key: cord-0708820-xlaq1lq7
authors: Kim, Yu-Jin; Venturini, Victor; de la Torre, Juan C.
title: Progress in Anti-Mammarenavirus Drug Development
date: 2021-06-22
journal: Viruses
DOI: 10.3390/v13071187
sha: 8dfebe13aea7911f3d92ff9a365836fb1745fbcb
doc_id: 708820
cord_uid: xlaq1lq7

Mammarenaviruses are prevalent pathogens distributed worldwide, and several strains cause severe cases of human infections with high morbidity and significant mortality. Currently, there is no FDA-approved antiviral drugs and vaccines against mammarenavirus and the potential treatment option is limited to an off-label use of ribavirin that shows only partial protective effect and associates with side effects. For the past few decades, extensive research has reported potential anti-mammarenaviral drugs and their mechanisms of action in host as well as vaccine candidates. This review describes current knowledge about mammarenavirus virology, progress of antiviral drug development, and technical strategies of drug screening.

Members of the family Arenaviridae are classified into four genera based on phylogenetic analysis of their RNA-directed RNA polymerase (L protein) and nucleoprotein (NP) sequences: Antennavirus, Hartmanivirus, Mammareanvirus, and Reptarenavirus [1] . Antennaviruses (2 species) were discovered in actinopterygian fish by next-generation sequencing, and no biological isolate has been reported yet. Hartmaniviruses (4 species) and reptarenaviruses (5 species) infect captive snakes, and some of them have been associated with boid inclusion body disease (BIBD). Mammarenaviruses (39 species) infect mainly rodents, and the infection is generally asymptomatic. Current knowledge about the biology of snake and fish arenaviruses is very limited, and their zoonotic potential unknown. In contrast, some mammarenaviruses have been found to infect and cause disease in humans.

Mammarenaviruses are enveloped viruses with a bi-segmented single-stranded negativesense RNA genome [1] . Mammarenaviruses cause chronic infections of their rodent natural reservoirs across the world, but some of them have zoonotic potential. Human infections occur through mucosal exposure to aerosols or by direct contact of abraded skin with infectious materials [2] . Based on their antigenic properties, mammarenaviruses have been classified into two distinct groups, Old world (OW) mammarenaviruses, aka Lassa-lymphocytic choriomeningitis virus serocomplex," including viruses present in Africa and the worldwide distributed lymphocytic choriomeningitis virus (LCMV), and the New World (NW) mammarenaviruses, aka "Tacaribe serocomplex", including viruses indigenous to the Americas [3] . Both OW and NW mammarenaviruses include several species members that can cause severe hemorrhagic fever (HF) diseases in humans that are associated with high morbidity and significant mortality; these viruses include Lassa (LASV), Junin (JUNV), Machupo (MACV), Guanarito (GTOV), Sabia (SABV), Chapare (CHPV), and Lujo (LUJV) [4] . Concerns posed by human pathogenic mammarenaviruses are exacerbated by the overall lack of FDA-licensed vaccines and current anti-mammareanavirus therapy being limited to off-label use of ribavirin that is only partially effective, has a narrow therapeutic window, and can be associated with side effects [5] . The only mammarenavirus vaccine tested in humans is the live-attenuated Candid#1 strain of Both genome segments contain non-coding cis-regulatory elements required for replication and transcription of the virus genome, including the 5′ and 3′ untranslated regions (UTR) present at the ends of both RNA segments and the non-coding intergenic regions (IGR) separating the two open reading frames present in each genome segment [36] . Mammarenaviruses have highly conserved sequences at the 3′-end of the L and S RNA genome segments that contain the genome promoter recognized by the virus polymerase to initiate RNA synthesis [37] . Genomes and antigenome RNAs are highly complementary between their 5′-and 3′-termini, with both L and S genome segments, predicted to form panhandle structures, which is consistent with EM images showing the existence of circular RNP complexes within mammarenavirions. Mammarenavirus IGRs are predicted to fold into stable hairpin structures that constitute bona fide transcription termination signals. There are significant differences in sequence and predicted folded structure between the S and L IGR, but among isolates and strains of the same arenavirus, the S and L IGR sequences are highly conserved. The IGR has been also implicated in virus assembly or budding, or both, being required for the generation of infectious particles [38, 39] .

Mammarenavirus enter susceptible cells mainly via receptor-mediated endocytosis. The acidic environment of the late endosome facilitates a pH-dependent conformational change in the GP complex that induces a GP2-mediated fusion step between viral and cell membranes. Following fusion, the viral RNP is released into the cytoplasm, where it directs both replication and transcription of the viral genome ( Figure 2 ). Different cell surface receptors are used by Old World (OW) and New World (NW) mammarenaviruses. OW mammarenaviruses, including lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV), as well as clade C NW mammarenaviruses, use α-dystroglycan as the major high-affinity receptor to which GP1 attaches [40, 41] . In contrast, pathogenic clade B NW mammarenaviruses including Guanarito (GTOV), Sabia (SABV), Junin (JUNV), Machupo (MACV) viruses use human transferrin receptor 1 (hTfR1) as the main receptor for Both genome segments contain non-coding cis-regulatory elements required for replication and transcription of the virus genome, including the 5 and 3 untranslated regions (UTR) present at the ends of both RNA segments and the non-coding intergenic regions (IGR) separating the two open reading frames present in each genome segment [36] . Mammarenaviruses have highly conserved sequences at the 3 -end of the L and S RNA genome segments that contain the genome promoter recognized by the virus polymerase to initiate RNA synthesis [37] . Genomes and antigenome RNAs are highly complementary between their 5 -and 3 -termini, with both L and S genome segments, predicted to form panhandle structures, which is consistent with EM images showing the existence of circular RNP complexes within mammarenavirions. Mammarenavirus IGRs are predicted to fold into stable hairpin structures that constitute bona fide transcription termination signals. There are significant differences in sequence and predicted folded structure between the S and L IGR, but among isolates and strains of the same arenavirus, the S and L IGR sequences are highly conserved. The IGR has been also implicated in virus assembly or budding, or both, being required for the generation of infectious particles [38, 39] .

Mammarenavirus enter susceptible cells mainly via receptor-mediated endocytosis. The acidic environment of the late endosome facilitates a pH-dependent conformational change in the GP complex that induces a GP2-mediated fusion step between viral and cell membranes. Following fusion, the viral RNP is released into the cytoplasm, where it directs both replication and transcription of the viral genome ( Figure 2 ). Different cell surface receptors are used by Old World (OW) and New World (NW) mammarenaviruses. OW mammarenaviruses, including lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV), as well as clade C NW mammarenaviruses, use α-dystroglycan as the major high-affinity receptor to which GP1 attaches [40, 41] . In contrast, pathogenic clade B NW mammarenaviruses including Guanarito (GTOV), Sabia (SABV), Junin (JUNV), Machupo (MACV) viruses use human transferrin receptor 1 (hTfR1) as the main receptor for entry [42] . A cell entry receptor for clade A NW mammarenaviruses has not been identified yet [43] . The OW hemorrhagic fever mammarenavirus Lujo virus (LUJV) LUJV was shown to use neuropilin (NRP)-2 as its main cell entry receptor [44] . Interestingly, NRP-2 is highly expressed on microvascular endothelial cells and alveolar macrophages, which may explain the extent of coagulopathy observed in LUJV-induced clinical disease and aerosol transmission of LUJV [45] [46] [47] . Once GP1 binds to the cellular receptor, the virion is endocytosed following different pathways by NW and OW mammarenaviruses. NW mammarenaviruses are internalized via clathrin-dependent endocytosis [48, 49] , whereas OW arenaviruses proceed through a clathrin-independent pathway that involves PI3Kmediated formation of multivesicular bodies (MVB) during late endocytosis and the endosomal sorting complex required for transport (ESCRT) proteins [50] . Notably, completion of the cell entry process for LASV and LUJV requires a late endosomal receptor switch mechanism. For LASV, the lysosome-associated membrane protein 1 (LAMP1) mediates a cholesterol-dependent pH-receptor switch mechanism [51] , which stimulates GP1 dissociation from the GP complex and a conformational change in GP2 that drives the fusion of viral and cell membranes [20, 52] . In the case of LUJV, this process is mediated by the tetraspanin CD63 [44] .

Viruses 2021, 13, x FOR PEER REVIEW 4 of 18 entry [42] . A cell entry receptor for clade A NW mammarenaviruses has not been identified yet [43] . The OW hemorrhagic fever mammarenavirus Lujo virus (LUJV) LUJV was shown to use neuropilin (NRP)-2 as its main cell entry receptor [44] . Interestingly, NRP-2 is highly expressed on microvascular endothelial cells and alveolar macrophages, which may explain the extent of coagulopathy observed in LUJV-induced clinical disease and aerosol transmission of LUJV [45] [46] [47] . Once GP1 binds to the cellular receptor, the virion is endocytosed following different pathways by NW and OW mammarenaviruses. NW mammarenaviruses are internalized via clathrin-dependent endocytosis [48, 49] , whereas OW arenaviruses proceed through a clathrin-independent pathway that involves PI3Kmediated formation of multivesicular bodies (MVB) during late endocytosis and the endosomal sorting complex required for transport (ESCRT) proteins [50] . Notably, completion of the cell entry process for LASV and LUJV requires a late endosomal receptor switch mechanism. For LASV, the lysosome-associated membrane protein 1 (LAMP1) mediates a cholesterol-dependent pH-receptor switch mechanism [51] , which stimulates GP1 dissociation from the GP complex and a conformational change in GP2 that drives the fusion of viral and cell membranes [20, 52] . In the case of LUJV, this process is mediated by the tetraspanin CD63 [44] . (2). The acidic environment of the late endosome promotes fusion between viral and host cell membranes (3) . Following the pH-dependent membrane fusion event, the vRNP is released into the cytoplasm, where it directs the biosynthetic processes of replication and transcription of the viral genome (4, 5) . Viral assembly takes place in the cell cytoplasm, and virions bud from the plasma membrane. Z plays critical roles in both these processes (6).

Following its release from the late endosome into the cytoplasm, the vRNP directs the biosynthetic processes of replication and transcription of the viral genome RNAs ( Figure 2 ). L and NP are the minimal viral trans-acting factors required for these processes [53, 54] , whereas Z has been shown to exhibit a dose-dependent inhibitory effect on the (1) . Virus uptake into the cell is mediated by endocytosis (2) . The acidic environment of the late endosome promotes fusion between viral and host cell membranes (3) . Following the pH-dependent membrane fusion event, the vRNP is released into the cytoplasm, where it directs the biosynthetic processes of replication and transcription of the viral genome (4, 5) . Viral assembly takes place in the cell cytoplasm, and virions bud from the plasma membrane. Z plays critical roles in both these processes (6).

Following its release from the late endosome into the cytoplasm, the vRNP directs the biosynthetic processes of replication and transcription of the viral genome RNAs ( Figure 2 ). L and NP are the minimal viral trans-acting factors required for these processes [53, 54] , whereas Z has been shown to exhibit a dose-dependent inhibitory effect on the vRNP activity, likely by interacting with the viral L polymerase and disabling its catalytic activity [31, 53] .

Mammarenaviruses generate three different RNA species during replication and transcription: genomic (gRNA) and antigenomic (agRNA) RNA species and sub-genomic viral mRNA species. Synthesis of viral mRNAs starts at the genome promoter and terminates within the distal region of the hairpin-structured IGRs [4] . Due to the ambisense coding strategy that characterizes mammarenavirus genome, NP and L mRNAs are transcribed from the genomic RNA, while GPC and Z mRNAs are transcribed from the corresponding antigenome RNA species. This ambisense coding strategy has been suggested to produce a hierarchal pattern of gene expression where NP and L proteins are produced at very early times of infection, and GPC and Z proteins at later times [4, 55] .

Mutation-function analysis using cell-based minireplicon assays for several mammarenaviruses indicated that the activity of the mammarenavirus genomic promoter requires both sequence specificity within the highly conserved 3 -terminal 19 nucleotides of arenavirus genomes and the integrity of the predicted panhandle structure formed via sequence complementarity between the 5 -and 3 -termini of viral genome RNAs [37, 56] . IGRs serve as bona fide transcription termination signals, but the synthesis of translationcompetent viral mRNAs does not strictly require the presence of the IGR [38, 39, 57] .

Mammarenavirus transcription initiation uses a cap-snatching mechanism, where short 5 -capped primers derived from host-cell mRNAs are used by the virus polymerase complex to prime the synthesis of viral mRNAs [4, 58] . The 5 -capped primers are generated via cleavage of cellular mRNAs by the endonuclease activity associated with the N-terminus of L [59] . The 5 -end of mammarenavirus genome and antigenome RNAs each contain a non-templated G residue that likely reflects a prime-and-realign mechanism for RNA replication mediated by L [60, 61] . Mammarenavirus genome and antigenome, sequence terminal complementarity combined with the prime-and-realign mechanism for replication initiation generates doublestranded RNAs with overhanging 5 -ppp nucleotides. These structures can act as RIG-I decoys, thereby diminishing RIG-I-mediated interferon induction in mammarenavirusinfected cells [62] . NP mRNA of the NW mammarenavirus Tacaribe (TCRV) was detected at early times of infection and in the presence of inhibitors of protein synthesis, suggesting that, unlike the closely related bunyaviruses, but similarly to orthomyxoviruses, rhabdoviruses, and paramyxoviruses, primary transcription directed by the incoming mammarenavirus vRNP can proceed in the absence of translation [4, 63] .

As with many other enveloped NS RNA viruses, mammarenavirus assembly and budding require the participation of the matrix protein Z [64] . Z interacts with GP2 [33] and NP [65] , which facilitates vRNP-GP complex interactions required for the assembly of mature viral particles ( Figure 2 ). As with other matrix proteins, Z has the capability to selfassemble and direct budding of VLPs [35] . Z budding activity is driven by canonical viral late (L) domains within the C-terminus of Z, which mediate Z interaction with components of the cellular ESCRT complexes [66] known to play essentials roles in the budding process of different viruses [67, 68] .

Completion of mammarenavirus cell entry requires a GP2-mediated fusion event between viral and cellular membranes, a process triggered by the acidic environment of the late endosome. Therefore, targeting the GP2-mediated fusion event is an attractive strategy to inhibit mammarenavirus infection. Accordingly, a high-throughput screen (HTS) of a small molecule library identified a series of molecules (ST-193, ST-294, and ST-336) that target GP2 and inhibit viral entry [69, 70] . has been shown to confer significant protection against LASV infection in a guinea pig model [71] , and the optimized chemical analog of ST-193 compound, LHF-535, was shown to be a potent broad-spectrum inhibitor of HF mammarenaviruses via targeting the GP2-mediated fusion step of virus cell entry [72] . Another GP2-mediated fusion inhibitor, F3406, was shown to inhibit LCMV multiplication [73] . AVP-p, a peptide derived from the Pichinde virus (PICV) GP2 subunit, was found to bind the prefusion state of the GP complex, which arrests the GP2-mediated fusion event [74] . Arbidol (umifenovir) was developed as an inhibitor of influenza virus infection, but it has been shown to exhibit broad-spectrum antiviral activity against different viruses by interfering with different steps, including cell entry of the virus life cycle [75] . Arbidol inhibits both LASV and Ebola virus (EBOV) GP-mediated fusion events required to complete the virus cell entry process [76, 77] . Screening of a natural product library identified tangeretin as a cell entry inhibitor of seven different HF-causing viruses [78] .

Several compounds have been shown to inhibit mammarenavirus cell entry via offtarget activities distinct from their intended therapeutic effects. The clotrimazole-derivative TRAM-34 is an ion channel blocker that antagonizes the calcium-activated potassium channel KCa3.1. TRAM-34 specifically inhibited mammarenavirus GP2-mediated fusion, and this anti-mammarenaviral effect was independent of its channel blocking activity [79] . Losmapimod, an p38 MAPK inhibitor, was developed as a therapeutic drug for chronic obstructive pulmonary disease (COPD) and has been reported to inhibit LASV entry by blocking the pH-dependent GP2-mediated fusion without requiring inhibition of p38 MAPK [80] . NH125, a selective eEF-2 kinase inhibitor, inhibited cell entry of recombinant VSVs expressing envelope glycoproteins of avian influenza virus, EBOV, and LASV due to the compound lysosomotropic properties and independently of eEF-2 kinase inhibition [81] . ZCL278 was identified as an inhibitor of Cdc42, a small GTPase that regulates actin polymerization [82] . Subsequently, ZCL278 was shown to exhibit broad-spectrum anti-viral activity, including against mammarenaviruses, by redistributing viral particles from endosomal to lysosomal membranes, and this antiviral activity was not dependent on the downregulation of Cdc42 activity [83] . The antifungal drug isavuconazole, which was approved by the FDA as an orphan drug for aspergillosis, mucormycosis, and candidiasis, was shown to inhibit viral fusion, targeting the SSP-GP2 interface of LASV [84] . The adamantyl diphenyl piperazine 3.3 was developed to target the lysosome-associated membrane protein 1 (LAMP1) by competing with cholesterol, preventing the interaction between LAMP1 and LASV GP [85] .

The nucleoside analog ribavirin has been shown to inhibit mammarenavirus replication in cell culture systems, as well as to have clinical benefits when used to treat LF cases at early times after the onset of clinical symptoms [86, 87] . Several different mechanisms of action have been proposed to account for the antiviral activity of ribavirin, including up-regulation of interferon responses [88] , GTP pool depletion by inhibiting IMP dehydrogenase (IMPDH), and direct inhibition of viral RNA-directed RNA polymerase (RdRp) activity [89] . Due to its broad-spectrum antiviral activity against multiple RNA and DNA viruses, most likely different mechanisms of action, and combination of them, contribute to the antiviral activity of ribavirin. Notably, a recent study has shown that the main mechanism of the antiviral activity of ribavirin against LASV, is by protecting LASV-infected cells from death [90] .

Favipiravir was initially developed as an antiviral drug targeting the influenza virus polymerase and subsequently shown to exhibit a broad-spectrum activity against different RNA viruses, including mammarenaviruses, bunyaviruses, flaviviruses, alphaviruses, picornaviruses, and noroviruses [91, 92] . BCX4430 is another broad-spectrum inhibitor of many types of RNA viruses, including mammarenaviruses, which interferes with the activity of the viral RdRp by acting as an RNA chain terminator [93] .

Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO), designed to target conserved regions within mammarenavirus genome RNA, were effective against multiple mammarenaviruses, including JUNV, LCMV, TCRV, and PICV [94] . The inhibitors of DEDDh family of 3 -5 exonucleases aurintricarboxylic acid (ATA) and pontacyl violet 6R (PV6R) have been reported to inhibit the 3 -5 exonuclease activity of LASV NP [15] , which in addition to its role counteracting the host cell IFN-I response, has been shown to play a critical role in viral fitness in IFN-I deficient cells [95] . As metal-chelating pharmacophores, diketo acids, polyphenols, and N-hydroxyisoquinoline-1,3-diones were able to inhibit the endonuclease activity of arenavirus L protein, which resulted in inhibition of the capsnatching mechanism used by mammarenavirus polymerases to initiate transcription of Viruses 2021, 13, 1187 7 of 18 viral mRNAs [96] . A carboxamide-derivatized disulfide, NSC4492, was reported to show antiviral activity against JUNV and TCRV [97] and this compound was shown to impair viral RNA synthesis of JUNV via targeting the replication complex [98] . KP-146 was shown to have dual roles in its antiviral activity against LCMV, not only interfering with vRNP activity responsible for directing LCMV genome replication and gene transcription but also inhibiting Z protein-mediated budding process [95] .

The mammarenavirus GPC precursor is co-translationally cleaved by cellular signal peptidases to generate a 58 amino acid-long stable signal peptide (SSP) and the immature GP1/GP2 precursor [99] . Subsequent processing of GP1/GP2 by the cellular subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) into GP1 and GP2 is required for the production of infectious progeny [17, 19] . GP1, GP2, and the SSP form the mature trimeric GP spike complex [99] . Decanoyl-RRLL-chloromethylketone (dec-RRLL-CMK) was developed as a SKI-1/S1P inhibitor based on the cleavage recognition site present within LASV GPC [100] and was shown to exert a potent antiviral activity against LCMV as well as additive antiviral drug activity in combination with ribavirin [101] . PF-429242, a small molecule inhibitor of S1P, was shown to interfere with the proteolytic processing of GPC, which correlated with the compound's ability of inhibiting multiplication of LCMV and LASV in cultured cells [102] . In S1P-deficient cells, wild-type LCMV consistently underwent extinction without emergence of S1P-independent escape variants [101] . Moreover, PF-429242 efficiently and rapidly cleared persistent virus from infected cells, and interruption of drug treatment did not result in re-emergence of infection, indicating that PF-429242 treatment resulted in virus extinction [103] . These findings indicate a high genetic barrier for the emergence of viral variants capable of using an alternative host cellular protease for the processing of GPC, thus making S1P a very attractive target for the development of antiviral drugs against mammarenaviruses.

Assembly and cell release of infectious mammarenavirus progeny involves Z-L, Z-NP, and Z-GP interactions to facilitate the co-localization of all viral proteins for the assembly of mature infectious particles [30] . Functional studies have shown that the matrix Z protein plays a key role in mammareanvirus budding, a process mediated by the interaction of Z late (L) domain motifs, PTAP and PPPY, with components of the cellular ESCRT complexes [104] . Z-mediated budding also requires myristoylation of the Z protein at a glycine (G) in position 2 to target Z to the plasma membrane, the location of arenavirus budding [105] . Accordingly, treatment with 2-hydroxymyristic acid, an inhibitor of the N-myristoyltransferase (NMT), impaired Z budding activity and production of mammarenavirus infectious progeny [105] . Valproic acid (VPA), a short-chain fatty acid, used in anti-epileptic therapy [106] , was shown to inhibit Z-mediated budding of LCMV, likely due to VPA-mediated alteration of the lipid composition of cellular membranes, which is critical in virus budding [107] . Compound 0013, identified as a potent inhibitor of the interaction between the PTAP L domain and Tsg101, a member of the host cell ESCRT complex proteins, was shown to inhibit viral budding by blocking the Z-Tsg101 interaction [108] . The ubiquitin ligase Nedd4 E3 is also a component of the ESCRT complex [109] . A small molecule termed compound1 was identified as an inhibitor of Z-Nedd4 interaction, resulting in inhibition of viral budding [110] . BEZ-235, a phosphatidylinositol 3-kinase (PI3K) inhibitor, was shown to inhibit Z protein-mediated budding of LCMV and LASV by a mechanism of action yet to be determined [111] . In addition to driving virion assembly and release, Z proteins have been reported to modulate various host mechanisms such as repression of translation by binding and counteracting eIF4E [26] and suppression of host innate immune responses [112] , suggesting potential host targets for developing antiviral drugs. Recent Z interactome study has identified human proteins that interact with arenavirus Z and validated potential host targets for antiviral therapeutics, including ADP ribosylation factor 1 (ARF1), ATP synthase, H transporting mitochondrial F1 complex beta polypeptide (ATP5B), ATPase H transporting lysosomal 38-kDa V0 subunit d1 (ATP6V0D1), inosine monophosphate dehydrogenase 2 (IMPDH2), peroxiredoxin 3 (PRDX3), and Ras-related protein Rab5c [113] .

Monoclonal antibody-based therapies represent an attractive strategy to treat infections by highly pathogenic mammarenaviruses. A monoclonal antibody specific for human transferrin receptor 1 (hTfR1), the receptor used by pathogenic NW mammarenaviruses, inhibits viral entry of several NW mammarenaviruses, including JUNV, GTOV, CHAV, SABV, and MACV [114] . In addition, monoclonal antibodies targeting mammarenavirus GP have been reported to have potent neutralizing activity against MACV and LASV that correlated with inhibition of virus multiplication [115, 116] . A recent study documented the isolation of human monoclonal antibodies from LF survivors and characterized their epitope and neutralization profiles, showing that 80% of the monoclonal antibodies with neutralizing activity targeted complex epitopes involving LASV GP1 and GP2 subunits [117] . Importantly, this study identified several human monoclonal antibodies with neutralizing activities against members of the main four lineages of LASV, and some of them showed cross-reactivity to LCMV, LUJO, and MACV. This finding has provided insights to develop therapeutic strategies based on the use of broadly reactive monoclonal antibodies.

As strict parasites, viruses rely on many host cell factors to complete their life cycles. Therefore, there is increasing interest in targeting host cell factors required for virus multiplication as an antiviral drug strategy. Direct-acting antivirals (DAAs) that target specific viral gene products and functions are likely to be well tolerated by the infected host cell. Still, they are limited by the common problem in antiviral therapy posed by the emergence of drug-resistant variants. In contrast, the emergence of viral variants resistant to host-targeting antivirals (HTAs) is usually significantly reduced or entirely absent, but HTAs can be associated with significant side effects. However, side effects associated with the use of HTAs might be manageable in the case of acute infections, such as HF disease caused by arenaviruses, where the duration of the treatment would be rather short.

Dihydroorotate dehydrogenase (DHODH) small molecule inhibitors A3 and A77172 interfere with de novo pyrimidine biosynthesis and exhibit potent antiviral activity against LCMV and JUNV [118, 119] . Likewise, de novo purine biosynthesis is a potential cellular target for the development of HTAs. Inhibition of inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the purine biosynthesis pathway, was shown to be associated with a broad-spectrum antiviral activity against RNA viruses including JUNV [120, 121] . S-adenosylhomocysteine hydrolase (SAHH) is an important cellular enzyme for regulating viral mRNA capped structures, and inhibition of SAHH activity by 3-deazaneplanocin was associated with potent antiviral activity against TCRV and PICV [122] . ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1) and prohibitin (PHB) were identified as mammarenavirus NP-interacting host cell proteins and act as pro-viral factors that promote mammarenavirus multiplication. Accordingly, the ATP1A1 inhibitors, ouabain and bufalin, as well as the PHB inhibitor, rocaglamide A, exhibit potent antiviral activity against LCMV and LASV infection [123] .

Anti-mammarenaviral drugs discussed in this section have been summarized in Table 1 . 

The development of mammarenavirus reverse genetics (RG) systems has provided investigators with a novel and powerful approach for the investigation of the cis-acting sequences and trans-acting factors that control arenavirus replication, gene expression, and budding, as well as the rescue of infectious mammarenaviruses from cloned cDNAs. These advances in mammarenavirus molecular genetics have facilitated the generation of recombinant mammarenaviruses expressing reporter genes of interest that have enabled the development of cell-based assays and HTS strategies to identify novel anti-mammarenaviral drugs and assess how they target each of the different steps of the virus life cycle [36] . Different strategies, including the development of tri-segmented mammarenaviruses [36] and the use of the self-cleaving P2A linker to facilitate expression of a reporter gene and NP from the same bicistronic NP mRNA [73] , have been used to generate these recombinant mammarenaviruses.

However, the use of recombinant infectious HF mammarenaviruses expressing reporter genes for antiviral drug screening campaigns or the investigation of drug mechanism of action would face the complications imposed by the requirement of BSL4 biocontainment. Hence, the advantage of the implementation of RG approaches to develop non-infectious cell-based assays recreating each of the key steps of the virus life cycle and that is amenable to HTS, which can be used without the need of a high-level biocontainment facility. Thus, a number of different platforms have been developed to screen for compounds capable of inhibiting cell entry mediated by GPs of different HF mammarenaviruses, using pseudotype viruses [124] [125] [126] . Likewise, the biosynthetic processes of replication and transcription of HF mammarenavirus genomes can be mimicked using cell-based minireplicon, or minigenome (MG) systems [127] . These cell-based MG systems are based on the intracellular reconstitution of a functional vRNP directing expression of a reporter gene whose expression level serves as a surrogate of the vRNP activity. For this, cells are transfected with plasmids expressing the viral trans-acting factors L and NP, together with a plasmid that allows for intracellular synthesis of an RNA containing the open reading frame of a reporter gene under the control of the cis-acting regulatory sequences of the S or L genome RNA [54] . In addition to these transient transfection-based MG systems, cell lines have been generated to constitutively express a functional, non-infectious vRNP of LCMV or LASV [128] . This overcomes some technical complications related to transient transfection in the context of HTS. Thus, cell lines expressing LCMV and LASV functional vRNPs were successfully used to screen different compound libraries, resulting in the identification of a number of hits that were confirmed to exhibit antiviral activity against infectious LCMV and LASV [128] .

Generation of infectious virus-like particle (iVLP) systems containing functional virus MGs allows for modeling of not only viral genome replication but also cell entry and budding [129] . A chimeric protein consisting of a secretion deficient form of Gaussia luciferase (GLuc) fused to the C-terminus of LASV Z protein was successfully used to develop a cell-based assay to quantify Z-mediated budding activity. This assay has features compatible with its use in HTS [130] , as levels of GLuc in the tissue culture supernatant serve as an accurate surrogate of Z budding activity.

The Discovery and development of novel drugs require significant investments and resources and an average processing time for market authorization of 10 to 17 years [131] . The rapid development of antiviral therapeutics is important to combat emerging viruses. Finding novel applications of clinically approved drugs can accelerate the drug development process and significantly reduce risks during clinical trials assessing the new drug application. Accordingly, repurposing existing drugs is considered an attractive strategy to combat emerging viral infections [132] . This has been illustrated by efforts to combat the current COVID-19 pandemic, where screening of libraries of already approved drugs resulted in the rapid identification of anti-SARS-CoV-2 drug candidates that were very rapidly advanced to clinical trials [133] . Among the listed compounds in Table 1 , ribavirin, arbidol and favipiravir are currently being tested in COVID-19 patients in clinical trials [133] .

Screening of a library of FDA-approved drugs using VSV pseudotyped with LASV GP identified a number of inhibitors of LASV GP-mediated cell entry [125, 134] . Likewise, screening of the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library identified several potent anti-mammarenaviral compounds [135] . Importantly, selected hits initially identified based on their anti-LCMV activity, which were confirmed to show potent antiviral activity against the HF causing mammarenaviruses LASV and JUNV. These compounds exerted their antiviral activity via targeting host cellular factors, including enzymes required for pyrimidine and purine biosynthesis, regulators of apoptosis, and the mitochondrial electron transport complex III [135] . Recently, this ReFRAME library was used to screen for antiviral drugs against SARS-CoV-2, and the existing pharmacological and safety data on the identified hits will facilitate their rapid testing in the clinic [136] . In addition to HTS formats to rapidly identify novel targets and antiviral drug candidates, function-focus based assays have also been successfully used to identify compounds that could be repurposed as antiviral drugs. For example, screening of a collection of kinase inhibitors identified several cellular kinases that were involved in LASV GP-mediated viral entry, including protein kinase C, phosphoinositide 3-kinase, and human hepatocyte growth factor receptor (HGFR), which is a receptor tyrosine kinase [137] .

As documented in this review, significant efforts have been dedicated to finding effective antiviral drugs against human pathogenic mammarenaviruses. Different screening platforms have identified a number of antiviral drug candidates with potent activity in cell-based infection assays. However, for the majority of the identified hits, there is only very limited information regarding their in vivo efficacy. To advance the development of novel effective antiviral drugs, further validation should be conducted using appropriate in vivo models of mammarenavirus disease, including non-human primates.

Drug repurposing approaches have identified a number of host cell factors as attractive antiviral targets for which drugs with a good safety profile have been already documented, which should facilitate the assessment of their efficacy in vivo using appropriate animal models of mammarenavirus induced disease. Since different viruses may share some key host cell functions to complete their life cycle, a host-targeting strategy would be an attractive approach for the development of broad-spectrum antiviral therapeutics. Synergistic antiviral effects have been documented in combination therapies of approved drugs, as illustrated by the results of combination therapy of ribavirin and favipiravir against LASV infection in pre-clinical [87] and clinical [138] studies. Likewise, combination therapy of arbidol with aripiprazole or sertraline resulted in synergistic inhibition of pseudotyped viruses with GPs from LASV and JUNV [139] . Synergistic effects are likely to be facilitated by combination therapy with drugs targeting different steps of the virus life cycle. To identify combinations for antiviral therapeutics, modern computational approaches, including available data libraries and analytical resources [140] , would be promising tools by which data and text mining could identify potential drug combinations for further experimental validations. 

Arenaviridae: The viruses and their replication

Hemorrhagic Fever-Causing Arenaviruses: Lethal Pathogens and Potent Immune Suppressors

Human hemorrhagic Fever causing arenaviruses: Molecular mechanisms contributing to virus virulence and disease pathogenesis

Treatment of Arenavirus Infections

Candid No. 1 Argentine hemorrhagic fever vaccine protects against lethal Junin virus challenge in rhesus macaques

Protective efficacy of a live attenuated vaccine against Argentine hemorrhagic fever. AHF Study Group

Junin virus pathogenesis and virus replication

Complementarity in the supramolecular design of arenaviruses and retroviruses revealed by electron cryomicroscopy and image analysis

Induction and inhibition of type I interferon responses by distinct components of lymphocytic choriomeningitis virus

Transcription and replication mechanisms of Bunyaviridae and Arenaviridae L proteins

Roles of Arenavirus Z Protein in Mediating Virion Budding, Viral Transcription-Inhibition and Interferon-Beta Suppression

Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike

Multifunctional nature of the arenavirus RING finger protein Z. Viruses

Structure of the Lassa virus nucleoprotein reveals a dsRNA-specific 3' to 5' exonuclease activity essential for immune suppression

Inhibition of the type I interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus

The Lassa virus glycoprotein precursor GP-C is proteolytically processed by subtilase SKI-1/S1P

Identification of an N-terminal trimeric coiled-coil core within arenavirus glycoprotein 2 permits assignment to class I viral fusion proteins

Mechanisms for lymphocytic choriomeningitis virus glycoprotein cleavage, transport, and incorporation into virions

Acidic pH triggers LCMV membrane fusion activity and conformational change in the glycoprotein spike

X-ray structure of the arenavirus glycoprotein GP2 in its postfusion hairpin conformation

The role of single N-glycans in proteolytic processing and cell surface transport of the Lassa virus glycoprotein GP-C

The signal peptide of the Junin arenavirus envelope glycoprotein is myristoylated and forms an essential subunit of the mature G1-G2 complex

The RNA synthesis machinery of negative-stranded RNA viruses

The small RING finger protein Z drives arenavirus budding: Implications for antiviral strategies

The lymphocytic choriomeningitis virus RING protein Z associates with eukaryotic initiation factor 4E and selectively represses translation in a RING-dependent manner

Structural characterization of the Z RING-eIF4E complex reveals a distinct mode of control for eIF4E

An arenavirus RING (zinc-binding) protein binds the oncoprotein promyelocyte leukemia protein (PML) and relocates PML nuclear bodies to the cytoplasm

Role of the promyelocytic leukemia protein PML in the interferon sensitivity of lymphocytic choriomeningitis virus

Molecular mechanism of arenavirus assembly and budding

Arenavirus Z protein controls viral RNA synthesis by locking a polymerase-promoter complex

RING finger Z protein of lymphocytic choriomeningitis virus (LCMV) inhibits transcription and RNA replication of an LCMV S-segment minigenome

Arenavirus Z-glycoprotein association requires Z myristoylation but not functional RING or late domains

The C-terminal region of lymphocytic choriomeningitis virus nucleoprotein contains distinct and segregable functional domains involved in NP-Z interaction and counteraction of the type I interferon response

Lassa virus Z protein is a matrix protein and sufficient for the release of virus-like particles

Replicating-Competent Recombinant Arenaviruses. Viruses

Characterization of the genomic promoter of the prototypic arenavirus lymphocytic choriomeningitis virus

Dual role of the lymphocytic choriomeningitis virus intergenic region in transcription termination and virus propagation

The High Degree of Sequence Plasticity of the Arenavirus Noncoding Intergenic Region (IGR) Enables the Use of a Nonviral Universal Synthetic IGR To Attenuate Arenaviruses

New World arenavirus clade C, but not clade A and B viruses, utilizes alpha-dystroglycan as its major receptor

Identification of alpha-dystroglycan as a receptor for lymphocytic choriomeningitis virus and Lassa fever virus

Transferrin receptor 1 is a cellular receptor for New World haemorrhagic fever arenaviruses

NRP2 and CD63 Are Host Factors for Lujo Virus Cell Entry

Neuropilins in neoplasms: Expression, regulation, and function

Specific Neuropilins Expression in Alveolar Macrophages among Tissue-Specific Macrophages

Neuropilin-2 regulates airway inflammatory responses to inhaled lipopolysaccharide

Different mechanisms of cell entry by human-pathogenic Old World and New World arenaviruses

Cellular entry of lymphocytic choriomeningitis virus

Old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport

Virus entry. Lassa virus entry requires a trigger-induced receptor switch

Kinetics and pH dependence of acid-induced structural changes in the lymphocytic choriomeningitis virus glycoprotein complex

Transcription and RNA replication of tacaribe virus genome and antigenome analogs require N and L proteins: Z protein is an inhibitor of these processes

NP and L proteins of lymphocytic choriomeningitis virus (LCMV) are sufficient for efficient transcription and replication of LCMV genomic RNA analogs

Visualization of Arenavirus RNA Species in Individual Cells by Single-Molecule Fluorescence In Situ Hybridization Suggests a Model of Cyclical Infection and Clearance during Persistence

Replicon system for Lassa virus

A single stem-loop structure in Tacaribe arenavirus intergenic region is essential for transcription termination but is not required for a correct initiation of transcription and replication

Nontemplated bases at the 5' ends of Tacaribe virus mRNAs

An N-terminal region of Lassa virus L protein plays a critical role in transcription but not replication of the virus genome

Biochemical characterization of the Lassa virus L protein

Arenavirus reverse genetics: New approaches for the investigation of arenavirus biology and development of antiviral strategies

Short double-stranded RNAs with an overhanging 5' pppnucleotide, as found in arenavirus genomes, act as RIG-I decoys

Participation of eIF4F complex in Junin virus infection: Blockage of eIF4E does not impair virus replication

Late budding domains and host proteins in enveloped virus release

A role for the C terminus of Mopeia virus nucleoprotein in its incorporation into Z protein-induced virus-like particles

Cellular factors required for Lassa virus budding

Virus budding and the ESCRT pathway

The Interplay between ESCRT and Viral Factors in the Enveloped Virus Life Cycle

Identification and characterization of potent small molecule inhibitor of hemorrhagic fever New World arenaviruses

Identification of a broad-spectrum arenavirus entry inhibitor

Evaluation of Lassa antiviral compound ST-193 in a guinea pig model

A potent Lassa virus antiviral targets an arenavirus virulence determinant

Identification and Mechanism of Action of a Novel Small-Molecule Inhibitor of Arenavirus Multiplication

Inhibition of arenavirus infection by a glycoprotein-derived peptide with a novel mechanism

Arbidol as a broad-spectrum antiviral: An update

The Synthetic Antiviral Drug Arbidol Inhibits Globally Prevalent Pathogenic Viruses

Arbidol and Other Low-Molecular-Weight Drugs That Inhibit Lassa and Ebola Viruses

Tangeretin, an extract from Citrus peels, blocks cellular entry of arenaviruses that cause viral hemorrhagic fever

Identification of Clotrimazole Derivatives as Specific Inhibitors of Arenavirus Fusion

Identification of a clinical compound losmapimod that blocks Lassa virus entry

1-Benzyl-3-cetyl-2-methylimidazolium Iodide (NH125) Is a Broad-Spectrum Inhibitor of Virus Entry with Lysosomotropic Features

Small molecule targeting Cdc42-intersectin interaction disrupts Golgi organization and suppresses cell motility

Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor

The antifungal isavuconazole inhibits the entry of lassa virus by targeting the stable signal peptide-GP2 subunit interface of lassa virus glycoprotein

Critical role for cholesterol in Lassa fever virus entry identified by a novel small molecule inhibitor targeting the viral receptor LAMP1

Effective therapy with ribavirin

Efficacy of Favipiravir Alone and in Combination With Ribavirin in a Lethal, Immunocompetent Mouse Model of Lassa Fever

Mechanism of action of interferon and ribavirin in treatment of hepatitis C

Mechanism of action of ribavirin in the combination treatment of chronic HCV infection

Determining Ribavirin's mechanism of action against Lassa virus infection

Favipiravir (T-705), a broad spectrum inhibitor of viral RNA polymerase

Favipiravir (T-705), a novel viral RNA polymerase inhibitor

Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430

Development of peptide-conjugated morpholino oligomers as pan-arenavirus inhibitors

Identification of Inhibitors for the DEDDh Family of Exonucleases and a Unique Inhibition Mechanism by Crystal Structure Analysis of CRN-4 Bound with 2-Morpholin-4-ylethanesulfonate (MES)

Metal chelators for the inhibition of the lymphocytic choriomeningitis virus endonuclease domain

Inhibition of arenavirus infection by thiuram and aromatic disulfides

Targeting of arenavirus RNA synthesis by a carboxamide-derivatized aromatic disulfide with virucidal activity

Cleavage of the Glycoprotein of Arenaviruses

The proprotein convertase SKI-1/S1P. In vitro analysis of Lassa virus glycoprotein-derived substrates and ex vivo validation of irreversible peptide inhibitors

Targeting the proteolytic processing of the viral glycoprotein precursor is a promising novel antiviral strategy against arenaviruses

Antiviral activity of a small-molecule inhibitor of arenavirus glycoprotein processing by the cellular site 1 protease

Evaluation of the anti-arenaviral activity of the subtilisin kexin isozyme-1/site-1 protease inhibitor PF-429242

Arenavirus budding

Myristoylation of the RING finger Z protein is essential for arenavirus budding

Life on the battlefield: Valproic acid for combat applications

Inhibition of multiplication of the prototypic arenavirus LCMV by valproic acid

A host-oriented inhibitor of Junin Argentine hemorrhagic fever virus egress

Functional involvement of a novel Nedd4-like ubiquitin ligase on retrovirus budding

Smallmolecule probes targeting the viral PPxY-host Nedd4 interface block egress of a broad range of RNA viruses

The PI3K/Akt pathway contributes to arenavirus budding

The Z proteins of pathogenic but not nonpathogenic arenaviruses inhibit RIG-I-like receptor-dependent interferon production

A Proteomics Survey of Junin Virus Interactions with Human Proteins Reveals Host Factors Required for Arenavirus Replication

An antibody recognizing the apical domain of human transferrin receptor 1 efficiently inhibits the entry of all new world hemorrhagic Fever arenaviruses

Treatment of Lassa virus infection in outbred guinea pigs with first-in-class human monoclonal antibodies

Monoclonal Antibodies with Neutralizing Activity and Fc-Effector Functions against the Machupo Virus Glycoprotein

Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

Inhibition of arenavirus by A3, a pyrimidine biosynthesis inhibitor

Antiviral activity of A771726, the active metabolite of leflunomide, against Junin virus

Inhibition of Junin virus RNA synthesis by an antiviral acridone derivative

A heterocyclic molecule with significant activity against dengue virus

l-like 3-deazaneplanocin analogues: Synthesis and antiviral properties

Interactome analysis of the lymphocytic choriomeningitis virus nucleoprotein in infected cells reveals ATPase Na + /K + transporting subunit Alpha 1 and prohibitin as host-cell factors involved in the life cycle of mammarenaviruses

Unique small molecule entry inhibitors of hemorrhagic fever arenaviruses

Screening and Identification of Lassa Virus Entry Inhibitors from an FDA-Approved Drug Library

Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2

Reverse Genetics Approaches to Control Arenavirus

A cell-based, infectious-free, platform to identify inhibitors of lassa virus ribonucleoprotein (vRNP) activity

High-throughput screening for negative-stranded hemorrhagic fever viruses using reverse genetics

A cell-based luciferase assay amenable to high-throughput screening of inhibitors of arenavirus budding

Drug repositioning: Identifying and developing new uses for existing drugs

Drug Repurposing for Viral Infectious Diseases: How Far Are We?

Potential therapeutic targets for combating SARS-CoV-2: Drug repurposing, clinical trials and recent advancements

A systematic screen of FDA-approved drugs for inhibitors of biological threat agents

The ReFRAME library as a comprehensive drug repurposing library to identify mammarenavirus inhibitors

Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing

Lassa Virus Cell Entry via Dystroglycan Involves an Unusual Pathway of Macropinocytosis

Favipiravir and Ribavirin Treatment of Epidemiologically Linked Cases of Lassa Fever

Inhibition of Arenaviruses by Combinations of Orally Available Approved Drugs

High-throughput methods for combinatorial drug discovery

The authors declare no conflict of interest.Viruses 2021, 13, 1187