The lipopeptide Pam3CSK4 inhibits Rift Valley fever virus infection and protects from encephalitis

Rift Valley fever virus (RVFV) is an encephalitic bunyavirus that can infect neurons in the brain. There are no approved therapeutics that can protect from RVFV encephalitis. Innate immunity, the first line of defense against infection, canonically antagonizes viruses through interferon signaling. We found that interferons did not efficiently protect primary cortical neurons from RVFV, unlike other cell types. To identify alternative neuronal antiviral pathways, we screened innate immune ligands and discovered that the TLR2 ligand Pam3CSK4 inhibited RVFV infection, and other bunyaviruses. Mechanistically, we found that Pam3CSK4 blocks viral fusion, independent of TLR2. In a mouse model of RVFV encephalitis, Pam3CSK4 treatment protected animals from infection and mortality. Overall, Pam3CSK4 is a bunyavirus fusion inhibitor active in primary neurons and the brain, representing a new approach toward the development of treatments for encephalitic bunyavirus infections.


Introduction
Rift Valley fever virus (Phlebovirus riftense, RVFV) is an emerging, arthropod-borne Phenuivirus that can cause encephalitis in humans.RVFV is endemic in sub-Saharan Africa and has spread since its discovery, causing outbreaks across the continent, the surrounding islands, and the Arabian Peninsula [1][2][3].The World Health Organization has designated RVFV as a research priority because of its broad vector tropism and potential to severely impact humans and agriculturally important animals [4].In ruminant animals, RVFV causes abortions and is highly pathogenic in newborns [5,6].Humans can become infected through contact with infected fluids, or by mosquito bite.Many human infections are asymptomatic or produce flulike illness, but 8-10% develop severe symptoms including eye disease, hemorrhage, and meningoencephalitis [6][7][8][9].Neurologic disease is characterized by symptoms such as disorientation, hallucinations, vertigo, and/or coma, which often present days or weeks after the onset of infection [7][8][9].There are no approved treatments for RVFV infection in humans, and few antivirals in development that target the neurologic stage of disease, although the delayed appearance of neurologic symptoms may provide a temporal window for the delivery of antivirals.
Antiviral therapeutics often target viral entry, such as neutralizing antibodies and fusion inhibitors that have been used to treat human immunodeficiency virus (HIV) and severe acute respiratory syndrome-coronavirus-2 [10][11][12].RVFV entry may also be a promising target for antiviral inhibition.RVFV enters host cells using the glycoproteins Gn and Gc [13,14].RVFV Gn on the virion surface can bind the host protein low-density lipoprotein receptor-related protein 1 (LRP1) [15].Next, RVFV is endocytosed, and membrane fusion is initiated when the endosomal pH drops below ~5.5, triggering the rearrangement of RVFV Gc, a class II fusion protein [13,14].Treatments which block RVFV fusion can inhibit infection in vitro, including endosomal acidification inhibitors [13,16,17], Gc binding peptides [18], virion membraneintercalating amphipathic compounds [19], and fusion-inhibiting antibodies [20].However, it is unknown whether such antivirals can decrease RVFV infection in neurons or ameliorate encephalitic disease.
Neurons, a primary target of RVFV infection in the brain [21], are essential, terminally differentiated, long-lived, and largely non-replenished cells.The brain is protected by the bloodbrain barrier, which restricts the ingress of cells and pathogens from the blood, yet RVFV can surmount this barrier.In neurons, as in all cells, the innate immune system detects and responds to infections.Innate immunity is characterized by two canonical pathways: the inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and antiviral interferon (IFN) pathway.RVFV infection of the brain induces NF-κB and type-I IFN responses [7,21], but it is unclear if innate immunity is protective in this tissue, as inflammation in the brain can lead to encephalitic death [22].It is hypothesized that innate immune responses in neurons are regulated or executed differently to avoid damage [23,24].Indeed, while IFN pretreatment is broadly antiviral, IFN-exposed neurons remain vulnerable to viral infections [25][26][27][28].Although little is known about neuronal immunity during RVFV infection, the distantly related Peribunyavirus, La Crosse virus (LACV, Orthobunyavirus lacrosseense) readily infects neurons in the mouse brain, even while neighboring astrocytes and microglia
We set out to identify immune stimuli that inhibited neuronal RVFV infection.We found that treating neurons with IFNs led to the transcription of interferon stimulated genes (ISGs) including antiviral effectors which directly antagonize viral processes like interferon induced protein with tetratricopeptide repeats 1-3 (IFIT1-3) [33], as well as chemokines such as CXCmotif ligand 10 (CXCL10, also known as IP-10) [22].However, IFN pretreatment weakly blocked RVFV replication in neurons compared to non-neuronal cells.These data suggest that IFN signaling is not sufficient to control RVFV infection in neurons.
To identify alternative immune agonists with anti-RVFV activity in neurons, we screened a diverse library of innate immune ligands.We identified two Toll-like receptor 2 (TLR2) agonists that were protective against RVFV infection.We found that the synthetic lipopeptide Pam 3 CSK 4 blocked infection by RVFV and LACV in neurons and non-neuronal cells.TLR2 inflammatory signaling was not required for Pam 3 CSK 4 antiviral activity.Rather, we found that Pam 3 CSK 4 inhibited RVFV fusion.Pam 3 CSK 4 was active in vivo, as treatment prevented RVFV infection in the mouse brain and protected from mortality.These findings reveal a novel function of Pam 3 CSK 4 , a molecule that has potential in the control of encephalitic bunyavirus infection.

Interferons do not efficiently protect neurons from RVFV infection
Previous studies of RVFV have shown that type I interferon (IFN) responses are important in controlling infection peripherally [34][35][36].However, it has not been tested if IFNs are protective in the brain, as IFN-treated neurons remain susceptible to some viral infections [25,26,28,30,37].We tested this in vitro using the biosafety level two strain RVFV MP-12, which is attenuated by nine amino acid substitutions across all three genomic segments [38].At baseline, RVFV MP-12 readily infected primary rat (Rattus norvegicus) cortical neurons and human-derived U2OS osteosarcoma cells, a non-neuronal cell (S1A and S1B Fig) .We pretreated neurons or U2OS cells for four hours with several doses of universal IFNα [39] or vehicle (water) before infecting with RVFV, and quantified infection by immunofluorescence microscopy 24 hours post infection (hpi; Fig 1A -1D).A high dose of 12,000 Units/mL of IFNα blocked infection of U2OS, but had little effect in neurons, suggesting that IFN treatment of neurons is not protective against RVFV (Fig 1A -1D).In U2OS, the inhibitory concentration 50% (IC50) of universal IFNα was 16.9 U/mL, while in neurons 12,000 U/mL of IFNα failed to reduce infection to 50% of control (Fig 1C and 1D).We observed no IFN-induced toxicity in either cell type (Fig 1C and 1D).
We complemented these microscopy-based studies with plaque assays and real-time, reverse transcription quantitative polymerase chain reaction (qPCR) to measure viral RNA.In U2OS cells, a four hour pretreatment with 12,000 U/mL of universal IFNα reduced RVFV titers from 9.3 x 10 5 to 6.8 x 10 4 plaque forming units (PFU) / mL at 16 hpi, and decreased RVFV RNA at 24 hpi by ~30 fold (Fig 1E and 1F).In neurons, the same treatment reduced RVFV titers from 8.7 x 10 3 to 6.1 x 10 3 PFU / mL, and decreased RVFV RNA ~2-fold compared to vehicle (Fig 1I and 1J).While these decreases were statistically significant, the small magnitude of change in neurons suggests that IFN treatment is not as potent in neurons as in other cell types.
To evaluate whether neurons and U2OS respond to IFN, we stimulated with 12,000 U/mL of universal IFNα or vehicle for four hours and measured ISG transcription by qPCR.We measured IFIT1 in U2OS cells as it is well known to be transcribed and translated in these cells following IFN stimulation [40].In neurons, we measured Ifit2 because it has been reported that Ifit1 basal expression can be elevated in some subpopulations of mouse neurons [28].In both U2OS and rat neurons, we observed multi-log increases in the RNA levels of ISGs (Fig 1G , 1H, 1K and 1L).We conclude that both cell types detect and respond to IFNα.
To test whether species-specific IFNs would be more active than universal IFNα, we pretreated rat cortical neurons with rat IFNα, IFNβ, or IFNγ and evaluated RVFV infection by microscopy and qPCR.None of the rat IFNs tested decreased infection below 50% of control levels at 12,000 U/mL (Fig 1M ), or significantly reduced RVFV RNA (S1C Fig) .These data support our finding that IFN responses are induced but insufficient to control RVFV infection in neurons.Therefore, we set out to identify alternative innate immune ligands that could block neuronal RVFV infection.

Pam 3 CSK 4 is antiviral against Phenuiviridae and Peribunyaviridae in vitro
We validated that Pam 3 CSK 4 had antiviral activity against RVFV using plaque assays, dose titrations, and qPCR.For plaque assays, we added Pam 3 CSK 4 four hours before RVFV infection, and washed off the drug and viral inoculum two hours after infection.Pam 3 CSK 4 reduced titers from 8.7 x 10 3 to 2.6 x 10 3 PFU / mL at 16 hpi (Fig 3A).In U2OS, titers were reduced from 5.2 x 10 5 to 1.0 x 10 5 PFU / mL (Fig 3B).Further, we found that there was a dose-dependent antiviral effect in rat neurons and U2OS cells, as measured by microscopy (neuron IC50 = 3.8 μg/mL; U2OS IC50 = 2.1 μg/mL) (Fig 3B).We also tested mouse cortical neurons and observed antiviral activity with no cytotoxicity, although the potency was lower (IC50 = 8.6μg/mL) (Figs 3B and S2D).These data demonstrate that Pam 3 CSK 4 is antiviral against RVFV in primary rodent neurons and a human cell line.
We next tested the breadth of Pam 3 CSK 4 antiviral activity.We treated rat neurons with several doses of Pam 3 CSK 4 four hours before infection with the Peribunyavirus La Crosse virus (LACV) and found that Pam 3 CSK 4 also inhibited LACV (Fig 3D).This led us to test Pam 3 CSK 4 against two additional related viruses: the Peribunyavirus California Encephalitis virus (CEV, Orthobunyavirus encephalitidis) in the same group as LACV, and the Phenuivirus Punta Toro virus-Balliet (PTV, Phlebovirus toroense) in the same group as RVFV.CEV causes rare cases of encephalitis in humans, while PTV causes febrile illness but not encephalitis [49,50].By plaque assay and qPCR, we found that Pam 3 CSK 4 decreased RVFV, LACV and CEV infection in neurons (Figs 3A, 3F, 3G, 3J, 3K, S2F-S2G).PTV was not tested in neurons because infection was poor.In U2OS, Pam 3 CSK 4 had antiviral activity against RVFV, LACV, and PTV, but not CEV (Figs 3B, 3C, 3I, 3L-3N, S2H-S2J).We also tested whether an unrelated negative sense RNA virus, the Rhabdovirus vesicular stomatitis virus (VSV, Vesiculovirus Indiana,) was sensitive to Pam 3 CSK 4 treatment.VSV RNA replication was unchanged by Pam 3 CSK 4 treatment, and the IC50 of Pam 3 CSK 4 against VSV was >10μg/mL (Figs 3F and  S2E).This suggests that Pam 3 CSK 4 can inhibit bunyaviruses in the Phenuiviridae and Peribunyaviridae, though not all RNA viruses.S3).TLR2 ligands can engage distinct receptor complexes, as Pam 2 CSK 4 binds TLR2/6 heterodimers [42,51], while Pam 3 CSK 4 binds TLR2/1 heterodimers [52].TLR2/1 and TLR2/6 complexes signal through the same adaptor proteins [53,54].Therefore, we explored whether Pam 2 CSK 4 could have been a false negative in the screen.However, we found that treatment of neurons or U2OS with Pam 2 CSK 4 had no antiviral effect on RVFV, LACV, or VSV, as observed by qPCR and microscopy (Fig 3D -3H).These data suggest that TLR2/1 and TLR2/6 signaling may differ in neurons, or that the structural differences between Pam 3 CSK 4 and Pam 2 CSK 4 may directly contribute to antiviral activity.  .Metascape pathway analysis [55] revealed that both treatments activated similar sets of pathways, and for both ligands, the most strongly enriched Gene Ontology (GO) term clusters were (inflammatory response) and (innate immune response) (S4C Fig).Thus, Pam 3 CSK 4 and Pam 2 CSK 4 induce similar transcriptional responses largely consisting of NF-κB-regulated genes in rat neurons.We confirmed that Tnfa, Cxcl1, and Cxcl10 are induced by both treatments using qPCR (Fig 4C -4E).We also found that both treatments induced TNFα and CXCL10 in U2OS cells, though with a smaller fold change (S4F and S4G Fig).While these ligands do not canonically induce ISGs, we also tested if Ifit2 was induced by Pam 3 CSK 4 treatment at 4h or 24h.We saw no induction of Ifit2 in neurons, or IFIT1 in U2OS cells (S4D and S4E Fig).These data show that Pam 3 CSK 4 and Pam 2 CSK 4 induce a similar set of NF-κBdependent genes, and since only Pam 3 CSK 4 is antiviral, our data suggests that the activity is likely independent of this pathway.

Pam 3 CSK 4 -induced inflammatory signaling is not sufficient to control RVFV infection
To directly test if Pam 3 CSK 4 antiviral activity requires NF-κB signaling, we took advantage of two inhibitors of NF-κB activation [54].IKK 16 is an inhibitor of multiple IkBa kinases (IKKs) [56] while TPCA-1 inhibits IKK2 [57].Both block the degradation of IκB, preventing NF-κB activation after an inflammatory stimulus.As expected, we observed blunted transcription of the NF-κB target genes Tnfa and Cxcl1 in neurons that were treated with IKK 16 or TPCA-1 prior to Pam

Structure-activity relationship
Since we observed little difference in gene expression but striking differences in antiviral activity between Pam 3 CSK 4 and Pam 2 CSK 4 , we set out to define a structure-activity relationship.Thus, we tested a more extensive panel of related lipopeptides.Pam 1 CSK 4 has the same peptide motif as Pam 3 CSK 4 , but only one 15-carbon palmitoyl group [52]

Pam 3 CSK 4 antiviral activity does not require TLR2
Although the preceding data suggested that Pam 3 CSK 4 does not require inflammatory signaling for antiviral activity, this did not rule out a role for TLR2.Therefore, we cultured cortical neurons from TLR2 knock out (TLR2 KO) or wild type C57BL/6 mice [61].We first tested whether the deletion of TLR2 prevented inflammatory signaling.We measured Cxcl1 and C-C motif chemokine ligand 2 (Ccl2, also known as Mcp-1) levels by qPCR after a four hour Pam 3 CSK 4 stimulation and found that Pam 3 CSK 4 induced expression of these genes in wild type neurons, but this response was ablated in TLR2 KO neurons (Fig 4K and 4L).Next, we tested whether Pam 3 CSK 4 had antiviral activity.In dose response microscopy studies, we found that Pam 3 CSK 4 protected both WT and TLR2 KO neurons from RVFV infection (Wild type IC50 = 7.0μg/mL, TLR2 KO IC50 = 7.7μg / mL; Fig 4M).Pam 3 CSK 4 pretreatment decreased RVFV titers from 1.6 x 10 5 PFU / mL to 3.7 x 10 4 PFU / mL in wild type neurons, and 1.2 x 10 5 PFU / mL to 3.2 x 10 4 PFU / mL in TLR2 KO neurons (Fig 4N).Similar patterns were observed for RVFV RNA, although the decrease was not significant in WT neurons due to variability (S5C Fig) .We conclude that Pam 3 CSK 4 has antiviral activity against bunyaviruses that is independent of TLR2 or downstream signaling.

Pam 3 CSK 4 inhibits RVFV entry by blocking viral fusion
As our data suggested that Pam 3 CSK 4 -induced inflammatory signaling was not antiviral, we sought to identify the step in the viral lifecycle inhibited by Pam 3 CSK 4 .We began by performing a time of addition assay, where Pam 3 CSK 4 was added to neurons four hours before infection (t = -4), at the time of infection (t = 0), or two-four hours afterwards, and quantified infection by microscopy at 24 hpi.We found that the potency of Pam 3 CSK 4 treatment decreased over time, with infection reduced to 22% of control at t = -4, 40% at t = 0, 59% at t = +2, and 64% at t = +4, suggesting that Pam 3 CSK 4 may act on an early stage of viral infection (Fig 5A).Our plaque assays also supported this, as Pam 3 CSK 4 decreased RVFV titers even though it was washed off two hours after infection (Fig 3A and 3B).We then performed a series of experiments to define the step in the entry process that is blocked by Pam 3 CSK 4 , using RVFV MP-12.First, we tested whether Pam 3 CSK 4 directly neutralizes RVFV virions.We mixed 10μg/mL of Pam 3 CSK 4 , vehicle, or the RVFV-neutralizing monoclonal antibody 4D4 [62] with 6.7 x 10 6 PFU of RVFV (neutralization condition) and incubated at 37˚C for four hours.We then used this mixture to infect the cells at a final MOI of 0.4, which diluted the Pam 3 CSK 4 to 0.1μg/mL, below the active concentration.We compared this neutralization condition to our established Pam 3 CSK 4 pretreatment (stimulation condition), where we treated the cells with 10μg/mL Pam 3 CSK 4 for four hours, and then infected with 0.4 MOI of virus.At 24 hours, we quantified infection by microscopy, and observed that Pam 3 CSK 4 failed to neutralize RVFV but was antiviral when preincubated with the cells (Fig 5C).4D4 prevented infection under both conditions [62] (Fig 5C).These findings suggest that Pam 3 CSK 4 does not directly inactivate or neutralize virions but works at a step downstream.
To test whether Pam 3 CSK 4 impacts RVFV attachment to cells, we used qPCR to measure cell-associated virions.We treated either neurons or U2OS cells with Pam 3 CSK 4 or vehicle and infected with RVFV at 15˚C, which allowed for binding but prevented internalization.
After one hour we washed the cells to remove unbound virus and measured bound viral RNA by qPCR.As a positive control we trypsinized the cells to remove bound virions, and indeed, we were able to strip bound virus (S6A and S6C Fig) Next, we investigated whether Pam 3 CSK 4 treatment decreased viral internalization.After binding RVFV at 15˚C, we shifted neurons or U2OS to 37˚C for two hours to allow endocytic uptake to resume and virions to become internalized.We removed the virions that remained on the surface with trypsinization, and quantified internalized viral RNA by qPCR.We found that trypsinization modestly reduced the levels of RVFV RNA, suggesting that virions were efficiently internalized by neurons at this time point (S6B Fig) .We observed no difference in internalized RVFV RNA in the presence of Pam 3 CSK 4 (Figs 5E, S6B and S6D).Together, these assays suggest that Pam 3 CSK 4 inhibits RVFV entry at a step following viral attachment and uptake.The final step in the entry process involves the fusion of the virion and endosomal membranes [13].Thus, we tested whether RVFV fusion is inhibited.First, we performed an acid bypass assay, which bypasses endocytosis and forces fusion at the plasma membrane [13].This allowed us to determine whether Pam 3 CSK 4 blocked a step in endocytic entry or blocked viral fusion directly.To validate the assay, U2OS cells were pretreated with vehicle or the endosomal acidification inhibitor bafilomycin A1 (BafA) and the cells were then cooled to 15˚C, to allow viral binding but not uptake.RVFV was bound for one hour and then the cells were pulsed for 10 minutes with media at neutral pH (pH 7.6) or acidic pH (pH5.2), as RVFV fusion is induced at pH 5.2 [13,63].Cells were washed and incubated in media at neutral pH for 24h and viral RNA was measured by qPCR.As expected, we found that BafA treatment blocked infection at pH 7. We performed a lipid dye dequenching assay [64,65] to verify that Pam 3 CSK 4 blocks viral fusion.RVFV virions were labelled with the lipophilic fluorescent dye, 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine Perchlorate (DiD, 30μM).At this concentration, the virions self-quench and do not fluoresce.However, once virions fuse with the larger endocytic membrane, the dye diffuses and can be measured by fluorescence microscopy.Thus, quantification of DiD puncta is a measurement of viral fusion.We again validated the assay using BafA, which blocks viral fusion.DiD labeled RVFV (RVFV-DiD, MOI 15) was bound to U2OS cells in the presence of vehicle or BafA at 15˚C to allow binding but not uptake.Cells were fixed after binding (t = 0) or shifted to 37˚C for one hour to allow viral uptake and fusion.

Pam 3 CSK 4 protects mice against encephalitic RVFV infection
As Pam 3 CSK 4 decreased RVFV infection in primary neurons, we tested whether Pam 3 CSK 4 was protective in a mouse model of RVFV encephalitis.Three to six week-old C57BL/6 mice were inoculated intracranially with RVFV ZH501, mixed with vehicle or Pam 3 CSK 4 .This route of inoculation allowed us to control the timing and input of virus into the central nervous system.10% of mice infected with 5 PFU survived to day 3, and 5% of mice infected with 1 PFU survived to 6 dpi, demonstrating that mice are reliably infected at these doses (Fig 6A).However, Pam 3 CSK 4 was protective, with greater than 50% of treated mice surviving 5 PFU and 80% surviving 1 PFU, while all mice treated with Pam 3 CSK 4 alone survived (Fig 6A).Consistent with this drastic increase in survival, we found that Pam 3 CSK 4 treatment decreased viral replication in the brain by more than 5 logs when mice were infected with 5 PFU (Fig 6B We also monitored NF-κB-dependent inflammatory responses in the brain at 3 days.RVFV infection with 1 PFU induced Cxcl1 approximately 10-fold while 5 PFU induced these transcripts by ~100-fold, consistent with previous studies of RVFV encephalitis [66,67] (Fig 6D and 6E).In uninfected animals, Pam 3 CSK 4 induced Cxcl1 approximately 10-fold, similar to the 1 PFU infection and reflecting our RNAseq results.However, Pam 3 CSK 4 treatment alleviated inflammation caused by severe RVFV infection (5 PFU), as we observed ~10-fold decreased Cxcl1 levels relative to infected, vehicle-treated animals (Fig 6D and 6E).We also monitored Ccl2 mRNA, as this cytokine has also been associated with RVFV encephalitis and was differentially expressed in our RNAseq experiments [66,67].We observed a similar pattern, as Pam

Discussion
RVFV infection leads to acute disease, but in a subset of individuals, severe disease including delayed-onset meningoencephalitis can result in long term neurologic sequelae or death [6,21].As these symptoms present in the later stages of infection, there is potentially a window of time to deliver therapeutics.However, there are no approved treatments for humans and little is known about preventing RVFV infection in neurons, a vulnerable and essential cell type.We thus explored RVFV infection of primary neurons as these are the natural target cells infected in the human and rodent central nervous system [21].Previous studies found that neurons have an altered response to type I IFN stimulation, which can leave them vulnerable to RNA virus infection [25][26][27][28].Indeed, IFNα, IFNβ, or IFNγ-stimulated neurons were not strongly protected from RVFV infection.
We sought to identify alternative innate immune agonists with antiviral activity.Of the 75 ligands we tested, five were antiviral without cytotoxicity in primary neurons, including two TLR2 ligands.However, several other TLR2 ligands had no antiviral activity, suggesting that TLR2 stimulation is not sufficient to inhibit infection.We focused on the well-defined synthetic lipopeptides Pam 3 CSK 4 and Pam 2 CSK 4 which are structurally similar but had divergent effects on infection: Pam 3 CSK 4 was antiviral while Pam 2 CSK 4 was not.Validation experiments recapitulated this pattern across multiple cell types and species.Further, Pam 3 CSK 4 limited infection with other members of the Peribunyaviridae and Phenuiviridae, but not the unrelated negative sense Rhabdovirus VSV.In U2OS, Pam 3 CSK 4 had no antiviral activity against CEV, which may be related to the high MOI of 5 needed for robust infection in these cells.Overall, Pam 3 CSK 4 may have a specific antiviral effect against viruses within the Phenuiviridae and Peribunyaviridae families.Other families of the Bunyavirales, and other encephalitic viruses remain untested.
As TLR2 canonically activates NF-κB, and Pam 3 CSK 4 reportedly antagonizes Hepatitis B virus through NF-κB signaling [68], we investigated whether RVFV was similarly controlled.Previous work has shown that TLR2 activating lipopeptides induce inflammatory pathways in the brain, but these responses have largely been attributed to microglia or astrocytes [69][70][71].Thus, we used transcriptomics to define the genes induced by Pam 3 CSK 4 and Pam 2 CSK 4 in primary neurons.We found that both ligands induced canonical NF-κB-dependent inflammatory pathways, demonstrating that neurons respond to TLR2 ligands.It was not surprising that these responses were alike, as TLR2/1 and TLR2/6 heterodimers signal through the same adaptor proteins [53].Moreover, these findings suggested that the antiviral effect of Pam 3 CSK 4 is not mediated by NF-κB-induced gene expression, which was also supported by NF-κB inhibitor experiments.In fact, we showed that the antiviral activity of Pam 3 CSK 4 is independent of TLR2, as TLR2 knock out neurons were protected by Pam 3 CSK 4 .
To further define the structural requirements for antiviral activity, we took advantage of a panel of molecules related to Pam 3 CSK 4 .PHCSK 4 , a similar molecule with a modified backbone, activated inflammatory gene transcription but had no antiviral activity against RVFV, which indicates that the backbone of the hydrocarbon chains is a determinant of antiviral activity.Next, we tested stereoisomers of Pam 3 CSK 4 , as the R isomer binds more strongly to TLR2/1 than the S isomer [43,72].Although S-Pam 3 CSK 4 stimulated chemokine transcription less potently than R-Pam 3 CSK 4 , it had a stronger antiviral effect, demonstrating that the orientation of the molecule matters for the control of infection.
Pam 3 CSK 4 is cationic, and has been shown to impact viral binding to target cells in some circumstances [73].Pam 3 CSK 4 increases respiratory syncytial virus (RSV), measles virus, human metapneumovirus, and HIV binding and infection in primary airway epithelial cells and lymphoid cells, potentially through enhancing binding to glycosaminoglycan attachment factors [73].Furthermore, this activity is specific and independent of TLR2, as Pam 2 CSK 4 does not impact RSV infection [73].As these effects were attributed to a change in surface binding in the presence of Pam 3 CSK 4 , we tested whether Pam 3 CSK 4 impacts bunyavirus binding to target cells.
Unlike prior studies we found that Pam 3 CSK 4 did not alter RVFV binding or uptake in target cells.However, by comparing VSV and VSV-RVFV infection, we showed that Pam 3 CSK 4 blocked viral entry.Indeed, we found that Pam 3 CSK 4 blocked viral fusion both in endosomes and at the plasma membrane, suggesting specific interactions independent of the compartment.We infer that Pam 3 CSK 4 does not prevent endosomal acidification, as RVFV and VSV rely on a similarly low pH but Pam 3 CSK 4 had no activity against VSV [18,63,74].As our data show that the palmitoyl chains of Pam 3 CSK 4 are important for antiviral activity, these hydrophobic groups may interact with the host and/or viral membrane to disrupt the function of the Gc fusion peptide.It has been found that the addition of lipid groups to some fusion-inhibiting peptides can enhance antiviral activity by increasing peptide concentrations on the membranes where fusion occurs [11,75].We suggest that the palmitoyl groups of Pam 3 CSK 4 have a similar function.
Amphipathic fusion inhibitors, which intercalate into virion membranes and irreversibly inactivate them, inhibit several enveloped viruses including RVFV [19,76,77].Structurally, these molecules resemble a wedge, with narrow hydrophobic domains linked to bulky polar regions.The hydrophobic domains insert into viral membranes, stabilizing the pre-fusion curvature of the viral membrane [19,76].While the Pam 3 CSK 4 shares some structural similarities with this class of inhibitors, Pam 3 CSK 4 does not irreversibly inactivate RVFV particles, demonstrating a distinct mechanism (Fig 5C ).
Lastly, we tested whether Pam 3 CSK 4 had antiviral activity in vivo in the central nervous system.We found a robust antiviral effect in the mouse brain, as the delivery of Pam 3 CSK 4 reduced viral titers by several logs, and pathogenesis was dramatically reduced.RVFV encephalitis was highly inflammatory in the mouse brain at three dpi.Pam 3 CSK 4 treated, infected mice had less inflammatory gene expression than infected, untreated mice, likely due to the reduction in viral replication.In addition to the direct effect of Pam 3 CSK 4 on viral fusion in neurons, it is possible that TLR2-expressing cells responded to Pam 3 CSK 4 .Whether this is protective or pathogenic is unclear.It is known that TLR2 signaling in the brain can activate microglia and astrocytes, and recruit lymphocytes [70,78,79], and thus it is possible that these responses are indirectly impacting infection or pathogenesis.Further work is required to explore the impact of Pam 3 CSK 4 on diverse cell types during severe neurologic bunyaviral infection.As we showed that S-Pam 3 CSK 4 had limited inflammatory properties but retained antiviral activity, this stereoisomer could decouple these effects.We suggest that S-Pam 3 CSK 4 would block viral fusion without inflammatory responses which may be beneficial.One challenge in using fusion inhibitors to treat acute infections is time, as they are most effective when present early in infection.RVFV meningoencephalitis is a late-presenting stage of disease, and fusion inhibitors may be useful in preventing infection in the brain.While it is unknown whether Pam 3 CSK 4 can be used therapeutically in the brain, or whether it has antiencephalitic activity when delivered systemically, these questions should be addressed in future studies.It will be important to test whether peripherally-delivered Pam 3 CSK 4 can enter the brain, as delivery into the central nervous system is a challenge.However, encephalitis can result in blood-brain barrier permeability, which may enable Pam 3 CSK 4 to enter.
In sum, this work describes a role for Pam 3 CSK 4 as an inhibitor of viral fusion, which may have value in treating neurologic bunyavirus infections.

Ethics statement
All mouse experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee, protocol #23083513.No human participants or samples were used in this work.
were quantified using Molecular Devices MetaXpress (version 6) modules including Cell Scoring, Multiwavelength Cell Scoring, and Granularity.For confocal microscopy experiments, the final PBS wash was replaced with H 2 O and coverslips were mounted in Vectashield (Fisher Scientific NC9265087) and sealed with clear nail polish.Coverslips were imaged on a laser scanning Leica TCS SPE-II with 40x or 63x objective lenses with 1.5x zoom and a pinhole of 1 at the highest wavelength.Sites were selected based on the nuclear channel alone, and the same settings were used within each experiment.The following fluorophores were used: Hoechst 33342, AlexaFluor 488, AF594, Cy5, and DiD (excitation at 633 nm).Images were captured at a resolution of 1024x1024 pixels.Images were equally levelled for display using ImageJ, and granularity was analyzed using MetaXpress, with a minimum granule size of 3μm and a maximum size of 15μm.
For hematoxylin and eosin (H & E) staining and immunofluorescent imaging of tissues, samples underwent cryopreservation through 24 hour incubations in 20% sucrose in PBS, then 40% sucrose in PBS prior to freezing in mounting optimal cutting temperature (OCT) compound (Fisher) and long-term storage at -80˚C.Tissues were sliced to 5 μm sections using the Histocore Autocut CryoStar X7-CryoStat (ThermoFisher Scientific) and stored at -80˚C until staining was performed.Tissues were stained by standard H & E methods and images were taken at 20x magnification on an Olympus CX41 microscope with a Levenhuk microscope digital camera (M base series).For immunofluorescent imaging, cryo-sections were rehydrated with PBS containing 0.5% bovine serum albumin (PBB), and then blocked in 5% normal goat serum in PBB.The tissues were washed, then permeabilized in 0.1% Triton X-100 detergent for 15 minutes.The tissues were washed, then probed with custom rabbit anti-RVFV nucleoprotein polyclonal antibody (1:50; Genscript), washed again, then incubated for 30 minutes with an anti-rabbit IgG-FITC conjugated secondary antibody (Invitrogen).The slides were washed in PBS, then counterstained with Hoechst, washed again, and then mounted with Gelvatol.Dried slides were imaged at 40x magnification using a Leica DM18 inverted fluorescence microscope and denoised using the Leica Application Suite X software.

Real-time reverse transcription quantitative PCR (qPCR)
For qPCR experiments, cells were grown in 6 well plates (Neurons: 9.6 x 10 5 cells/well.U2OS: 2 x 10 5 cells/well).Cellular RNA was collected in Trizol (Life Technologies 15596018).Nonneuronal RNA was purified using a RNA clean & concentrator-25 kit (Zymo research R1018).Neuron RNA was purified by chloroform precipitation with glycogen addition.1μg of RNA was used to synthesize complimentary DNA (cDNA) using Moloney Murine Leukemia Virus-Reverse Transcriptase (ThermoFisher scientific 28025013) and hexameric random primers (ThermoFisher scientific 48190011).cDNA was then diluted 1:5, and 5μL of diluted cDNA were loaded into 384 well qPCR plates in triplicate.Forward and reverse primers were diluted to 0.2μM in PowerSYBR Green master mix (Thermofisher scientific 4368577), and 5uL of the combined mix was added per well.A Thermofisher QuantStudio 6 RT-qPCR instrument was used to quantify cDNA amplification.Gene expression was normalized to GAPDH and relative fold change in expression was calculated using the ddCt method.Primer sequences are included in S1 Table.

Plaque assay
Neurons or U2OS were treated and infected as indicated.Two hours after infection, inoculum was removed and replaced with fresh media (U2OS), or conditioned media from uninfected neurons.Drugs were not replenished.Supernatants were collected at the indicated time points and stored at -80˚C.Infected supernatants or tissue homogenates were serially diluted in

RNAseq
Neurons (9.6 x 10 5 cells in duplicate wells) were treated with vehicle, 10μg/mL of Pam 3 CSK 4 or Pam 2 CSK 4 , for 6h.RNA was collected in Trizol, and purified using a Zymo RNA clean & concentrator-25 kit, with duplicate wells pooled during purification.cDNA libraries were created with a TruSeq RNA library prep kit (Illumina) and sequenced on an Illumina NextSeq 500.Reads were aligned to the Rnor_6.0genome with Kallisto [83], normalized with EdgeR [84], and differentially expressed genes were identified with Limma [85] using a linear model fit.Differentially expressed genes were used for pathway analysis, using Metascape [55].

Viral neutralization assay
Pam 3 CSK 4 was added to RVFV at 10μg/mL, and was incubated at 37˚C for 4h.At the time of infection, the mixture was diluted to infect neurons at 0.3 MOI.This diluted the virus and ligand by more than 1000x.24hpi, cells were fixed and infection was quantified by microscopy.As a positive control, a RVFV-neutralizing antibody (4D4) was added at 1:1000.

Viral entry assays
Drugs at the indicated concentrations were added to cells in 6 well plates, and incubated for 1hr.Plates were sealed in plastic bags and cooled to 15˚C for 30min.Virus was added at the indicated MOI, and was bound for 1hr for U2OS or 2h for neurons at 15˚C, where viral entry does not occur.For viral attachment assays, virus was then aspirated from the cells and they were washed 2x with cold DPBS.Total RNA was then collected, and quantified by RT-qPCR.For viral uptake assays, cells were treated and virus was bound as above.Cells were then transferred to a 37˚C incubator for 2hr to allow for viral entry.Non-internalized virus was removed by trypsinization (0.25% for 3 minutes at 37˚C), and cells were washed before RNA was collected.Viral RNA was measured by qPCR.For acid bypass assays, 1 x 10 5 U2OS cells in 12 well plates were pretreated with indicated drugs for 2 hours, before the addition of Bafilomycin A1 for 1hr.RVFV was bound to cells as above.Cells were washed 2x with DPBS, and then treated for 10 minutes with indicated treatments diluted in OptiMEM, pH adjusted to 7.6 or 5.2.Cells were washed, and complete media containing BafA or vehicle, and Pam 3 CSK 4 or vehicle was replaced for a 24h incubation.Infection was measured by qPCR.For RVFV-DiD assays, cells were plated on coverslips and treated and cooled to 15 degrees C. RVFV-DiD was bound in the presence of vehicle, Pam 3 CSK 4 , or bafilomycin A1 for 1hr at 15˚C.At this time point cells were either washed and fixed, or incubated at 37˚C for the indicated times.Fixed cells were washed 3x with PBS without detergent, and DNA was stained with Hoechst 33342.Coverslips were then imaged by confocal microscopy.

Animal studies
Female C57BL/6 (3-6 weeks old) were purchased from Jackson Laboratories and housed at the RBL, up to 5 to a cage in temperature-controlled rooms with a 12 hour day/ 12 hour night light schedule.To account for age-dependent effects, treatment groups of mice of similar age distributions were used for each experiment.Food (IsoPro Rodent 3000) and water were provided ad libitum.For Pam 3 CSK 4 therapeutic studies, mice were intracranially injected at the intersection of the coronal and sagittal sutures of the skull with 10 μL, containing 1 or 5 PFU of RVFV ZH501 and 100 μg of Pam 3 CSK 4 .For untreated controls, RVFV was combined with PBS.Virus was diluted in D2 medium (DMEM, 2% (v/v) FBS, 1% L-glutamine, and 1% penicillin-streptomycin) and media without virus was delivered to uninfected animals.Mice were weighed daily and closely monitored for development of clinical signs of disease.Endpoint criteria, which prompt immediate euthanasia, were defined based on weight, appearance, ataxia, and anemia scores.Unless mice met the euthanasia criteria explained above, they were euthanized on 3dpi or on 10 dpi.Mice were anesthetized by inhalation of vaporized isoflurane (Iso-Thesia, Henry Schein).For all experiments, necropsy was performed upon euthanasia to collect brain, liver, and serum samples from the mice.For tissues, half of the sample was immediately frozen at -80˚C for virological analysis by qRT-PCR and plaque assay, while the other half was fixed in fresh 4% paraformaldehyde (Sigma) for histological analyses.Liquid samples were immediately stored at -80˚C prior to downstream virological analyses.

Data analysis
Automated microscopy, plaque assay, and qPCR data were compiled and analyzed using Graphpad Prism 9.All statistical tests performed were two sided, and all replicate experiments represent independent biological replicates.RNAseq data were processed using RStudio 2021.09.02 and R 4.1.2.

Fig 1 .
Fig 1. Type I IFNs do not potently protect neurons from RVFV infection.(A-D) Human U2OS cells (A,C), or rat cortical neurons (B,D) were treated with vehicle or universal IFNα for 4h before infection with RVFV (24hpi, U2OS: MOI 1, neurons: MOI 0.3).Immunofluorescence and automated microscopy were used to detect RVFV Gn (green) or neuronal MAP2 (magenta).A and B show representative images of cells treated with vehicle or 1.2 x 10 4 Units / mL at 10x magnification, scale bars = 350μm.C and D show quantification of relative infection (red squares) and survival (black circles) in U2OS (C) or neurons (D).Automated image analysis was used to determine the percentage RVFV Gn positive U2OS cells, or the percentage of neurons (MAP2 positive cells) positive for RVFV Gn.Infection and survival normalized to % infected or total cell number in vehicle treated, infected cells.IC50 values were determined by

Fig 2 .
Fig 2. Pam 3 CSK 4 and LPS-Rs protect primary neurons from RVFV infection.(A) Diagram of screening workflow.(B,C) Results from microscopy-based screening of innate ligands in neurons (B) or U2OS (C) infected with RVFV (neuron MOI 0.1, U2OS MOI 0.35), as detailed in (A).Four images were captured per well and averaged.Each dot represents a ligand.Plots show infection relative to vehicle from duplicate screens.Dotted red lines indicate 60% of vehicle infection levels.Antiviral TLR2 ligands are labelled.(D) Representative images from neuron PAMP screens, showing vehicle (water), Pam 3 CSK 4 (10μg/mL), or LPS-Rs (100μg/mL) treated cells at 10x magnification.Neurons are MAP2 positive (magenta), while infected cells are positive for RVFV Gn (green).(E) A subset of screen data, showing relative RVFV infection in neurons treated with TLR2 ligands.Bars show mean of two replicates.LPS-Rs and Pam 3 CSK 4 are colored red, while Pam 2 CSK 4 is shown in orange.https://doi.org/10.1371/journal.ppat.1012343.g002

Pam 2 CSK 4 ,
a diacylated lipopeptide, is not antiviral against RVFV or LACV We screened several putative TLR2 ligands, including Pam 2 CSK 4 , which differs from Pam 3 CSK 4 by one palmitoyl group, yet only Pam 3 CSK 4 and LPS-Rs showed antiviral activity (Figs 2E and To compare the neuronal response to Pam 3 CSK 4 and Pam 2 CSK 4 , we used RNA sequencing (RNAseq) and transcriptomic analysis.Time course qPCR studies of Tnfa and Cxcl1 RNA levels after Pam 3 CSK 4 stimulation revealed rapid induction of both genes, with Tnfa peaking at four hours and Cxcl1 at eight hours (S4A and S4B Fig).Therefore, we stimulated neurons with vehicle, Pam 3 CSK 4 , or Pam 2 CSK 4 for six hours and identified differentially expressed genes (adjusted P value of <0.01, and a Log 2 fold change >2) compared to vehicle treated cells.In Pam 3 CSK 4 stimulated cells, 24 genes were significantly upregulated (Fig 4A).In Pam 2 CSK 4 stimulated cells, 53 genes were upregulated (Fig 4A).There were no significantly downregulated genes in either condition.Of the 24 Pam 3 CSK 4 induced genes, 23 were also significantly

Fig 3 .
Fig 3. Pam 3 CSK 4 is antiviral against bunyaviruses in vitro.(A,B) Neurons or U2OS were treated with vehicle (water) or 10μg/mL Pam 3 CSK 4 prior to RVFV infection (neurons: MOI 0.1, U2OS: MOI 1).Supernatants were collected at 15hpi and titers were determined by plaque assay.LoD = limit of detection.*P = .0373,**P = .0015(C) Rat (red squares) or mouse cortical neurons (blue triangles), or U2OS (black circles) were treated with the indicated dose of Pam 3 CSK 4 for 4h before infection with RVFV (Neurons: MOI 0.3, U2OS: MOI 1).At 24hpi RVFV infection was determined by immunofluorescence automated microscopy and automated analysis.n = 3 (neurons) or 4 (U2OS).IC50 values were determined by nonlinear regression.(D) Rat neurons were treated as in (C), and infected with RVFV (MOI 0.3) or LACV (MOI 0.01) for 24h.Data acquired and presented as in (C).(E) Rat neurons were treated with serial dilutions of vehicle or TLR2 ligands 4h before RVFV infection.Data acquired and presented as in (C).(F-H) Rat neurons were treated with 3 CSK 4 stimulation (S4H and S4I Fig).Despite this, neither inhibitor impacted the antiviral activity of Pam 3 CSK 4 , as RVFV RNA levels were significantly decreased by Pam 3 CSK 4 in the presence of TPCA-1 or IKK 16 (S4J Fig).These data further suggest that Pam 3 CSK 4 does not control RVFV through NF-κB-dependent transcriptional pathways.

Fig 4 .
Fig 4. Pam 3 CSK 4 -induced inflammation is not antiviral in neurons.(A) Rat cortical neurons were stimulated with vehicle or 10μg/mL of Pam 3 CSK 4 or Pam 2 CSK 4 for 6h, and transcriptomic analysis was used to identify differentially expressed genes (�2 Log 2 fold change compared to vehicle, adjusted P �0.01).Heatmap shows hierarchical clustering of DEG.Rows are independent replicates.Color represents row Z score.(B) Linear regression of DEG expression after Pam 3 CSK 4 or Pam 2 CSK 4 stimulation.Genes from (A) are plotted by average Counts Per Million under each condition.(C-E) Relative expression of Tnfa (C), Cxcl1 (D), and Cxcl10 (E) in rat neurons

Fig 5 .
Fig 5. Pam 3 CSK 4 blocks viral entry by reducing fusion.(A) Quantification of automated microscopy and analysis to detect RVFV infected rat neurons (MOI 0.3, 24hpi) when Pam 3 CSK 4 (10μg/mL) or vehicle were added at the indicated timepoints (pre-or post-infection).Infection = time 0. Infection calculated relative to -4h, vehicle treated cells.*P = .0169,**P = .0069,***P = .0002,****P< .0001.(B) Quantification of VSV N RNA in rat neurons treated with 10μg/ mL of TLR2 ligands 4h before VSV-RVFV infection (MOI 5, 14hpi).RNA levels measured by qPCR, relative to Gapdh.***P = .0001,vehicle vs. Pam 2 CSK 4 **P = .0061,Pam 3 CSK 4 vs.Pam 2 CSK 4 **P = .0042.(C) Quantification of automated microscopy and analysis of RVFV-infected rat neurons after Pam 3 CSK 4 neutralization.Vehicle, Pam 3 CSK 4 , or 4D4 anti-RVFV Gn were either added to cells (stimulation, solid bars) or mixed with stock RVFV (neutralization, checked bars) for 4h.RVFV or neutralized RVFV was used to infect the cells at a MOI of 0.3 for 24h.****P< .0001(D) RVFV (MOI = 5) was bound to rat neurons at 15˚C in the presence or absence of 10μg/mL Pam 3 CSK 4 .RVFV N RNA was measured by RT-qPCR, normalized to Gapdh, and set relative to vehicle.(E) RVFV was bound to neurons in the presence or absence of Pam 3 CSK 4 as in (D), and cells were shifted to 37˚C for 2h to allow viral uptake.Cells were trypsinized to remove remaining surface bound particles and RVFV RNA was measured, analyzed and shown as in Pam 3 CSK 4 did not change the amount of viral RNA bound to cells (Figs 5D, S6A and S6C).

(
D). (F) Quantification of RVFV N RNA in U2OS following acid bypass assay.Cells were pretreated with Pam 3 CSK 4 at 10μg/mL and bafilomycin A1 (0.05μM) or DMSO prior to RVFV binding at 15˚C (MOI 0.2).Cells were pulsed with neutral (pink) or acidic (yellow) OptiMEM containing water or Pam 3 CSK 4 , and then cells were incubated for 24h in the presence or absence of BafA.*P = .0100,***P = .0006,****P< .0001.(G) Confocal microscopy showing RVFV-DiD puncta (white) in U2OS cells (nuclei stained blue).60x magnification, scale bar = 25μm.Z-stacks were acquired and are shown as maximum projections.Images representative of five sites per condition, n = 4. (H-J) MetaXpress software was used for automatic quantification of 1h timepoint from (G), showing (H) the number of DiD puncta per cell, (I) the total area of DiD per cell, (J) the average intensity of DiD puncta within each cell.Each dot represents one cell.Vehicle: 322 cells analyzed; Pam 3 CSK 4 : 385 cells analyzed; BafA: 385 cells analyzed.For (I and J), cells with 0 puncta are not plotted due to the log scale of the Y axis.Dotted lines represent median of compiled experiments.****P< .0001.For A-F, bars represent mean, error bars = SEM.Statistical analyses were performed using two-way ANOVA with Dunnett's multiple comparisons test (A), one-way ANOVA with Tukey's multiple comparisons test (B,H-J), two-way ANOVA with S ˇı ´da ´k's multiple comparisons test (C, F), or Welch's t test (D,E).https://doi.org/10.1371/journal.ppat.1012343.g005 6 since endosomal entry was inhibited (Fig 5F).In contrast, BafA did not block infection in acid-washed cells, as fusion occurred at the cell surface (Fig 5F).In parallel, we treated the cells with Pam 3 CSK 4 , which inhibited RVFV infection in DMSO treated cells (Fig 5F).We found that Pam 3 CSK 4 also decreased RVFV infection in acid-washed, BafA-treated cells, suggesting that Pam 3 CSK 4 inhibited RVFV fusion at the cell surface (Fig 5F).
Few puncta were detected at t = 0 (Fig 5G), demonstrating that prior to fusion, RVFV-DiD particles do not fluoresce (Fig 5G).In contrast, after incubation at 37˚C, most cells showed multiple puncta in vehicle treated conditions (Fig 5G).As expected, BafA significantly decreased the number of puncta per cell, as well as the median area of DiD positive pixels per cell and the median intensity of puncta (Fig 5H-5J).Upon treatment with Pam 3 CSK 4 , we also observed decreases in number of puncta per cell, the median area of DiD positive pixels per cell, and the median intensity of puncta (Fig 5H-5J), demonstrating again that Pam 3 CSK 4 blocks viral fusion.We performed a parallel experiment in primary neurons (S7 Fig).Although fewer puncta were observed per cell in control treated neurons (median = 2 in neurons vs. median = 9 in U2OS cells), the number, intensity, and area of DiD puncta were decreased in neurons after BafA or Pam 3 CSK 4 treatment (S7A-S7D Fig).These data further demonstrate that Pam 3 CSK 4 inhibits RVFV fusion.

3 CSK 4 induced
Ccl2 expression in uninfected animals, but decreased Ccl2 levels in the context of 5 PFU infection.Hematoxylin and eosin staining of mouse brains and livers showed that Pam 3 CSK 4 treatment reduced hemorrhages caused by RVFV in the liver (S8B Fig).Immunofluorescence for RVFV nucleoprotein also showed that Pam 3 CSK 4 decreased viral replication in the brain and liver at 3 dpi (S8C Fig).Altogether, Pam 3 CSK 4 protected mice from neuronal RVFV infection, and reduced spread from the central nervous system to peripheral tissues.