Rottlerin inhibits La Crosse virus-induced encephalitis in mice and blocks release of replicating virus from the Golgi body in neurons

Ojha, Durbadal; Winkler, Clayton W.; Leung, Jacqueline M.; Woods, Tyson A.; Chen, Catherine Z.; Nair, Vinod; Taylor, Katherine; Yeh, Charles D.; Tawa, Gregory J.; Larson, Charles L.; Zheng, Wei; Haigh, Cathryn L.; Peterson, Karin E.

doi:10.1038/s41564-021-00968-y

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Published: 21 October 2021

Rottlerin inhibits La Crosse virus-induced encephalitis in mice and blocks release of replicating virus from the Golgi body in neurons

Nature Microbiology volume 6, pages 1398–1409 (2021)Cite this article

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Abstract

La Crosse virus (LACV) is a mosquito-borne orthobunyavirus that causes approximately 60 to 80 hospitalized pediatric encephalitis cases in the United States yearly. The primary treatment for most viral encephalitis, including LACV, is palliative care, and specific antiviral therapeutics are needed. We screened the National Center for Advancing Translational Sciences library of 3,833 FDA-approved and bioactive small molecules for the ability to inhibit LACV-induced death in SH-SY5Y neuronal cells. The top three hits from the initial screen were validated by examining their ability to inhibit virus-induced cell death in multiple neuronal cell lines. Rottlerin consistently reduced LACV-induced death by 50% in multiple human and mouse neuronal cell lines with an effective concentration of 0.16–0.69 µg ml⁻¹ depending on cell line. Rottlerin was effective up to 12 hours post-infection in vitro and inhibited virus particle trafficking from the Golgi apparatus to trans-Golgi vesicles. In human inducible pluripotent stem cell-derived cerebral organoids, rottlerin reduced virus production by one log and cell death by 35% compared with dimethyl sulfoxide-treated controls. Administration of rottlerin in mice by intraperitoneal or intracranial routes starting at 3 days post-infection decreased disease development by 30–50%. Furthermore, rottlerin also inhibited virus replication of other pathogenic California serogroup orthobunyaviruses (Jamestown Canyon and Tahyna virus) in neuronal cell lines.

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Main

La Crosse virus (LACV), a California serogroup (CSG) mosquito-borne arbovirus endemic to North America, can induce a rare, life-threatening neurological disease in children^1,2. First isolated in Wisconsin, USA, in the early 1960s^3,4, LACV encephalitis (LACV-E) cases have been found in more than 20 states⁵. The life cycle of LACV is maintained between the vector, the eastern tree-hole mosquito Aedes triseriatus and vertebrate hosts including eastern grey squirrels and chipmunks^6,7.

Humans are ‘dead-end’ hosts as LACV does not reach sufficient viremia levels to be picked up by mosquitos⁸. Most LACV infections in humans are asymptomatic or induce mild clinical symptoms of fever, headache, lethargy and vomiting⁹. However, in some cases LACV causes severe neuroinvasive disease requiring hospitalization². Most LACV-E cases occur in children under 16 years of age¹⁰. To date, there is neither a licensed vaccine¹¹ nor an antiviral therapy approved by the Food and Drug Administration (FDA) to prevent or treat LACV-E⁹.

Mouse models, neuronal cell lines (NCLs) and human cerebral organoids (hCOs) have provided insights into the mechanisms of LACV-induced neuronal damage. Mice demonstrate an age-related resistance to LACV-induced neurological disease (LACV-ND), with mice 3 weeks old or younger being highly susceptible to disease development. Virus is detected in the brain by 3 days post-infection (dpi), and pathogenesis appears to be primarily driven by virus infection and apoptosis of neurons^12,13,14,15.

Human neuronal cells also undergo apoptosis following LACV infection as observed in human NCLs¹⁶, neuron–astrocyte co-cultures¹⁷ and cerebral organoids¹⁸. In recent studies with hCOs, LACV induced changes in the structural density of hCOs starting at 3 dpi. These hCOs contain neurons at different stages of differentiation, but not microglia, astrocytes or oligodendrocytes. Thus, they are useful models to study infection of human neurons in a complex, structured environment¹⁸ and provide a system with heterogeneous human neural-lineage cell types to analyse therapeutics and other reagents to prevent LACV-induced neuronal death.

Therapeutics for LACV-E need to be effective when administered after viral encephalitis has been diagnosed. At this time, LACV has entered the central nervous system (CNS), infecting and inducing apoptosis of neurons. Thus, an effective drug needs to inhibit virus replication in neurons or virus-induced neuronal cell death. LACV enters cells through clathrin-mediated endocytosis¹⁹ and traffics to early endosomes, where uncoating of the viral genome occurs followed by viral RNA transcription in the cytoplasm^19,20. Viral proteins are transported to the Golgi for viral assembly and mature viruses are trafficked by Golgi-derived vesicles to the plasma membrane for release²⁰. Compounds that inhibit virus entry, uncoating, transcription, translation, assembly or trafficking without causing neuronal damage would be ideal therapeutics for LACV-E.

In this study, we conducted a high-throughput screen of approved drugs and bioactive small molecules to identify compounds that inhibit LACV-induced neuronal death. We identified 10 potential compounds and further analysed the three most promising candidates. One compound, rottlerin (RTL), showed strong therapeutic potential in vitro, in hCOs and in a mouse model.

Results

Screen of compounds for inhibition of LACV-induced cell death

A total of 3,833 compounds from the library of the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection of FDA-approved drugs and the library of pharmacologically active compounds (LOPAC1280) were screened using the human SH-SY5Y NCL infected with LACV at 0.01 multiplicity of infection (MOI) (Extended Data Fig. 1a). Cell viability was measured using a high-throughput ATP-content assay to identify compounds that inhibited LACV-mediated cell death. These screening data are available as PubChem bioassay assay ID 1508616. A total of 4,096 compounds were tested, with 263 duplicate compounds and 3,830 compounds listed in the PubChem library. Of these, 32 compounds showed >80% protection. Ten of these 32 compounds were positive in a confirmation run of the same assay (Extended Data Fig. 1b–k). The top three hits (NBI 27914, RTL and tyrphostin AG879) were selected for further analysis.

Analysis of selected compounds in multiple cell lines

Toxicity analysis of the top three compounds was completed in Vero cells, two rodent-derived NCLs (N2a, C17.2) and SH-SY5Y cells. For each compound and cell line, a dose–response curve without virus infection was plotted to determine the 50% cellular toxicity (CC₅₀) as summarized in Table 1. A similar assay, with the addition of LACV infection, was used to determine the 50% effective concentration (EC₅₀), the concentration of the drug that inhibited LACV-induced cell death by 50% (Table 1 and Extended Data Fig. 2a). The ratio of CC₅₀ to EC₅₀ provided the selectivity index (SI), the difference in the drug concentration that is toxic to cells versus the concentration that inhibits virus-induced cell death²¹. Of the three drugs examined, only RTL had a high SI for all cell lines (Table 1). The other two drugs, NBI 27914 and tyrphostin AG879, were inconsistent in inhibiting virus-induced death between the cell lines (Table 1). T-705 was also tested as a positive control²² and showed a high SI in Vero cells but not in neuronal cells (Table 1 and Extended Data Fig. 2b). RTL was then further tested in human neural stem cells (hNSCs) and mouse primary neurons, with an SI of 12.67 and 5 in these cells, respectively (Table 1 and Extended Data Fig. 2a). Thus, RTL inhibited LACV-induced cell death in all cell lines tested, indicating a consistent antiviral drug against LACV.

Table 1 Cytotoxicity and anti-LACV activity of three different bioactive compounds with T-705 (FPV) as positive control by MTT assay

Full size table

Effect of RTL on virus replication

We next examined whether RTL inhibited LACV infection, replication or release by measuring virus titres from cell supernatants. Treatment with RTL at doses of EC₅₀ and 2 × EC₅₀ significantly reduced virus levels in cell supernatants by up to three logs in all neuronal cell cultures (Fig. 1a–d). Quantitative real-time (qRT)-PCR analysis of RNA levels of the viral S gene segment (Extended Data Fig. 2c–f) as well as the M and L segments (Supplementary Fig. 1) showed that RTL decreased viral RNA production by up to two logs. Immunofluorescence analysis of infected N2a or SH-SY5Y cell monolayers at 24 hours post-infection (hpi) showed that RTL significantly reduced the number of infected cells compared with dimethyl sulfoxide (DMSO)-treated cells (Fig. 1e–h). Thus, RTL treatment significantly lowered the percentage of infected cells, which was associated with reduced viral RNA and infectious virus particles.

**Fig. 1: Anti-LACV activity of RTL in multiple NCLs.**

Analysis of RTL activity at early time points in virus replication

As RTL had similar effects on all cell lines, we focused on SH-SY5Y and hNSCs to study the mechanism by which RTL inhibits LACV replication. To determine whether RTL inhibited LACV before or after viral entry, cells were treated with RTL before infection (pre-treatment), during infection (co-treatment), after infection (post-treatment) or by continuous treatment (CT) at all three time points. Neither pre- nor co-treatment of SH-SY5Y cells or hNSCs with RTL inhibited LACV-induced neuronal death (Fig. 1i). RTL did not affect virus attachment (Extended Data Fig. 3a,b) and pre-incubation of RTL with virus did not affect LACV replication (Extended Data Fig. 2c), indicating that RTL did not inhibit virus entry. However, RTL inhibited cell death when added post-infection (PI) or maintained continuously (Fig. 1i). Thus, RTL reduced LACV-induced cell death when added after the initial stage of infection in SH-SY5Y cells and hNSCs.

Kinetic determination of RTL inhibition

To determine how late PI RTL could be added to inhibit LACV, we added RTL at different time intervals. Inhibition of cell death (Fig. 1j), virus production in the supernatant (Extended Data Fig. 3d) and viral RNA levels within the cells (Fig. 1k and Extended Data Fig. 3e,f) was observed at all time points, with a slight drop-off from 8–12 hpi. Similar results were observed with different MOIs of virus (Extended Data Fig. 3g,h). Thus, RTL was effective when added PI, with no observable difference when treatment was delayed to up to 8 hpi. This time point also correlated with the normal production of large amounts of virus from neuronal cells (Extended Data Fig. 3i). Together, these data suggest that RTL may inhibit a later stage of LACV replication in the cell, possibly virus assembly or release. Due to the similar effects of RTL on SH-SY5Y cells and hNSCs, further studies were done with hNSCs alone, as hNSCs are not an immortalized cell line and were more amenable to immunohistochemistry (IHC) and transmission electron microscopy (TEM) analysis.

Analysis of RTL effect on LACV localization within the cell

Orthobunyavirus replication includes virus assembly inside the Golgi complex and release with the help of Golgi vesicles²⁰. To determine whether RTL affected LACV replication in the Golgi, we compared RTL to the Golgi inhibitor brefeldin A (BFA). BFA did not have any synergistic effect with RTL on inhibiting LACV replication, suggesting that RTL acts at a similar or later stage of virus replication (Extended Data Fig. 4a). Analysis of virus replication in hNSCs showed that association of LACV proteins with giantin (medial Golgi)-positive membranes was minimal at 6 hpi, but pronounced at 8–12 hpi, with large clusters of virus particles appearing to leave the Golgi at this time (Extended Data Fig. 4b–e). We next analysed LACV-infected hNSCs that were treated with RTL at 8 hpi by IHC at 24 hpi. Cells were labelled with cis-Golgi (Fig. 2a), medial Golgi (Fig. 2b) or trans-Golgi (Fig. 2c) markers and anti-LACV antibody. LACV proteins were more closely associated with markers for the cis-, medial and trans-Golgi compartments in cells treated with RTL compared with the DMSO-treated cells (Fig. 2a–c, Extended Data Fig. 4f and Supplementary Videos 1, 2). The majority of LACV signal (79%) was retained within the medial Golgi in RTL-treated cells, compared with 38% in DMSO-treated controls (Fig. 2d). No gross differences in morphology were observed in the medial Golgi of uninfected RTL-treated cells compared with DMSO-treated controls (Fig. 2e,f), suggesting that the effect of RTL on LACV localization was not due to a change in Golgi architecture.

**Fig. 2: LACV localization in Golgi after RTL treatment of LACV-infected hNSCs.**

**Fig. 3: TEM images of RTL (2 × EC₅₀ dose)-treated LACV-infected hNSCs.**

As viral protein translation initiates in the endoplasmic reticulum (ER)²⁰ and RTL has been reported to modulate the morphological integrity of the ER–Golgi intermediate compartment (ERGIC)²³, we also analysed these organelles. However, no visible differences in LACV localization were observed for either the ER or ERGIC in RTL-treated versus control cells (Extended Data Fig. 4g,h). Thus, RTL treatment resulted in the accumulation of LACV signal inside the Golgi, suggesting that virus release from the Golgi was impaired.

To examine virus particles inside the cell more closely, we utilized TEM. LACV-infected hNSCs were treated with RTL or DMSO at 8 hpi and then examined at 24 hpi. Uninfected RTL-treated cells (Fig. 3b) showed no difference from untreated uninfected cells in Golgi structure (Fig. 3a). In control DMSO-treated LACV-infected hNSCs, virus was primarily observed extracellularly, but was also found inside the cell (Fig. 3c,d). Intracellular virus particles in both DMSO- and RTL-treated hNSCs were found in vesicles (Fig. 3d–f). However, in RTL-treated cells, these virus-enclosing vesicles were generally larger and often contained multiple virus particles (Fig. 3e,f). Thus, LACV-infected cells treated with RTL had virus particles in vesicles near the Golgi that appeared aberrant compared with DMSO-treated cells.

Effect of RTL on LACV infection of hCOs

We next examined whether RTL could inhibit neuronal death in a more complex system, inducible pluripotent human stem cell-derived COs. In our previous studies, LACV infection of 3-week-old hCOs resulted in neuronal apoptosis and decreased organoid viability¹⁸. hCOs infected with LACV were treated at 24 hpi with 0.3 µg ml⁻¹ RTL and followed for viability and virus production. RTL-treated hCOs had increased cell survival (Fig. 4a) and reduced virus production (Fig. 4b) compared with DMSO-treated controls. IHC staining for viral antigen correlated with these results (Extended Data Fig. 5a,b), although the varied structure, size and areas of virus infection did not allow for clear quantification of infection or apoptosis. Analysis of older 11-week-old hCOs showed some dose-dependent low-level toxicity of RTL (Extended Data Fig. 5c) of up to 12% with 0.3 µg ml⁻¹ RTL and up to 20% with 1 µg ml⁻¹ RTL. However, even with the added toxicity, RTL treatment still significantly increased cell survival following LACV infection (Fig. 4c) and reduced virus production (Fig. 4d) in the 11-week-old hCOs in a dose-dependent manner. Thus, RTL inhibited LACV-induced cell loss in the complex structure of 3- and 11-week-old hCOs.

**Fig. 4: RTL treatment inhibits LACV infection in hCOs.**

Effect of RTL on LACV-ND in mice

To examine whether RTL could inhibit LACV pathogenesis in vivo, we utilized a mouse model of LACV-ND¹². RTL toxicity was assessed by intraperitoneal (IP) administration at 20 mg per kg body weight daily in 24-day-old Macrophage Fas-Induced Apoptosis (MAFIA) mice for 7 d or by intracranial (IC) inoculation at 1 mg per kg body weight in 25- and 29-day-old MAFIA mice. MAFIA mice express green fluorescent protein (GFP) under the control of the Csf1r promoter, allowing for easy analysis of myeloid cell activation, but are otherwise fully immunocompetent. Most animals showed no response to RTL administration; however, a few mice treated by IC inoculation had short-term inactivity. Therefore, the IC dose was reduced fourfold to 250 µg per kg body weight for therapeutic studies. No negative effect was observed with IP inoculations, consistent with previous studies²⁴. Histopathological analysis of kidney, liver and spleen in RTL-treated animals showed normal histology and no increased GFP expression in brain tissue compared with controls (Supplementary Fig. 2). Thus, RTL did not appear to have toxic effects in young mice.

In most instances of LACV-E, the disease is not detected or diagnosed until neurological signs occur, indicating virus in the CNS. Therefore, we started treatment at 3 dpi, a time when LACV is consistently found in infected neurons of the brain¹⁴. LACV-infected mice were administered either 20 mg per kg RTL per day by IP inoculation at 3–10 dpi or 250 µg per kg RTL by IC inoculation at 4 and 7 dpi. LACV-infected mice treated with vehicle control (DMSO) by either the IP or IC route had a clinical disease rate of 78–81% (Fig. 5a,b). Incidence of disease in DMSO-treated mice was slightly less than in untreated animals, suggesting that DMSO alone may also have a slight protective effect. RTL treatment via either the IP or IC route significantly decreased the incidence of neurological disease compared with DMSO-treated mice (Fig. 5a,b). Thus, RTL treatment inhibited LACV pathogenesis in vivo.

**Fig. 5: RTL treatment increases survival of LACV-infected mice and reduces viral load in brain tissue.**

We also studied whether RTL protected against LACV-ND when administered through the oral route. Unfortunately, oral administration of RTL was not as effective as IP or IC inoculation, with no statistical difference in disease relative to controls (Extended Data Fig. 6a). Analysis of T-705 in vivo showed a slight, but not significant, decrease in neurological disease. Thus, RTL but not T-705 treatment was effective in inhibiting LACV-ND in mice (Extended Data Fig. 6b). RTL was most effective when administered by the IC or IP route, but not when administered orally.

Analysis of viral RNA (Fig. 5c,d) and replicating virus (Fig. 5e,f) showed a slight but not significant decrease in virus levels in RTL-treated mice at 5 dpi, 1–2 d after RTL treatment was initiated. However, by 8 dpi, a significant decrease was observed in RTL-treated mice, with non-clinical mice having reduced viral RNA and replicating virus in the brain (Fig. 5c–f). IHC analysis of non-clinical mice at 8 dpi showed widespread viral infection throughout the brain in DMSO-treated mice (Fig. 5g–i) compared with mock-infected controls (Extended Data Fig. 6c,d). In contrast, RTL-treated mice appeared to have more focal distribution of virus (Fig. 5h–j). Although the virus distribution was not quantifiable, it may suggest less spread within areas of infection in the RTL-treated mice. Similar findings were observed in clinical mice. Although high amounts of virus were found in all clinical animals, there was focal distribution in RTL-treated clinical mice (Extended Data Fig. 6e–h). Thus, RTL inhibition of LACV-ND was associated with a decreased amount of CNS virus at later stages of infection.

Effect of RTL on other CSG orthobunyaviruses

We also examined the ability of RTL to inhibit other CSG orthobunyaviruses, namely Jamestown Canyon (JTCV) and Tahyna virus (TAHV). RTL inhibited both JTCV- and TAHV-induced cell death in all cell lines tested (Supplementary Table 1 and Extended Data Fig. 7a,b). RTL treatment significantly reduced JTCV and TAHV viral RNA expression and viral production in SH-SY5Y cells (Extended Data Fig. 7c–f) following the same protocols as for LACV infection (Fig. 1). Thus, RTL inhibited virus replication for multiple CSG orthobunyaviruses. Further studies will be needed to determine whether this inhibition extends to other bunyaviruses as well.

Discussion

In this study, we utilized a high-throughput screen of FDA-approved drugs and small molecules to identify a compound, RTL, that effectively inhibited LACV replication and neuronal damage. RTL treatment in mice showed that RTL could inhibit LACV-ND even after the virus was already present in the CNS. Thus, RTL appears to inhibit CSG virus-mediated damage of neurons and could be an effective therapeutic for inhibition of CSG virus-induced encephalitis.

The Golgi apparatus has a crucial role in the bunyavirus replication cycle^20,25,26. Following translation of viral mRNA, the two surface glycoproteins, Gn and Gc, associate in heteromultimeric form after precursor polyprotein cleavage in the ER^27,28. The transmembrane domains in Gn help to localize these Gn–Gc heteromultimers to the Golgi apparatus^20,25,29,30. The Golgi Gn–Gc heteromultimers are then inserted into ribonucleoproteins, which are bound to the three segmented viral genomes to form mature virus particles that migrate towards the cell membrane using Golgi-derived vesicles²⁰. Our current studies indicate that RTL inhibits migration of LACV at the exit from the Golgi complex, as observed by confocal and TEM analysis. This correlates with the more focal infection in brain tissue observed in RTL-treated mice, suggesting that RTL may work in vivo by preventing neuron-to-neuron transmission of the virus. However, RTL may also limit virus-induced damage by other mechanisms such as by directly interfering with neuronal apoptosis or by affecting glial responses that influence neuronal apoptosis.

The hCO model of LACV infection may provide insight into how RTL could directly inhibit LACV-induced neuronal death in a complex system. We utilized hCOs that lack astrocytes and other glial cells, which allows the study of virus-induced neuronal damage without the impact of glial cells. RTL treatment reduced virus titres and limited virus-induced growth inhibition of hCOs. Because the hCOs lack consistent identical structures, we were unable to quantify cellular differences in these organoids. However, the effect of RTL on hCOs appears to be at the level of inhibiting virus replication. There was decreased virus replication at early time points PI, which continued over the duration of the experiment. Future studies of LACV-infected hCOs would be useful to determine whether subpopulations of neurons can be more protected by specific drugs.

RTL is a polyphenol (secondary metabolite) isolated from Mallotus philippinensis, which was used as an ancient medicament for its antihelminthic activity in the Ayurvedic medicinal system³¹. RTL has primarily been studied for its ability to inhibit different types of cancer³², including glioblastomas and breast cancer. Other studies have indicated potential anti-inflammatory³³, anti-oxidant³⁴, anti-allergic³³ and antibacterial³⁵ effects of RTL. Although originally thought to be a selective inhibitor of protein kinase C (PKC)-δ^36,37, in more recent studies RTL inhibited multiple kinases^23,38 and directly uncoupled mitochondrial respiration, leading to decreased cellular ATP levels^38,39. ATP is required for multiple steps in vesicular transport. Decreased cellular ATP may be a mechanism by which RTL inhibits anterograde and retrograde transport via the Golgi, rather than through direct inhibition of PKC-δ²³. Another drug that disrupts ATP synthesis, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), inhibited Semliki Forest virus and Sindbis virus membrane glycoproteins from migrating from the Golgi apparatus to the cell surface⁴⁰. Similarly, RTL inhibition of LACV viral transport from the Golgi may be due, in part, to RTL inhibition of mitochondrial respiration and ATP production.

There were eight compounds in the drug screen that can affect mitochondrial respiration and ATP production. Six of these drugs, metformin, aspirin, sorafenib, tamoxifen, cisplatin and atovaquone, did not significantly inhibit LACV-induced cell death. Two others, itraconazole and ivermectin, had an effect and were in the confirmed screen. Ivermectin was positive in the confirmation run (Extended Data Fig. 1f) but was not among the top three compounds. Ivermectin has potent antiviral activity against multiple viruses^41,42, although the exact mechanisms remain unknown. The ability of three of the eight compounds that can affect mitochondrial respiration and ATP production to inhibit LACV-induced death of neurons suggests that this may be an important pathway for drug targeting. However, it does not demonstrate that RTL functions through this pathway.

Only a small fraction of LACV infections cause severe neuroinvasive disease². Thus, conducting clinical trials assessing the efficacy of putative therapeutics is limited by patient access. Furthermore, the early common signs of disease such as fever, headache, lethargy and vomiting make early diagnosis of encephalitis difficult². A useful therapeutic drug would need to treat LACV-E at later stages when a clinical diagnosis is made, which would require that the therapeutic cross the blood–brain barrier (BBB). RTL treatment was effective at reducing LACV disease in mice, even though we delayed treatment until 3 dpi, a time where LACV can be found within the CNS¹⁴. There are no reports that RTL crosses the BBB, although its ability to treat disease would suggest that it does. Alternatively, RTL may cross the BBB following LACV-induced vascular leakage¹⁴. Possibly, virus-induced damage to the BBB may allow RTL or other therapeutics access to the CNS, which is needed to treat encephalitis.

Other molecules, including the nucleoside analogues ribavirin and T-705, have shown antiviral activity against LACV in vitro^22,43,44. Interestingly, in our study, T-705 was effective in Vero cells but was not very effective in neuronal cells (Table 1 and Extended Data Fig. 2b) or in mice (Extended Data Fig. 6b). The lack of antiviral activity of T-705 may be due to inadequate conversion to its active triphosphate state from the ribonucleotide monophosphate in neuronal cells⁴⁵. In contrast, RTL was highly efficient at inhibiting LACV infection in neuronal cells. The ability of a drug to inhibit virus replication in neurons may help determine whether specific drugs could be useful for the treatment of viral encephalitis, particularly for later stages of disease when infection is primarily found in the CNS.

Methods

Cell lines

The human neuroblastoma cell line SH-SY5Y (American Type Culture Collection, ATCC) was cultured in Eagle’s minimum essential medium (EMEM) and F-12K (Kaighn’s modification of Ham’s F-12) medium in equal amounts (1:1) supplemented with 10% FBS. These cells have previously been reported in the literature⁴⁶ to grow in clusters as is observed in Supplementary Fig. 2g,h. H9 human embryonic stem cell-derived hNSCs (ThermoFisher Scientific) were propagated in KnockOut DMEM/F-12 media containing StemPro neural supplement, GlutaMAX-I supplement, fibroblast growth factor and epidermal growth factor. The H9 cells were registered with the National Institutes of Health (NIH) Human Embryonic Stem Cell Registry, and the NIH Guidelines for Human Stem Cell Research were followed. The culture vessels were pre-coated with human plasma fibronectin (Sigma-Aldrich) for growth and experiments of hNSCs. The mouse neuroblastoma cell lines Neuro-2a (N2a) (ATCC) and C17.2 (Sigma-Aldrich) were cultured and maintained in Dulbecco’s modified Eagle medium (DMEM) containing 4,500 mg l⁻¹ d-glucose, l-glutamine and 110 mg l⁻¹ sodium pyruvate supplemented with 10% fetal bovine serum (FBS). Vero cells (African green monkey kidney cells, ATCC) were also used for the cell viability study utilizing the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay and plaque assays, as well as for viral maintenance and stock production.

Primary neuronal culture

Primary cortical neurons were isolated from the fetuses of pregnant Inbred Rocky Mountain White mice at 14–16 d gestation as previously described⁴⁷. Calcium-free, magnesium-free Hank’s balanced salt solution containing 0.125% trypsin was used to digest the mouse cortices, which were dissociated by repeated pipetting and plated in amine-coated plates (BD Biosciences). After attachment, the neuronal plating medium was replaced with neurobasal medium containing 2% B-27 and 0.5 mM glutamine and cells were cultured for at least 12 h before use in experiments.

Small-molecule screening and confirmation

SH-SY5Y cells were seeded (6,000 per well) in 12 µl of growth medium in 384-well plates (Greiner Bio-One), incubated for 24 h, then 23 nl of compounds in DMSO was dispensed per well using pintool transfer before 12 µl per well of LACV in growth medium was added (0.01 MOI) and incubated with cells for 6 d. After the 6-d incubation, LACV-induced cytotoxicity was assayed via an ATP-content assay by adding 24 µl per well of ATPLite (PerkinElmer) and reading the luminescence signal on a ViewLux (PerkinElmer). A quantitative high-throughput screening (qHTS) was performed on the following libraries: (1) the LOPAC, a collection of 1,280 small molecules with characterized biological activities, purchased from Sigma-Aldrich; and (2) the NCATS Pharmaceutical Collection of small-molecule drugs that were approved in the United States, Canada, the European Union and Japan⁴⁸. For the primary screen, each compound was assayed at four concentrations (9.6 µM, 1.9 µM, 383 nM and 77 nM) and percent activity was normalized as 100% for uninfected SH-SY5Y control and 0% for SH-SY5Y with virus (Extended Data Fig. 1a). Subsequent selection as detailed in the results section identified NBI 27914, RTL and tyrphostin AG879 as top candidates. All three compounds were purchased from Santa Cruz Biotechnology and the stock solutions were prepared by dissolving 10 mg of each compound in 1 ml DMSO (Sigma-Aldrich) and stored at −80 °C for further use.

Cytotoxicity assay

Cells were plated in 96-well plates (10⁴ cells per well) and different concentrations of each drug were added to each well, in triplicate. DMSO (0.1%) and T-705²² were used as negative and positive controls, respectively. At 48-h time intervals, cell viability (MTT) assays were carried out according to the manufacturer’s instructions (Invitrogen, ThermoFisher Scientific). This colorimetric assay was used to estimate the cell viability or cytotoxicity and is visualized by NAD(P)H-dependent oxidoreductase enzymes of metabolically active cells converting yellow tetrazolium salt to purple formazan crystals. These formazan crystals were dissolved using DMSO and the formazan concentrations were measured using the Synergy 4 cell plate reader (absorbance at 540 nm; BioTek) driven by Gen5 v1.09.7 software¹⁵. All absorbances were calculated to measure cell viability using the formula: (sample absorbance/DMSO absorbance) × 100%. The CC₅₀ (50% cytotoxic concentration) was determined by extrapolating the dose–response curve.

Antiviral efficacy study

Cells (10⁴ cells per well) were grown in 96-well plates, infected with LACV (0.1 MOI) and treated with different concentrations of test drugs, using DMSO (0.1%) and T-705 (0–125 µg ml⁻¹) as controls. At specific time intervals, the MTT assay was carried out. For SH-SY5Y cells and hNSCs, the MTT assay was measured at 48 hpi, while for mouse primary neurons and N2a and C17.2 cells, the MTT assay was performed at 72 hpi. The EC₅₀ of the drug was calculated by plotting dose-dependent curves.

Viral load determination

Cells were cultured in 24-well plates (2 × 10⁵ cells per well) and infected with LACV (0.1 MOI). At 1 hpi, medium was removed and fresh medium containing EC₅₀ and 2 × EC₅₀ concentrations of RTL or DMSO (0.1%) was added. At specific time intervals (6, 12 and 24 h for RNA expression, and 12, 24 and 48 h for plaque assay), cell supernatant was removed, centrifuged to eliminate cell debris and frozen at −80 °C for plaque assay following the method of Evans et al.¹⁶, and cells were subjected to RNA isolation for qRT-PCR to determine the viral load inside the cells. Data were analysed using Microsoft Excel v.2102 and converted into graphs, and statistical analysis was completed using GraphPad Prism v.7.04 or v.8.2.0.

RNA isolation, cDNA synthesis and qRT-PCR

RNA was isolated from each well using an RNA isolation kit (Zymo Research). cDNA was synthesized from the RNA samples using an iScript reverse transcription kit (Bio-Rad Laboratories). A fivefold dilution of cDNA samples in RNase-free water was used for qRT-PCR analysis using SYBR Green SuperMix (Bio-Rad Laboratories) using a ViiA 7 instrument and software (v1.2.2) following the method of Butchi et al.⁴⁷. The sequences of the forward and reverse primers were as follows: LACV ‘S’ gene forward (5′-ATTCTACCCGCTGACCATTG-3′), reverse (5′-GTGAGAGTGCCATAGCGTTG-3′); LACV ‘M’ gene forward (5′-TTGCCTGGCTGTAAGTGATG-3′), reverse (5′-CAGTCACTTCCTCGGTCTCC-3′); LACV ‘L’ gene forward (5′-TCTGTGGGTGATTGTGCCTA-3′), reverse (5′-TGCTATGGACACCCATGCTA-3′); JCTV forward (5′-TATGGTTCCCCGGTAGTGTG-3′), reverse (5′-TAACATGGTGCTTCTCGTGC-3′); TAHV forward (5′-AGGTCCTACATTGCCGTTCA-3′), reverse (5′-TGGTCTACAGGTGCTAGCTC-3′); mouse Gapdh forward (5′-AACGACCCCTTCATTGAC-3′), reverse (5′-TCCACGACATACTCAGCA-3′); human GAPDH forward (5′-TCGTGGAAGGACTCATGACC-3′) and reverse (5′-ATGATGTTCTGGAGAGCCCC-3′). Each primer set was reported with the mean of three mock controls. Each viral RNA sample was quantified as the percent variance in cycle threshold (CyT) value = CyT of GAPDH − CyT of viral RNA. Data were analysed using Microsoft Excel v.2102 and converted into graphs, and statistical analysis was completed using GraphPad Prism v.7.04 or v.8.2.0.

Confocal microscopy

Cells were grown on eight-well chambered cover glasses (Nunc Lab-Tek, ThermoFisher Scientific) and infected with LACV at 0.1 MOI. Cells were treated with either RTL (EC₅₀ or 2 × EC₅₀ concentrations) or vehicle (DMSO) control at the indicated time points. At 24 hpi, cells were washed with PBS and fixed with 4% paraformaldehyde in PBS. Cells were then permeabilized with 0.01% Triton X-100 and 0.1% saponin and blocked with 5% donkey serum in PBS (‘blocking buffer’). Primary antibodies (Supplementary Table 2) were diluted in blocking buffer and incubated at room temperature for 1 h. Cells were labelled with secondary antibodies (Supplementary Table 2) for 1 h and Hoechst 33342 (Tocris Bioscience) for nuclei staining, and washed with PBS. Samples were imaged using a Zeiss laser scanning confocal microscope (LSM 880) driven by ZEN v.2.3 software (Carl Zeiss Microscopy).

Image processing and analysis

For Fig. 1, image stacks were imported directly into Imaris ×86_64 v. 9.5.1 (Bitplane) from ZEN software. The LACV and Hoechst (cell nuclei) signals were segmented using the Surfaces module, and infected cells were identified using a distance transformation filter to the closest LACV signal. For Fig. 3, image stacks were exported from ZEN software and deconvolved with Huygens Professional v.19.04 (Scientific Volume Imaging) using the CMLE algorithm, with SNR = 20 and a maximum of 40 iterations. The deconvolved datasets were imported to Imaris for segmentation, surface rendering using the Surfaces module and quantification. The Spots module was used to calculate the total integrated intensity of LACV signal in LACV-infected cells, and a distance transformation filter was applied to generate the subset of LACV integrated intensity within the medial Golgi. The sets of integrated intensity values (n = 397 cells analysed for vehicle control, and n = 316 cells analysed for 8 hpi RTL from three independent biological replicates) were imported into Microsoft Excel for data compilation and graphing, and analysed using GraphPad Prism v. 8.2.0.

Time of drug addition assay

SH-SY5Y cells or hNSCs were treated with RTL (2 × EC₅₀) as a pre-treatment, co-treatment, post-treatment or CT²¹. For pre-treatment, cells were incubated with RTL for 2 h, washed with PBS and then infected with LACV (0.1 MOI) for 1 h, washed again and incubated with normal medium. For co-treatment, cells were simultaneously infected with LACV and treated with RTL for 1 h, after which cells were washed in PBS and fresh medium was added. For post-treatment, cells were infected with LACV for 1 h, washed with PBS and then fresh medium containing RTL was added. For CT, RTL was added 2 h before infection and then continuously maintained in the culture. For all conditions, cells were incubated at 37 °C for 2 d and then MTT assays were carried out as described above. For further analysis of post-addition time points²¹, cells were infected with LACV (0.1 MOI) for 1 h, washed with PBS and then cultured with fresh medium. At intervals of 2, 4, 6, 8 and 12 hpi, this medium was replaced with medium containing RTL (2 × EC₅₀). Cells were analysed at 24 hpi for qRT-PCR or at 48 hpi for MTT assay.

Attachment assay

Cells grown in 24-well plates (2 × 10⁵ cells per well) were pre-chilled at 4 °C for 1 h and subsequently challenged with LACV (0.1 MOI) in the presence of RTL (2 × EC₅₀) or heparin sodium salt (HPR; 1,000, 200 and 40 µg ml⁻¹) or DMSO for 3 h at 4 °C. After infection, the wells were washed twice with ice-cold PBS to remove unbound virus and fresh medium was added. At 1 or 24 hpi, qRT-PCR was carried out.

Virus inactivation assay

LACV (10⁶ PFU ml⁻¹) was treated with RTL (2 × EC₅₀ concentration), hyperimmune serum from infected adult mice that had high levels of neutralizing antibodies (Nab; 2.5, 0.25 and 0.025 µl) or DMSO at 37 °C for 1 h and then the mixture was diluted 100-fold with fresh medium to yield a sub-therapeutic concentration of the test compound. The virus inocula were then added to the SH-SY5Y cell monolayer. The 100-fold dilution served to titrate RTL below effective doses and prevent meaningful interactions with the host cell surface. After adsorption for 1 h at 37 °C, the diluted inocula were discarded, the cells were washed with PBS twice and fresh medium was added. At 24 hpi, qRT-PCR was carried out.

TEM

hNSCs were cultured on Thermanox coverslips, infected with LACV (0.1 MOI) and treated with RTL or DMSO at 8 hpi. At 24 hpi, cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. All subsequent steps of post-fixation were carried out in a Pelco Biowave microwave (Ted Pella) at 250 W. The cells were dehydrated in a graded ethanol series, infiltrated with Spurr’s resin and polymerized in a 60 °C oven. Ultrathin sections were cut using a Leica UC6 ultramicrotome (Leica Microsystems) and imaged on a Hitachi H7800 instrument (Hitachi High-Technologies) using a Hamamatsu Orca HR camera (Advanced Microscopy Techniques).

hCOs

hCOs were generated, maintained and infected with 1 × 10³ PFU LACV in 24-well culture plates under previously described conditions¹⁸. At 24 hpi, hCOs were treated with RTL at a concentration of 0.1, 0.3 or 1.0 µg ml⁻¹ or DMSO in 1 ml of CO culture medium. Culture supernatant from each organoid was collected every 24 h through the end of the experiment (6 d) and analysed for infectious virus via plaque assay as previously described. hCOs were also assayed for cell viability daily using a resazurin reduction-based colorimetric assay dye (PrestoBlue, Molecular Probes, ThermoFisher Scientific) as previously described¹⁹. Data are reported as the percent fluorescence at 590 nm relative to the 0 dpi baseline taken for each hCO. IHC analysis of hCOs was done at 3 or 6 dpi as described previously¹⁸. Data were analysed using Microsoft Excel v.2102 and converted into graphs, and statistical analysis was completed using GraphPad Prism v.7.04 or v.8.2.0

Mouse studies

All animal experiments were conducted in compliance with protocols 2016-016 and 2019-23, which were approved by the NIH/NIAID/RML Institutional Animal Care and Use Committee. For this study, MAFIA mice (C57BL/6-Tg(Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6)2Bck/J stock no. 005070) with fluorescently tagged microglia cells (Csf1r expressing) were used to allow for easy detection of microglia activation. A mix of male and female mice were used for all studies and were divided evenly between groups. Group size was determined using GraphPad StatMate with an 80% power to detect a 40% increase or decrease in disease development. Mice in different litters were randomly assigned to different groups. Mice were housed with a 12 h light/dark cycle at a temperature range of 21–24 °C and a humidity range system set to 50% ± 10%, and were maintained on 2016 Teklad global rodent chow. At 22–24 days old, mice were infected with 1 × 10³ PFU LACV by IP route. At 3 dpi, some mice were treated IP with RTL (20 mg per kg), T-705 (250 mg per kg) or DMSO for eight consecutive days. For IC treatment, mice were injected with RTL (250 µg ml⁻¹) or DMSO at 4 and 7 dpi using a Hamilton syringe (700 Series syringes, 10-µl volume) with a 33-gauge needle⁴⁹. For treatment via oral administration, mice were given RTL (50 mg per kg) or DMSO once daily for seven consecutive days, starting at 3 dpi, using a straight 24-gauge ball-tip disposable feeding needle (Instech). Mice were observed twice daily for 26–30 d for clinical signs of neurologic disease, which primarily included obvious ataxia, limb paralysis and weakness or repeated seizures. Mice showing any of these clinical symptoms were scored as clinical and euthanized for tissue removal. A total of 12 mice were excluded from this study due to issues with either a mistake in virus dose or development of unrelated disease. One experiment was removed completely due to a mistake in the dose of virus inoculation (too low), which included four control mice and five RTL-treated mice with no signs of neurological disease in either group. One mouse was excluded due to genital tract infection before infection and two mice (one DMSO treated and one FPV treated) were removed due to hydrocephaly early on after infection.

Viral load in the CNS

Brain tissues were removed from mice at the initial appearance of clinical signs (as described above) or at 5 and 8 dpi. Half of a sagittal section of the brain tissue was stored at −80 °C to be used for either RNA or plaque assay. The other half of the sagittal section was immediately placed in 10% neutral buffer formalin solution and used for IHC to evaluate virus infection. Sectioning of the brains and IHC were performed as previously described¹⁶. In brief, fixed tissue was embedded in paraffin and sliced in 5-µm sections. Slides were deparaffinized, rehydrated and antigen retrieval was conducted in a Decloaking chamber (Biocare Medical) using pH 6 citrate target retrieval solution (0.1 M citric acid and 0.1 M sodium citrate dehydrate) for 20 min at 120 °C. Slides were blocked in PBS containing 0.3 M glycine and 5% donkey serum. Slides were incubated with primary antibodies diluted in blocking buffer at 4 °C overnight. Slides were then labelled with secondary antibodies for 1 h and Hoechst 33342. Slides were scanned with a slide scanner (Axio Scan.Z1) using ZEN Blue 3.1 software (Carl Zeiss Microscopy). RNA isolation and qRT-PCR were carried out as described in Woods et al.⁴⁹. For plaque assay, brain tissues were homogenized at 5,300 r.p.m. for 25 s in a homogenizer (Bead Mill 24, Fisher Scientific) in the presence of DMEM. Samples were then centrifuged at 5,000g for 10 min and supernatants were transferred into a new tube. Samples were then diluted to 10⁻¹ to 10⁻⁷ in DMEM containing 10% FBS before being used in a plaque assay¹⁶.

Statistical analysis

For in vitro studies, no statistical methods were used to pre-determine sample sizes, but our sample sizes are similar to those reported in previous publications^15,16. All in vitro experiments were set up in triplicate samples with two to three repeated experiments. This provides controls for both in-experiment and between-experiment variation. For mouse studies, StatMate was used to calculate the number of animals to complete the studies using a predicted 80% of mice developing clinical signs of disease, and an 80% power to detect a 30% decrease in survival proportion with a significance level of 0.05 (two-tailed analysis in case the results were opposite to what we expected). All post-study statistical analysis was performed using GraphPad Prism v.7.04 and v.8.02. In each case, P values are represented as *P < 0.05, **P < 0.01, ***P < 0.001 and **** P < 0.0001. Specific analysis information is described in the figure legends. Exact P values are presented in Supplementary Table 3.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. The primary screening data have been made publicly available as PubChem bioassay assay ID (AID) 1508616. Source data are provided with this paper.

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Acknowledgements

We thank P. Policastro and A. B. Evans for assistance with cells, virus cultures and antibody preparation, and T. Starr for confocal microscopy assistance; A. Mora, R. Kissinger and the graphics art unit at RML for graphics assistance and figure preparation; B. Caughey, L. H. Evans, A. Chong and A. I. Chiramel for review of the manuscript; D. Long for assistance with tissue section preparations and H&E staining, and L. Lubke for light microscopy imaging; and the Rocky Mountain Veterinary Branch for their excellent animal husbandry and assistance. This work was supported by the Division of Intramural Research, National Institutes of Health, National Institute of Allergy and Infectious Diseases, 1ZIAAI001102-09 (K.E.P.). The funder had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA
Durbadal Ojha, Clayton W. Winkler, Jacqueline M. Leung, Tyson A. Woods, Vinod Nair, Katherine Taylor, Charles L. Larson, Cathryn L. Haigh & Karin E. Peterson
National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA
Catherine Z. Chen, Charles D. Yeh, Gregory J. Tawa & Wei Zheng

Authors

Durbadal Ojha
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Clayton W. Winkler
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Jacqueline M. Leung
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Tyson A. Woods
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Catherine Z. Chen
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Vinod Nair
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Katherine Taylor
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Charles D. Yeh
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Gregory J. Tawa
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Wei Zheng
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Cathryn L. Haigh
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Contributions

K.T., D.O. and K.E.P. conceived the idea and designed the experiments. C.Z.C., C.D.Y., G.J.T. and W.Z. completed the initial and secondary screens of the compounds. D.O., C.W.W., J.M.L., T.A.W., V.N., C.L.L. and C.L.H. completed the final screens, examined the mechanisms of RTL inhibition and completed the organoid and animal studies. D.O. and K.E.P. wrote the manuscript. D.O., C.W.W., J.M.L., T.A.W., C.Z.C., V.N., C.L.H. and K.E.P. helped revise and edit the manuscript.

Corresponding author

Correspondence to Karin E. Peterson.

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The authors declare no competing interests.

Additional information

Peer review information Nature Microbiology thanks Juan Carlos de la Torre, Masayuki Saijo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1

(a) Schematic diagram of detailed drug screening methods. (b-k) Anti-LACV activity of (b) tyrphostin AG879, (c) rottlerin, (d) NBI 27914, (e) cycloheximide, (f) ivermectin, (g) SMER 28, (h) ebselen, (i) hexachlorophene, (j) clofucarban and (k) disulfiram on SH-SY5Y cells. SH-SY5Y cells were grown on 384-well plates. Cells were pretreated with experimental compounds or control. Approximately 1 h after addition of compounds, LACV (0.01 MOI) was added to each well. After 6 days of incubation, ATPlite™ reagent was added to assay ATP content as a measure of cell viability. The % activity plotted was calculated as: % activity = (Signal – Min) / (Max – Min) x100; where Max = SH-SY5Y without virus; Min = SH-SY5Y infected with virus. Each symbol represents the mean ± SD of six wells from two independent experiments.

Source data

Extended Data Fig. 2

(a, b) Dose-dependent anti-LACV activity of (a) RTL and (b) T-705 on Vero, N2a, C17.2, SH-SY5Y, hNSCs, and mouse primary neurons. Cells (10⁴ cells/well) were plated in 96-well plates and infected with LACV (MOI 0.1) for one hour. Cells were then treated with different concentrations of (a) RTL and (b) T-705 using DMSO (0.1%) as control. At specific time intervals (72 h for rodent-derived cells and 48 h for human-derived cells), an MTT assay was carried out as described in the methods. Each symbol represents the mean ± SD of nine wells combined from three independent experiments except for mouse primary neurons where error bars represent three individuals well of a single experiment. (c-f) Anti-LACV activity of RTL in multiple neuronal cell lines. Viral RNA expression in (c) N2a, (d) mouse primary neuron, (e) SH-SY5Y and (f) hNSCs. Cells were infected with LACV for 1 hour followed by RTL treatment at EC₅₀ and 2xEC₅₀ doses. At 6, 12 and 24 hpi, RNA was isolated from cells for qRT-PCR analysis. Each symbol represents the mean ± SD of nine individual wells per cell type combined from 3 independent experiments, except for primary neurons where data are from three individuals well of a single experiment. *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001; Statistics were done with a One-way ANOVA at each time point by Dunnett’s multiple comparisons test. The percentage viral RNA expression of cell control (CC; mock control) were between 10^-1 to 10^-3. (g, h) Immunofluorescence labelling with (g) MAP2 and (h) NeuN of uninfected untreated N2a, SH-SY5Y, hNSCs cells. MAP2 and NeuN signal are shown in yellow and host cell nuclei is shown in cyan. Scale bar, 50 µm. Images are representative of 2 replicate experiments.

Source data

Extended Data Fig. 3 Time kinetics study of RTL and virus replication kinetics.

(a, b) Effect of RTL on virus attachment in SH-SY5Y cells. Prechilled SH-SH5Y cells were infected with LACV (MOI 0.1) mixed with RTL, heparin sodium salt or DMSO. HPR and DMSO were used as the positive and negative control respectively. After 3 h at 4 °C incubation, the cells were washed twice with ice-cold PBS and fresh media added. After (b) 1 or (a) 24 hpi qRT-PCR was carried out. Line with error bars indicates the mean ± SD for nine data points (symbols) of three independent experiments for each group. **P < 0.01 and ****P < 0.0001; by one-way ANOVA using Dunnett’s multiple comparisons test. (c) Virus inactivation assay of RTL on SH-SY5Y cells. RTL (2xEC₅₀ concentration), neutralizing antibody (NAb; 2.5, 0.25, and 0.025 µl) or DMSO was preincubated with LACV (10⁶ PFU) at 37 °C for 1 h. NAb and DMSO were used as a positive and negative control respectively. Then the mixture was diluted 100-fold and added to the SH-SY5Y cells monolayer. After 1 h at 37 °C incubation, the cells were washed with PBS twice and fresh media added. After 24 hpi incubation qRT-PCR was carried out. Line with error bars indicates the mean ± SD for nine data points (symbols) of three independent experiments for each group. ****P < 0.0001; by One-way ANOVA using Dunnett’s multiple comparisons test. (d) Extracellular virus production in cell supernatant in RTL treatment after 2, 8 and 12 hpi. Cells were infected with LACV (MOI 0.1) for 1 h, washed with PBS, and then cultured with fresh media. At intervals of 2, 8 and 12 hpi, this media was replaced with media containing RTL (2xEC₅₀). At 24 hpi, cell supernatants were harvested and titered by plaque assay. Line with error bars indicates the mean ± SD for nine data points (symbols) of three independent experiments for each group. ***P < 0.001 by Two-way ANOVA using Sidak’s multiple comparisons test. (e, f) Time of RTL addition on post-infection at different time intervals (2 h, 4 h, 8 h and 12 h) measuring (e) M and (f) L segment RNA expression. Experiments were performed following the same protocols as for Fig. 1k. Line with error bars indicates the mean ± SD for nine data points (symbols) of three independent experiments for each group. ****P < 0.0001; by One-way ANOVA using Dunnett’s multiple comparisons test. CC indicates cell control (mock control) and VC indicates vehicle control. (g, h) Time of RTL addition on post-infection at different MOI (1 to 100) as well as different time interval (2 h, 4 h, 8 h and 12 h) by MTT assay. (g) SH-SY5Y cells and (h) hNSCs were infected with LACV at 1, 10 and 100 MOI for 1 h, washed with PBS, and then cultured with fresh media. At intervals of 2, 4, 8 and 12 hpi, media was replaced with media containing RTL (2xEC₅₀). At 48 hpi MTT assay was carried out and cell viability was measured following previously described methods. Data are shown for six individual data points from two independent experiments with the mean ± SD. ***P < 0.001; by two-way ANOVA with Tukey’s multiple comparisons. (i) Virus release curves at different time intervals. hNSCs and SH-SY5Y cell monolayers (1×10⁵ cells/well) were infected with LACV (MOI 0.1) and allowed to absorb for 1 h. The inoculum was removed, and the cells were rinsed with PBS three times prior to the addition of fresh growth medium. At intervals post-infection, the media were collected separately, clarified by centrifugation, and frozen at -80 °C. The virus titre in each sample were determined by plaque assay. Data are shown for the individual samples from three independent experiments along with a line indicating the mean ± SD bars.

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Extended Data Fig. 4

(a) Effect of RTL or Brefeldin A separately or in combination on LACV RNA expression by qRT-PCR. To examine if RTL mediated virus inhibition through the same pathway as Brefeldin A, we analysed whether the two compounds had a synergistic or additive effect. hNSCs were cultured in 24-well plates (2×10⁵ cells/well) and infected with LACV (MOI 0.1). At 1 hpi, cells were washed with PBS and fresh media containing different concentrations of Brefeldin A or RTL or both were added. At 24 hpi, cells were lysed, and RNA was collected for qRT-PCR analysis. Each data set represents the mean ± SD of six replicate wells of two individual experiments. ****P < 0.0001; by one-way ANOVA using Dunnett’s multiple comparisons test. (b-e) LACV co-localization with medial Golgi in hNSCs at different time intervals. hNSCs were grown in 8-chamber slide and infected with LACV. At different time intervals post-infection, infected cells were fixed with 4% PFA and labelled using primary antibodies against LACV (magenta) and Giantin (yellow). (b) Cell control; (c) 6 hpi; (d) 8 hpi; (e) 12 hpi. Scale bar, 10 μm. Images are representative of 2 replicate experiments. (f) 2X images from Fig. 2bmedial Golgi images. (g-h) Staining of (g) ER and (h) ERGIC in LACV-infected, RTL-treated hNSCs. LACV infected hNSCs were treated with either DMSO or RTL at the dose of 2xEC₅₀ at 8 hpi. After 24 hpi, cells were immunostained with anti-LACV and either anti-Calnexin (ER) or anti-ERGIC antibody. Cells were imaged as described for Fig. 2. Scale bars (for all confocal images), 5 µm. Images are representative of 2-3 replicate experiments.

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Extended Data Fig. 5 Immunolabelling of LACV-infected hCOs treated with RTL.

LACV-infected 3 week old hCOs were stained at (a) 3 dpi and (b) 6 dpi with LACV (magenta), Hoechst (cyan) and active caspase 3 (yellow). -LACV, +RTL indicates uninfected and RTL treated control; +LACV,-RTL (DMSO) indicates infection control treated with DMSO; +LACV, +RTL indicates infected and treated with RTL. Scale bars for all confocal images are 50 µm. Images are representative of 8-9 organoid per group. (c) Dose-dependent cell viability assay of RTL on 11 weeks old hCOs. Organoids were treated with 1, 0.3 and 0.1 µg/ml of RTL or DMSO up-to 6 dpi. Every day organoids were assayed for cell viability using a resazurin reduction-based colorimetric assay dye. Dye intensity was compared to the intensity on day 0 of the same organoid to calculate a percentage of viability compared to baseline. Each bar represents the mean ± SD of an independent experiments of 6 organoids in each group. ***P < 0.001; by two-way ANOVA with a Dunnett’s multiple comparisons tests.

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Extended Data Fig. 6

In-vivo efficacy of (a) oral treatment of RTL or (b) IP treatment of FPV(T-705) on LACV-induced neurological disease. (a, b) A mix of male and female MAFIA mice at 22 to 24 days of age were infected with 1 ×10³ PFU of LACV by intraperitoneal administration. (a) For oral treatment, mice were given RTL (50 mg/kg; n = 17) or DMSO (n = 17) daily for eight days starting at 3 dpi and then followed for signs of clinical disease. (b) For FPV IP treatment, mice were given FPV (250 mg/kg; n = 23) or DMSO (n = 20) daily for eight days starting at 3 dpi and then followed for signs of clinical disease. (c-h) Distribution of LACV antigen in brain sections of (c, d) uninfected RTL treated non-clinical mice and (e-h) LACV-infected (e, f) DMSO or (g, h) RTL IP-treated clinical mice. At 22-24 days old mice were inoculated (IP route) with LACV (1 ×10³ PUF/mice) or media (uninfected control). After 3 dpi, mice were treated IP either with RTL (20 mg/kg) or DMSO. Brains were removed from these mice at the initial appearance of clinical signs and processed for IHC. Virus is shown in magenta and nuclei are shown in cyan. (d, f, h) are cropped images of caudate and thalamus region of (c, e, g) respectively. White arrows indicate the virus spread. Scale bar, 1 mm. Images are representative of 2-4 mice per group.

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Extended Data Fig. 7

(a, b) Dose-dependent activity of RTL on (a) JTCV and (b) TAHV-induced cell death. Cells (10⁴ cells/well) were infected with either JTCV or TAHV (MOI 0.1). At 1 hpi, cells were treated with different concentrations of RTL or DMSO (0.1%) as a control. At specific time intervals post infection (48 hpi for SH-SY5Y, or 72 hpi for N2a and primary neurons), MTT assay was carried out and the percentage of cell viability was calculated as described in the methods. Each symbol represents the mean ± SD of the combined data of triplicate wells from one to three independent experiments. (c-f) Effects of RTL addition on (c, d) JTCV and (e, f) TAHV replication. SH-SY5Y cells (2×10⁵ cells/ well of 24 well plate) were infected with (c, d) JTCV or (e, f) TAHV (MOI 0.1) for 1 hour. Unbound virus was removed and RTL (EC₅₀ or 2xEC₅₀) or DMSO was added to appropriate wells. At indicated time points (c, e) cell supernatant was harvested for plaque assay or (d, f) RNA was isolated from cells for qRT-PCR. Each symbol represents the mean ± SD of six individual wells combined from two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001; by One-way ANOVA using Dunnett’s multiple comparisons test. In (d, f), the percentage viral RNA expression of cell control (CC; mock control) were between 10^-1 to 10^-3.

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Ojha, D., Winkler, C.W., Leung, J.M. et al. Rottlerin inhibits La Crosse virus-induced encephalitis in mice and blocks release of replicating virus from the Golgi body in neurons. Nat Microbiol 6, 1398–1409 (2021). https://doi.org/10.1038/s41564-021-00968-y

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Received: 08 October 2020
Accepted: 25 August 2021
Published: 21 October 2021
Issue Date: November 2021
DOI: https://doi.org/10.1038/s41564-021-00968-y

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Abstract

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Main

Results

Screen of compounds for inhibition of LACV-induced cell death

Analysis of selected compounds in multiple cell lines

Effect of RTL on virus replication

Analysis of RTL activity at early time points in virus replication

Kinetic determination of RTL inhibition

Analysis of RTL effect on LACV localization within the cell

Effect of RTL on LACV infection of hCOs

Effect of RTL on LACV-ND in mice

Effect of RTL on other CSG orthobunyaviruses

Discussion

Methods

Cell lines

Primary neuronal culture

Small-molecule screening and confirmation

Cytotoxicity assay

Antiviral efficacy study

Viral load determination

RNA isolation, cDNA synthesis and qRT-PCR

Confocal microscopy

Image processing and analysis

Time of drug addition assay

Attachment assay

Virus inactivation assay

TEM

hCOs

Mouse studies

Viral load in the CNS

Statistical analysis

Reporting Summary

Data availability

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