2</i> slightly increased TMEV replication, irrespective of ApoL9 overexpression. The antiviral activity of prohibitins against TMEV contrasts with the pro-viral activity of prohibitins observed for VSV and reported previously for Dengue 2 (DENV-2), Chikungunya (CHIKV) and influenza H5N1 viruses. ApoL9 is thus an example of ISG displaying a narrow antiviral range, which likely acts in complex with prohibitins to restrict TMEV replication.</p></div

IFN-α and IFN-λ responding cells in the kidney.

<p>Mx1 expression, detected by immunohistochemistry (white nuclear spots), 7 days after electroinjection of a plasmid coding for MuIFN-α6T or MuIFN-λ3. Sections of the kidney from: A. control Mx1/WT mouse electroinjected with the empty vector. Note that few cells (mostly endothelial cells) were weakly Mx1-positive. B. control Mx1/IFNAR1-KO mouse electroinjected with a plasmid coding for IFN-α6T. C–E–G: Mx1/WT mouse electroinjected with a plasmid coding for IFN-α6T. D–F–H: Mx1/IFNAR1-KO mouse electroinjected with a plasmid coding for IFN-λ3.</p

Quantification of IFN-λ, IFN-α5, and IFN-β transcripts in virus-infected brains and livers.

<p>Histograms show the number of IFN cDNA copies per 10⁶ β-actin cDNA copies, determined by real-time PCR, after reverse transcription of RNA extracted from the brain and liver of mice infected with different RNA viruses, in different experimental settings (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000017#ppat-1000017-t002" target="_blank">Table 2</a>). A, B, F, G, H: mean and standard deviation of groups of mice. C–E: data from individual mice. Background amplification in mock-infected mice (not shown) was less than 1 copy of IFN-β or IFN-λ, and less than 10 copies of IFN-α5 cDNA, per 10⁶ copies of β-actin cDNA.</p

Processing of the IFN-ε precursor in transfected cells.

<div><p>Western blot analysis of IFN-α and IFN-ε processing in total protein extracts of Neuro2A cells transfected for 24h with pcDNA3 derivatives expressing FLAG tagged IFNs.</p> <p>A. Mouse IFN-αA and IFN-ε detection in the presence or absence of brefeldin A. Arrowheads point to the two bands detected for IFN-ε.β-actin detection was used as loading control. B. Detection of mouse IFN-αA and IFN-ε along with corresponding proteins expressed without signal peptide (Δsp). C. Human IFN-ε and mouse IFN-β detection in 293T cells before and after treatment with N-glycosidase F.</p></div

Mx1 gene expression induced by systemic IFN-α and IFN-λ, in organs of IFNAR1-positive and IFNAR1-deficient mice.

<p>Mx1 gene transcription was analyzed by real-time RT-PCR, 7 days after electroinjection of plasmid coding for MuIFN-α6T, MuIFN-λ3 or the empty vector (mock) in 6 week-old Mx1-positive mice (2 BALB.A2G-Mx1 and 2 B6.A2G-Mx1 mice, grouped as Mx1/WT mice) or in 8 week-old Mx1/IFNAR1-KO mice. Results are expressed as Mx1 cDNA copies per β-actin cDNA copy. Graphs present results for individual mice and the mean for each group, for one representative experiment.</p

ApoL9 proteins are not-secreted, cytoplasmic proteins.

<p>A. Antiviral activity of N-terminally (FLAG-ApoL9b) and C-terminally (ApoL9b-FLAG) FLAG-tagged ApoL9b against TMEV. Histograms show the relative infection efficiency, of L929 cells that were transduced with theTM945 lentiviral vector expressing mCherry alone (vector), or with the MK82 or MK83 derivatives co-expressing mCherry and FLAG-ApoL9b or ApoL9b-FLAG, respectively. Infection efficiency was measured, by flow cytometry, after infection of cells for 10 hours with 0.5 PFU per cell of the GFP-expressing KJ26 virus. B. FLAG detection (green) and nuclear staining (blue) in HeLa-M cells transduced with the MK65 construct expressing FLAG-ApoL9b (scale bar: 10 um). Typical immunolabeling patterns are shown: left and central panels: one non-transduced cell (arrow) and transduced cells showing a diffuse cytoplasmic FLAG-ApoL9b localization; right panel: cells showing typical small and medium-sized FLAG-ApoL9b granules (arrows). C. Confocal images showing FLAG (green—left panel) and mitochondria (red—central panel) labelings and merged image (right panel). Nuclei were stained with Hoechst (Blue). D. Western blot detection of FLAG-tagged ApoL9b, ApoL9a and IFN-αA in 293T cells. Cells were mock-transfected, transfected with the empty pTM943 plasmid (vector), or with pTM943 derivatives coding for the indicated FLAG-tagged ApoL9 proteins. As a control cells were also transfected with plasmid pcDNA3-IFNαA-FLAG, which expresses the secreted FLAG-tagged mouse IFN-αA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133190#pone.0133190.ref012" target="_blank">12</a>]. 20 hours post-transfection, cells were mock-treated (-) or treated for 4 hours with 10ug/ml of Brefeldin A (+) prior to total protein extraction. FLAG and β-actin were detected by Western blot in cell lysates.</p