Ch25h is an IFN-dependent Gene with Antiviral Activity
In a microarray analysis of IFNα and IFNγ stimulated murine bone marrow-derived macrophages (BMMs), we found both types of IFNs induced expression of Ch25h within 3hrs (). Gene expression analysis by qPCR showed that Ch25h is induced by polyI:C (TLR3 agonist) in BMMs and dendritic cells; BMMs had higher induction in response to IFN ( upper panel). In mice infected with 56 pfu of VSV i.p., Ch25h was induced after 18 and 36h in lung, liver, and kidney, with highest induction in liver and kidneys ( lower panel). An RNAseq analysis showed toll-like receptor 4 (TLR4) induction of Ch25h was dependent on IFN receptor (IFNAR) but independent of IL-27, a cytokine that mediates IFN secondary gene expression, such as IL-10 (). This result was confirmed by qPCR showing that Ch25h expression was induced by TLR2, 3, 4, and 9 agonists, with highest expression induced by polyI:C (TLR3) and lipidA (TLR4). IFN receptor deficient (ifnar−/−) BMMs had abrogated Ch25h expression when treated with these agonists showing that Ch25h expression is IFN-dependent ().
Ch25h is an IFN-dependent Antiviral ISG
In a previous study, we performed a blinded, unbiased screen for antiviral ISGs against vesicular stomatitis virus co-expressing GFP (VSV-GFP) (Liu et al., 2012
). We co-transfected individual plasmids encoding an ISG with a plasmid encoding a red fluorescent protein, DsRed. Transfection proceeded for 36h before infection with VSV-GFP. At 9hpi, we quantified VSV-GFP with FACs by gating on DsRed-positive population, which are cells that highly express the ISG (). Expression of Ch25h inhibited VSV-GFP replication by ~89% at 9hpi (). IFN activators like Tbk1, Ifih1, and Irf1 strongly inhibited VSV as well as the RNA exonuclease, ISG20. To validate the antiviral effect of Ch25h, we generated a doxycycline-inducible Ch25h-flag construct co-expressing a flourescent-red mCherry (Ch25h-mCherry). Doxycycline addition to HEK293T expressing this construct increased CH25H-flag expression ( top) and mCherry expression in a dose-dependent manner (, bottom). When infected with VSV-GFP, HEK293T expressing Ch25h-mCherry and treated with doxycycline exhibited a dose-dependent inhibition of VSV-GFP compared to vector control (, bottom). Taken together, Ch25h is sufficient to inhibit VSV.
Ch25h-deficiency increases susceptibility to viral infection in vitro
Loss of function of Ch25h leads to Susceptibility to Viral Infections in vitro
We sought to determine whether Ch25h might play a necessary role in the viral infection. We generated Ch25h stable knockdown cell lines from murine macrophage cell line, RAW264.7, with two distinct shRNA sequences against Ch25h and confirmed the knockdown by qPCR (). Both knockdown cell lines demonstrated increased VSV replication compared to scramble control (). To further validate these results, macrophage and B-cell lines were derived from Ch25h-deficient (ch25h−/−) and matching wild-type (ch25h+/+) mice. In our conditions, we could not establish VSV infection in primary cell lines; hence we immortalized BMMs and B-cells with J2 and BCR-ABL oncogenic retroviruses, respectively. Ch25h−/− J2 BMMs displayed 5-fold increased susceptibility to VSV infection compared to ch25h+/+ J2 BMMs at 14hpi. In B-cells transformed with BCR-ABL, we observed about 100 fold increase in VSV-GFP replication in 3 different Ch25h−/− B-Cell clones at 48hpi compared to 2 ch25h+/+ B-cell clones (). These results show that Ch25h may be required for host antiviral immunity.
Ch25h produces a soluble antiviral factor that is not IFN
In overexpression studies described in , HEK293T were transfected with ISG plasmids and DsRed in 3:1 ratio such that DsRed-positive cells (DsRed+) should represent cells that highly expressed the ISG, whereas DsRed-negative (DsRed-) cells should represent low ISG expressers (). IFN activators, Tbk1, Irf1, and Ifih1, inhibited VSV-GFP expression in both populations showing DsRed+ cells confer viral resistance to DsRed-through a soluble factor, which is IFN (). In contrast, the cytoplasmic viral RNA exonuclease, ISG20, only inhibited VSV growth in DsRed+ population, but not DsRed-population. Overexpression of Ch25h also inhibited virus in both DsRed+ and DsRed-populations suggesting that Ch25h produced a soluble factor that act in trans to confer antiviral activity onto other cells.
Ch25h produces a soluble antiviral factor that is not IFN
To determine if Ch25h produced a soluble antiviral factor, we tested whether conditioned medium from cells overexpressing Ch25h had antiviral activity. HEK293T cells were transfected with vector, interferon activators (Tbk11, Irf1, and Ifih1), Ch25h, or ISG20, and the conditioned media was transferred onto freshly plated HEK293T cells for 8h before infection with VSV-GFP (0.01MOI). As expected, conditioned media from IFN activators inhibited VSV growth, but not ISG20 (). Conditioned medium from Ch25h generated ~60% VSV-GFP inhibition. We have also observed similar effect in several human and murine cell lines including HeLa, 3T3, BHK, Veros, MDCK, and Huh751 (Supp. Fig. S1A
). These results demonstrate that Ch25h produces a soluble antiviral factor.
IFN induces many ISGs that positively feedback and amplify its production, leading to the hypothesis that Ch25h-induced soluble factor is IFN. Ch25h conditioned medium, however, had no detectable IFNβ by ELISA and did not induce an IFN-stimulated responsive element (ISRE) luciferase reporter (Supp. Fig. S1 B and C
). More importantly, Ch25h-conditioned medium inhibited VSV replication in both ifnar−/−
fibroblasts and J2 BMMs. As positive control, conditioned media from IFN activators, Irf1, Ifih1, and Rig-I, were unable to confer antiviral activity to ifnar−/−
cell lines (). Taken together, Ch25h produces a soluble factor that is not IFN and can confer antiviral activity independent of IFN.
25-hydroxycholesterol (25HC), the product of CH25H, has antiviral activity
Ch25h catalyzes oxidation of cholesterol to 25-hydroxycholesterol (25HC), a soluble oxysterol that acts as an autocine and paracrine mediator (, top). We hypothesized that the soluble antiviral factor generated by Ch25h is 25HC. Treatment of HEK293T cells with 25HC for 8h inhibited VSV-GFP expression by FACs in a dose-dependent manner with IC50 of ~1µM (, bottom). Two other oxysterols, 22-(R)-hydroxycholesterol (22R-HC) and 22-(S)-hydroxycholesterol (22S-HC), had no effect on VSV. 22R-HC is also an agonist for the nuclear hormone receptor LXR and 22S-HC is an inactive ligand. Since 25HC has been implicated as a LXR agonist, these results also suggest that the antiviral effect was LXR-independent. In addition, 25HC treatment of ch25h+/+ and ch25h−/− J2 BMMs reduced VSV replication ().
Ch25h produces 25-hydroxycholesterol, an oxysterol with broad antiviral properties
The effect of 25HC on cell viability and toxicity was also assessed. 25HC treatment at 10 times IC50
(10µM) did not increase LDH in supernatants of cells after 16h of treatment; LDH level increased only after 30–40h treatment at 40µM of 25HC (Supp. Fig. S2A and B
). Similarly, Ch25h-conditioned medium did not alter cell viability as measured by cellular ATP levels (Supp. Fig. S2C
). Therefore, these results suggest that the antiviral activity of Ch25h is carried out through its enzymatic product, 25HC, which has specific antiviral effect.
CH25H and 25HC are broadly antiviral
To determine the breadth of antiviral activity of Ch25h, we tested the effect of Ch25h-conditioned medium and 25HC on various viruses. For HIV, primary peripheral blood mononuclear cells (PBMCs) were treated with conditioned medium or oxysterol and subsequently infected with HIV NL4-3. At 3dpi, Ch25h- and Irf1-conditioned media caused ~75% reduction of HIV NL4-3 p24 expression (). Similarly, 25HC (1µM) inhibited p24 expression by ~80% at 3dpi compared to vehicle treatment, whereas 22S-HC had no effect (). Ch25h-conditioned medium also inhibited herpes simplex virus 1 (HSV-1) by plaque assay () and expression of Ch25h in HEK293T also reduced murine gammaherpes virus (MHV68) infection by plaque assay ().
HIV, HSV-1, and MHV68 are viruses that achieve chronically persistent infections. To determine whether Ch25h-induced 25HC can inhibit acutely pathogenic viruses, we tested the effect of 25HC on live Ebola virus (EBOV-Zaire), Nipah virus (Bangladesh), Russian Spring-Summer Encephalitis Virus (RSSEV), and Rift Valley fever virus RVFV (wild-type strain ZH501 and vaccine strain MP12) under BSL4 conditions. show that 1µM of 25HC inhibited replication of these live viruses. 25HC also inhibited replication of Nipah and RVFV (MP12) in a dose-dependent manner (Supp. Fig. S2 D and E
). In contrast, a non-enveloped virus, adenovirus coexpressing GFP, was not affected by 25HC as measured by FACs (). Taken together, Ch25h-induced 25HC has antiviral activity against several types of enveloped DNA and RNA viruses, while it does not affect a non-enveloped virus.
25HC inhibits VSV entry
We took advantage of tools available for VSV and HIV to study the mechanism of Ch25h inhibition on the viral lifecycle. First, we utilized the pseudotyped VSVΔG-Luc reporter virus system that has the receptor-binding G gene (VSV-G) replaced with a luciferase reporter gene that is capable of single-round infection (Negrete et al., 2006
). Quantification of luciferase activity is indicative of viral lifecycle processes from entry to protein synthesis. Ch25h- and Irf1-conditioned media inhibited VSVΔG-Luc expression suggesting inhibition of viral replication at an early stage (). In a time-of-addition experiment, longer pre-treatment times correlated with greater inhibition of VSVΔG-Luc expression, compared to vehicle treated controls (). These results suggest that 25HC does not inhibit VSV during infection or after infection has taken place. Rather, it is likely that 25HC establishes an antiviral state prior to infection.
Ch25h-induced 25HC inhibits VSV Entry
Since these data implicate early viral lifecycle steps may be affected, we carried out experiments to determine whether 25HC affects attachment (Weidner et al., 2010
). HEK293Ts were treated for 8h with ethanol (EtOH), 25HC (1µM), CPZ (10µg/mL), an endocytosis inhibitor that would have no effect on binding. To measure binding, VSV (1MOI) was incubated with HEK293T at 4°C for 1h to allow for binding but not cell entry. After washing 3 times with cold PBS, quantification of VSV genomic RNA (gRNA) showed that 25HC did not inhibit viral binding significantly (P>0.05) ().
To determine if 25HC affects efficiency of fusion, we established a VSV-G β-lactamase (βla) entry assay based on the ability of VSV-G to be pseudotyped onto viral-like particles made from the Bla-Nipah virus matrix fusion protein, herein called VSV-G/βlaM (Wolf et al., 2009
). VSV-G mediated fusion will result in cytoplasmic delivery of Bla-M; by addition of lipophilic fluorescent CCF2-AM substrate, the βla activity can be measured by the green (525nm) to blue (485 nm) fluorescence shift as a result of CCF2-AM cleavage (Zlokarnik et al., 1998
). Hence, efficiency of virus-cell fusion can be measured by the increase in the ratio of blue to green (blue:green) fluorescence, which is reflective of the βla activity associated with βlaM that was been released into the cytoplasm after VSV-G mediated fusion (Cavrois et al., 2002
; Wolf et al., 2009
). Unlike the VSVΔG-Luc pseudotyped virus, this VSV-G/βlaM entry assay does not require transcription and translation of viral proteins for reporter gene expression. Fusion is proportional to βlaM concentration, which is estimated by the rate constant (k
) derived from the slope of the reaction during the linear phase of the reaction within the first hour.
HEK293T cells were transfected with several ISGs for 48 hours and infected with VSV-G/βlaM. showed that Ch25h reduced efficiency of VSV fusion. Compared to vector control, βlaM activity from Ch25h-transfected cells proceeded 48% of vector-transfected cell (compare rate constants in inset table) and plateaued at a lower level. The previously described entry inhibitor, Ifitm3, also reduced VSV-G/βlaM fusion (Brass et al., 2009
). ISG20, a viral RNA exonuclease, had no effect on viral entry. Irf-1 transfected cells also inhibited fusion, presumably by up-regulation of IFN. To show this, we separately confirmed that recombinant IFN inhibited VSV-G/βlaM fusion (Supp. Fig. 3A
). Ch25h-conditioned medium similarly inhibited VSV-G/βlaM entry, with a more pronounced effect than Irf1-conditioned medium (). Furthermore, treatment of 25HC at 1, 2.5, and 5µM inhibited VSV-G/βlaM activity, by 44%, 56%, and 70%, respectively (). These results demonstrate that the IFN, Ch25h, and its cognate product, 25HC, modulates the target cell membrane in a manner that inhibits efficiency of virus-cell fusion.
Since the viral entry step involves interactions between both the viral and cellular membranes, we then asked if the infectivity of the virions were affected when produced from 25HC treated cells. HEK293T were treated with and without 25HC (2.5µM) for 8h and infected with live VSV at 0.01 MOI for 1h. The cells were treated as before for 24h. Viral superntants were then purified by ultracentrifugation through a 20% sucrose cushion, which removed any residual 25HC. As expected, there was >60% reduction in the amount of VSV from 25HC treated samples versus control, as measured by qPCR for the number of viral genome copies (gRNA) (Supp. Fig. 3B
). To assess infectivity, we normalized viral titer from 25HC- or vehicle-treated cells based on gRNA and determined infectivity by plaque assay. After normalization, VSV from 25HC-treated cells formed equivalent PFU titer as viruses from vehicle-treated cells (Supp. Fig. 3C
). These results show that 25HC exerts its antiviral effect by altering target cell membrane properties and does not alter membrane of virions produced from those cells.
25-hydroxycholesterol is a suppressor of SREBP2, which controls sterol biosynthesis and can alter membrane sterol composition. Hence, we tested the hypothesis that 25HC inhibits viral growth through suppression of SREBP2. We tested whether overexpression of active (cleaved) form of SREBPs in HEK293T would overcome the anti-viral effect of 25HC. Overexpression of active forms of SREBP1a, SREBP1-c, and SREBP2 in HEK293Ts, however, did not reverse 25HC antiviral effect (Supp Fig. S4A
). These data demonstrate that the antiviral effect of 25HC is SREBP independent.
25HC is known to down-regulate numerous sterol biosynthetic enzymes including HMG-CoA reductase, which produces the key intermediate mevalonate (Pezacki et al., 2009
) (Supp. Fig. S4B
). We hypothesized that mevalonate may reverse the antiviral effect of 25HC. Addition of exogenous mevalonate (300µM) to HEK293T before and during 25HC treatment, however, did not reverse the antiviral effect of 25HC (Supp Fig. S4C
25HC also may inhibit production another intermediate isopentenyl-pyrophosphate (Isopentyl-PP), which is substrate for prenylation of proteins on CAAX motif. This post-translational modification is mediated by farsenyl-transferase (FTase) and gernylgernyl-transferase (GTase). We tested the hypothesis that inhibition of prenylation can inhibit VSV growth. HEK293T were treated with FTase and GTase inhibitors, FTI-276 and GGTI-298 (5–20µM), and infected with VSV-GFP. FTI-276 treatment had no effect on VSV, whereas GGTI-298 reduced viral growth at 10–20µM (Supp Fig. S4D
). GGTI-298, however, caused >10% reduction in cell viability by ATP content, whereas 25HC and FTI had no effect (Supp Fig. S4E
). Furthermore, FTI-276 and GGTI-298 did not reduce VSV fusion entry by VSV-G/βlaM assay (Supp Fig. 4F and G
). Taken together, while prenylation inhibition may have some antiviral effect, it seems to inhibit cell viability and not affect the viral entry as observed with 25HC.
Ch25h and 25HC inhibits HIV entry
We sought to validate Ch25h and 25HC antiviral mechanism on HIV. Unlike VSV, HIV is a retrovirus that undergoes pH-independent cellular entry. In CEM cells, 25HC inhibited >50% luciferase expression from single round infection of pseudovirus with HIV-IIIB envelope on a NL4-3 backbone coexpressing luciferase (pNL4-3.Luc.-R-E) (). AZT, an inhibitor of reverse transcription, served as positive control and inhibited expression by ~70%. Hence, these data also suggest 25HC inhibits viral lifecycle prior or at translation.
25HC inhibits HIV Entry and Viral-Cellular Membrane Fusion
HIV initiates reverse transcription of its genomic RNA to DNA immediately after entry. Hence, we examined the effect of 25HC on the production of full-length, reverse-transcribed DNA (lateRT). CEM cells were infected with pseudotyped HIV-IIIB and lateRT was measured by qRT-PCR. 25HC inhibited lateRT expression >99% at 2hpi and ~70% at 6hpi (). The HIV entry inhibitor, AMD3100, served as a positive control. Elvitegravir is used as a negative control because it inhibits HIV at the step of DNA integration after lateRT formation. These results show that 25HC inhibits a stage of the HIV life cycle before reverse transcription of its genome.
We next asked whether Ch25h inhibits HIV similar to VSV at the level of entry. We coexpressed pNL4-3 with Bla-VPR fusion gene to produced virions containing Bla-VPR (NL4-3/Bla). CEM cells treated with Ch25h conditioned medium exhibited ~65% reduction in viral entry compared to vector- and Isg20-conditioned medium. AMD3100 abrogated NL4-3/Bla entry (). We further confirmed our findings by FACS analysis and observed ~50% decrease in the number of cells expressing cleaved CCF2-AM substrate (blue population) in CEM treated with Ch25h conditioned medium compared to control (supp. Fig. S5A
). Treatment of CEM cells with 25HC (5µM) for 24h caused ~60% decrease in NL4-3/Bla blue-green ratio at endpoint () and >85% reduction in cells expressing cleaved CCF2-AM by FACS analysis ().
Since 25HC may have diverse cellular effects, we asked whether 25HC might affect other HIV life cycle processes. To assess 25HC effect on HIV transcription, we transfected HEK293Ts with pNL4-3 co-expressing GFP (NL4-3-GFP) and treated the cells with 25HC at 4h post transfection. The NL4-3-GFP expression after 24h was not suppressed suggesting that 25HC does not affect HIV transcription and translation (Supp. Fig. S5B
). Concurrently, treatment of with 25HC did not reduce budding of HIV virions from NL4-3-GFP transfected cells as measured by HIV p24 in the supernatants, while Nelfinavir, a known protease inhibitor that reduces subsequent budding, inhibited p24 expression by >50% at 24 and 48h post transfection (Supp. Fig. S5C
). Taken together, Ch25h and 25HC inhibits efficiency of HIV membrane fusion, while 25HC treatment does not seem to affect HIV transcription, translation, and budding processes.
25HC inhibits virus-cell membrane fusion
Although βla data demonstrate 25HC inhibits viral entry processes up to fusion, we sought to test whether 25HC inhibits the viral fusion process itself. Since 25HC inhibited live Nipah replication (), we took advantage of the robust system of Nipah fusion (F) and attachment (G) proteins to induce pH-independent cell-cell membrane fusion to form syncytias. Vero cells were transfected with recombinant Nipah F and G at equal ratios for 5h and refreshed with media containing 25HC or vehicle control. At 21h post transfection, cells were fixed and stained by Giemsa. Grossly, 25HC treatment led to less syncytia formation and fewer nuclei per syncytias compared to control (). In a blinded count of numbers of nuclei per syncytia, a standard measure of fusion, 2µM of 25HC reduced fusion by ~50% and 10µM by ~60% relative to control (). These data demonstrate that 25HC modifies the cellular membrane to inhibit viral membrane fusion.
25HC Directly Modifies Cell Membrane to Impede Viral Infection
We further explored whether 25HC can directly change membrane property to inhibit fusion. We hypothesized artificial liposomes with 7:3 phosphatitdylcholine:cholesterol ratio, which is similar in composition to cell membranes, would compete with the ability for 25HC to incorporate into cell membrane. HEK293T incubated with liposome alone had no effect on viral infection while treatment of 25HC with liposomes caused a dose-dependent reversal of 25HC anti-viral inhibition (). As a positive control, we demonstrated that liposome could compete with a known viral membrane fusion inhibitor, LJ001, as described previously (Wolf et al., 2010
). These results show that 25HC directly modify cellular membrane to inhibit viral fusion.
25HC reduces HIV infection in vivo
We used HIV infection in a humanized mouse model to determine the antiviral effect of 25HC in vivo. Humanized NOD-Rag1nullIl2rgnull mice (NRG-hu) were administered 25HC (50mg/kg) 12h prior to infection with HIV NL4-R3A by intraperitoneal (i.p) injection. 25HC or the vehicle, 2-hydroxypropyl-β-cyclodextrin (HβCD), was administered daily and the serum was collected 7dpi. Quantification HIV RNA in the serum from 2 combined experiments showed >80% reduction of HIV RNA (copies/mL) in 25HC-treated mice compared to vehicle-treated mice (P<0.0001) (). At termination of the experiment on 14dpi, HIV p24 was significantly lower in CD4 T-cells from spleens of 25HC treated mice than control (). Moreover, at 10dpi, 25HC prevented HIV-mediated CD4+ T-cell depletion compared to vehicle control in CD3+(live T-cell) population in peripheral blood leukocytes (P<0.05); this effect was less significant in the spleen (P=0.06) (). These data show that administration of 25HC can cause antiviral effect against HIV in vivo.
25HC inhibits HIV replication and Ch25h is required for antiviral immunity in vivo
Ch25h-deficient mice are more susceptible to viral infections
To determine whether Ch25h has a physiological role in host defense against viral infection, we tested whether ch25h−/− mice had increased susceptibility to matching wild-type mice (ch25h+/+). Since Ch25h expression inhibited MHV68 in vitro, we used MHV68 coexpressing luciferase (MHV68-Luc) to infect mice so that viral lytic growth kinetics could be measured in real time by bioluminescence. Eight-week old female ch25h+/+ and ch25h−/− mice (N=4 in each group) were infected with 500pfu of MHV68-Luc i.p. and imaged every day after 3dpi. Average and maximal luminescence intensities from ventral, right, left, and dorsal side of every mouse were measured. We observed significantly higher MHV68-Luc activity in ch25h−/− mice over ch25h+/+ mice starting 5 dpi and maximal differences between day 7 and 8 (). MHV68-Luc activity began to wane in both groups by 9dpi with significantly higher activity in Ch25h−/− mice. To validate the imaging results, Ch25h−/− spleens had approximately ~3.5 fold higher MHV68 genomic DNA than spleens of Ch25h+/+ mice at 10dpi (). These results show that Ch25h is a physiologically important antiviral factor.