Screening and Identification of Lujo Virus Inhibitors Using a Recombinant Reporter Virus Platform

Lujo virus (LUJV), a highly pathogenic arenavirus, was first identified in 2008 in Zambia. To aid the identification of effective therapeutics for LUJV, we developed a recombinant reporter virus system, confirming reporter LUJV comparability with wild-type virus and its utility in high-throughput antiviral screening assays. Using this system, we evaluated compounds with known and unknown efficacy against related arenaviruses, with the aim of identifying LUJV-specific and potential new pan-arenavirus antivirals. We identified six compounds demonstrating robust anti-LUJV activity, including several compounds with previously reported activity against other arenaviruses. These data provide critical evidence for developing broad-spectrum antivirals against high-consequence arenaviruses.


Introduction
Lujo virus (LUJV), the first highly pathogenic arenavirus identified in Africa in over 40 years, was recognized after a cluster of severe viral hemorrhagic fever (VHF) cases in Southern Africa in September and October of 2008 [1,2]. The index case was identified in Zambia, and four subsequent nosocomial cases were detected in associated healthcare workers in South Africa. The infections initially presented as a non-specific febrile illness, but progressed in severity over 10-13 days to respiratory distress, neurological signs, and circulatory collapse [1]. To date, no additional cases of LUJV infection have been reported. However, the lack of a defined etiology or source of exposure for the index case; the apparent ease with which primary, secondary, and tertiary contacts were infected; and the unusually high case fatality rate (80%) caused significant alarm.
The family Arenaviridae encompasses three genera: Mammarenavirus, Reptarenavirus, and Hartmanivirus [3]. With the exception of the bat-borne Tacaribe virus, rodents are the natural hosts of the mammalian-specific mammarenaviruses, with each viral species closely associated with a single rodent species reservoir [4,5]. Infected rodents develop a persistent infection and continually shed the virus in body secretions such as urine and feces; human transmission occurs via contact with these secretions, either directly or in contaminated food products [6]. Mammarenaviruses are divided into two groups based on the geographic, genetic, and epidemiological relationship with their respective rodent hosts: Old World (or lymphocyte choriomeningitis/Lassa complex) species and New World (or Tacaribe complex) species [7]. Several Mammarenavirus species in both these groups can cause severe VHF disease in humans. Highly pathogenic Old World complex viruses such as LUJV and Lassa virus (LASV) are found in Africa, whereas the New World pathogens such as Junin (JUNV), Guanarito (GTOV), Machupo, and Chapare viruses are found in

Rescue of Recombinant LUJV
Recombinant reporter LUJV expressing the green fluorescent protein ZsGreen1 (rLUJV/ZsG) was generated using a previously described reverse genetics system [12]. Briefly, to rescue recombinant LUJV, T7 promoter-based plasmids containing full-length antigenomic sense LUJV L and S genome segments (prototypic Zambian strain; Genbank NC_012777.1 and NC_012776.1, respectively) were transfected into BSR-T7/5 cells. The reporter rLUJV/ZsG was generated by substituting the full-length antigenomic sense S segment with a modified version encoding ZsG. At 5 days post-transfection, cell culture supernatants were harvested, clarified by low-speed centrifugation, and used to infect Vero-E6 cells. Virus stocks harvested 3-4 days post-infection were quantified in Vero-E6 cells by tissue culture infective dose 50 (TCID 50 ) assays, using either immunofluorescence (wt virus) or ZsG expression (reporter virus) [13].

Next-Generation Sequencing and Bioinformatics
RNA was extracted from cell culture supernatants (clarified by low-speed centrifugation) using the MagMAX-96 Total RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) on a 96-well Roche MagMAX extraction platform with a DNase-I treatment step according to manufacturer's instructions. RNA sequencing was performed using KAPA RNA HyperPrep kit (Roche, Basel, Switzerland) for library preparation and analyzed on the MiniSeq system (Illumina, San Diego, CA, USA).

Data Analysis
ZsG fluorescence values for compound-treated infected cells were normalized to mocktreated (DMSO only), infected cells and used to fit a 4-parameter equation to semi-log plots of the concentration-response data. From this, the compound concentrations inhibiting ZsG expression by 50% (EC 50 ) were interpolated. Cell viability was similarly calculated in compound-treated, mock-infected cells to determine the 50% cell cytotoxicity concentration (CC 50 ) of each compound. The selectivity index was calculated by dividing CC 50 by EC 50 . Data analysis was performed using GraphPad Prism v9 (GraphPad, San Diego, CA, USA). Suitability for high-throughput screening was determined using the Z prime (Z ) score, a measure of statistical effect size, calculated as Z = 1 − (3 × (σ P + σ N )/|µ P − µ N |, where σ P = standard deviation of positive control, σ N = standard deviation of negative control, µ P = mean of positive control, and µ N = mean of negative control. Z values between 0.5 and 1.0 were considered acceptable. Signal-to-noise ratio was determined as (µ P − µ B )/σ B , where µ P = mean signal of positive control, µ B = mean background signal, and σ B = standard deviation of background signal. Significance was calculated using a one-sample t-test.

Generation of a Recombinant LUJV Expressing ZsGreen1 Fluorescent Protein
LUJV contains two ambisense genome segments termed large (L) and small (S) with respect to their relative nucleotide length ( Figure 1A). Each genome segment encodes two genes: RNA-dependent RNA polymerase (RDRP) and Z protein on the L, and the nucleoprotein (N) and glycoprotein precursor (GPC) on the S ( Figure 1B). Transcription of monocistronic viral mRNA is initiated at the untranslated region (UTR) and terminates at the intergenomic region (IGR).  Previous attempts to generate mammarenaviruses expressing reporter plasmids have either incorporated additional genome segments, as done with tri-segmented recombinant lymphocytic choriomeningitis virus (LCMV) [14], or introduced additional coding sequences (CDS) into the S genome segment, as in a recombinant reporter LASV [15,16]. Here, to generate a recombinant LUJV expressing the fluorescent protein ZsG, we constructed a modified S genome segment in which the ZsG coding sequence was inserted upstream of the N coding sequence, with a porcine teshovirus-1 2A peptide linker sequence (P2A) inserted between ZsG and NP ( Figure 1B). This genetic format transcribes a single viral mRNA, but the two separate proteins are expressed via a ribosomal skipping event during P2A translation [17]. The modified S genome segment was incorporated into the previously described reverse genetics system for LUJV [12]. The recombinant reporter LUJV, able to express all parental viral proteins alongside the inserted ZsG reporter, was successfully rescued using this system and was termed rLUJV/ZsG.
To establish the suitability of rLUJV/ZsG as a surrogate for wt LUJV, we first investigated viral growth kinetics. Huh7 or Vero-E6 cells were infected with either wt LUJV or rLUJV/ZsG at MOI 0.1, and titers were determined at 12, 24, 48, 72, and 96 hpi. In both cell lines, growth kinetics of wt LUJV and rLUJV/ZsG were comparable at each time point assessed. In cells infected with rLUJV/ZsG, strong ZsG expression was observed in monolayers of both cell types at 96 hpi ( Figure 1C). To assess the stability of the ZsG insert, we passaged rLUJV/ZsG 10 times in Vero-E6 cells (in triplicate). At each passage point, strong ZsG expression was detected in the monolayer, indicating that the ZsG-P2A-NP modification in the S genome segment was stable ( Figure 1D). To further assess the genome stability, we used next-generation sequencing (NGS) to analyze the viral RNA from passages 1, 5, and 10, and compared the sequences to those of wt LUJV passaged concurrently ( Figure 1E). The ZsG CDS was retained over 10 passages and no mutations were seen in any of the replicates. Similarly, no mutations were detected in the L genome segment over 10 passages. In the S genome segment, no mutations were seen over 5 passages in either wt LUJV or rLUJV/ZsG. At passage 10, 2 of the 3 replicate samples of both viruses had accumulated one non-synonymous, non-identical mutation.

Suitability of the LUJV Reporter Virus For Use in an In Vitro Screening Assay
To determine the suitability of rLUJV/ZsG for in vitro screening assays, potential LUJV inhibitors were screened using rLUJV/ZsG in Huh7 cells, which have been previously used in the discovery and evaluation of antiviral compounds for other VHF pathogens [16,18,19]. The assays were optimized using ribavirin, a well-documented broad-spectrum antiviral. The Z score (0.89) and signal-to-noise ratio (190:1) were calculated using 50 µM ribavirin as the positive control and DMSO vehicle only as the negative control and indicated a robust assay. Ribavirin dose-response curves demonstrated a concentration-dependent reduction in ZsG fluorescence, with a calculated EC 50 value of 11.13 ± 1.4 µM and CC 50 of >50 µM ( Figure 1F). These values are broadly similar to those reported for other arenaviruses, including LASV (2.47 µM) and JUNV (18.3 µM) [16,20], indicating that rLUJV/ZsG is suitable for a screening assay that uses reduction in ZsG fluorescence to indicate inhibition of viral replication.

Antiviral Compound Screening
To identify anti-LUJV therapeutics, a wide range of compounds was identified from published data on arenavirus inhibitors, broad-spectrum antivirals, and preliminary inhouse screens using an FDA-approved library of compounds. Virus inhibition was initially determined for each of 83 candidate compounds at three concentrations (5000, 500, and 50 nM), with cell viability confirmed concurrently (Supplementary Table S1). Twenty compounds demonstrated robust anti-LUJV activity (ZsG fluorescence reduced to <30% of control) while maintaining >80% cell viability. These compounds were further evaluated using an extended concentration curve ( Table 1). Six of the twenty compounds had SI ≥ 10, demonstrating promising efficacy and applicability for therapeutic use; efficacy was further confirmed against wt LUJV.
The 20 compounds demonstrating robust anti-LUJV activity can be broadly grouped based on known or predicted modes of antiviral action as: (1) inhibitors of viral replication; (2) inhibitors of viral entry, intracellular trafficking, or virus egress; (3) protein kinase inhibitors; (4) selective estrogen receptor modulators (SERMs); or (5) miscellaneous compounds not falling into the four previous groups.

Additional Compounds with Anti-rLUJV/ZsG Properties
Four of the compounds selected from the initial screen did not fit into the four previous groups based on predicted mode of action ( Figure 4B). Three of these compounds are FDAapproved compounds: benztropine mesylate (EC 50 5.14 ± 1.52 µM, CC 50 > 25, SI > 4.9) is a neurotransmitter inhibitor (anticholinergic); clemastine fumarate (EC 50 3.75 ± 1.79 µM, CC 50 13.12 ± 2.33 µM, SI = 3.5) acts primarily as an H1 histamine antagonist; and loperamide HCl (EC 50 3.67 ± 0.99 µM, CC 50 12.29 ± 3.57 µM, SI = 3.3) is an opioid receptor that targets µ-opioid receptors in the large intestine and acts as an antidiarrheal agent by decreasing intestinal movement. All three were able to moderately inhibit rLUJV/ZsG and had SI values between 3 and 5. The final compound, obatoclax (EC 50 0.36 ± 0.1 µM, CC 50 1.5 ± 0.33 µM, SI = 4.2) inhibits the Bcl-2 family of proteins and has been investigated as an experimental anti-cancer drug. It inhibited rLUJV/ZsG at sub-micromolar EC 50 value, but was cytotoxic at a relatively low concentrations, which reduced SI to 4.2.

Additional Compounds with Anti-rLUJV/ZsG Properties
Four of the compounds selected from the initial screen did not fit into the four previous groups based on predicted mode of action ( Figure 4B). Three of these compounds are FDA-approved compounds: benztropine mesylate (EC50 5.14 ± 1.52 μM, CC50 > 25, SI > 4.9) is a neurotransmitter inhibitor (anticholinergic); clemastine fumarate (EC50 3.75 ± 1.79 μM,

Confirmatory Screening with wt LUJV
To confirm that the antiviral activity of the compounds was not specific to the ZsGexpressing recombinant virus, the top six candidates (SI ≥ 10) were selected to be screened using wt LUJV; these compounds were 2-dFC, apilimod, AVN-944, brequinar, BX-795, and favipiravir. These six drugs were tested using a shortened concentration curve (5 dilutions centered around their calculated EC 50 value), with cell viability determined concurrently. All compounds evaluated inhibited wt LUJV titers in a concentration-dependent manner, and all compounds reduced viral titers by approximately 4 logs while retaining >75% cell viability ( Figure 5). These results further confirm the suitability of using a reduction of ZsG fluorescence in rLUJV/ZsG-infected cells as a convenient readout in place of wt virus infection.

Discussion
Only one outbreak of LUJV has been recorded to date, with four out of five patients succumbing to fatal disease. The effectiveness of existing antivirals in treating LUJV patients is unknown. Ribavirin was administered to the sole surviving patient; however, based on this single treated case, it is not possible to determine the effect of treatment on the outcome. Furthermore, given our knowledge of ribavirin treatment in other arenavirus infections, in which it is only partially effective and associated with significant side effects, investigations into uncovering effective and safe arenavirus therapeutic options are badly needed [7]. High-throughput screening assays utilizing recombinant reporter viruses as surrogates for the wild-type parental virus have previously been successful in Figure 5. Ability of the most potent anti-LUJV compounds to inhibit wild-type virus replication. Titer reduction assays using wt LUJV were performed in Huh7 cells treated with varying concentrations of indicated compound 1 h prior to infection (MOI 0.1). For each compound concentration, inhibition of rLUJV/ZsG and cell viability were concurrently assessed. Four days post-infection, cell culture supernatants were collected and LUJV titers (TCID 50 /mL) were determined in Vero-E6 cells by immunofluorescence assays. For each concentration, LUJV titers (black) and cell viability (blue) are shown. Dotted lines represent the limit of detection for the TCID 50 assay (red) and the mean wt LUJV titer in mock-treated (DMSO only) cells (green). Each point represents the mean of quadruplicate wells, with error bars indicating standard deviation; graphs are representative of at least 3 independent experiments. ZsG fluorescence in rLUJV/ZsG-infected Huh7 cells at each compound concentration is also shown (10× magnification).

Discussion
Only one outbreak of LUJV has been recorded to date, with four out of five patients succumbing to fatal disease. The effectiveness of existing antivirals in treating LUJV patients is unknown. Ribavirin was administered to the sole surviving patient; however, based on this single treated case, it is not possible to determine the effect of treatment on the outcome. Furthermore, given our knowledge of ribavirin treatment in other arenavirus infections, in which it is only partially effective and associated with significant side effects, investigations into uncovering effective and safe arenavirus therapeutic options are badly needed [7]. High-throughput screening assays utilizing recombinant reporter viruses as surrogates for the wild-type parental virus have previously been successful in identifying several anti-mammarenavirus compounds [15,16,46,47]. Here, using a novel recombinant reporter LUJV, we were able to identify six compounds with high efficacy against LUJV that warrant further evaluation.
Given the genetic diversity among species, the large number of different arenavirus host reservoirs, and the geographic range of these pathogens, the emergence of new arenaviruses pathogenic to humans remains an ongoing public health concern [7]. Importantly, several of the compounds identified here as effective against LUJV (i.e., favipiravir, 2-dFC, and brequinar) have also been shown to inhibit other arenavirus species. The nucleoside analogs favipiravir and 2-dFC are both known to inhibit other arenaviruses including LASV, JUNV, Machupo virus, and GTOV [16,48,49]. The most promising inhibitor identified here was the dihydroorotate dehydrogenase inhibitor brequinar (EC 50 0.11 ± 0.02 µM, CC 50 > 12.5 µM, SI > 115), which has previously been shown to inhibit LASV, JUNV, and LCMV at similar sub-micromolar EC 50 values [50]. These broad-spectrum antivirals inhibit a wide range of highly pathogenic arenavirus species, presumably by targeting conserved viral processes, and are thus promising candidates for treating current and future emerging arenaviruses. It should be noted that inhibitors of pyrimidine biosynthesis can exhibit potent antiviral activity in cell culture, but be ineffective in animal models, which is likely due to pyrimidines in the animals' diet overcoming the antiviral effect [51].
While not yet approved, obatoclax is an inhibitor of the Bcl-2 family of proteins and an experimental anti-cancer drug that has undergone several phase II clinical trials; it has been shown to inhibit LCMV [28] as well as multiple other virus species including Rift Valley fever virus and HSV-2 [30]. The ability to rapidly screen large libraries of FDA-approved compounds or small molecules for antiviral efficacy is a huge advantage of our reporter virus systems, since comparable studies using wt virus are difficult to adapt for highthroughput screening and are prohibitively time-consuming, costly, and labor-intensive.
The six most promising candidates with both high efficacy and low cytotoxicity fell within three broadly defined groups based on modes of action: inhibiting viral replication; viral entry and trafficking; or protein kinases. The majority (four out of six) of these with the highest SI values (and therefore the most promising therapeutic candidates) were inhibitors of viral replication. Several other compounds that were identified demonstrated potent anti-LUJV activity, but had associated cytotoxicity, leading to low SI values and a narrow therapeutic window. Although this would likely make these compounds unattractive as single-drug treatment options, combination therapy using compounds with distinct modes of action may synergistically inhibit LUJV [19,27,55,56]. This would allow these more cytotoxic compounds to be used at far lower concentrations while maximizing their antiviral potential.
Here we describe the development of a recombinant LUJV engineered to express the fluorescent protein ZsGreen1 and validate its use in high-throughput screening assays for large or complex screens containing multiple compounds at various concentrations. We identified several highly efficacious anti-LUJV compounds with multiple predicted modes of action. Future studies are planned to investigate if potential synergistic actions increase the effectiveness of these compounds, as well as to advance the most promising candidates to studies using the available animal models of disease to evaluate in vivo effectiveness. Given the genetic diversity of mammarenaviruses and the high-consequence nature of their infections, it is promising to report that newly emerging species such as LUJV are similarly inhibited by several previously reported compounds. Identifying pan-arenavirus inhibiting compounds serves as a key step in the preparedness against new and emerging pathogenic threats.