key: cord-0262021-1lvagm7c authors: Guo, Jiao; Zhang, Guangshun; Liu, Yang; Cao, Junyuan; Zhang, Mengmeng; Lan, Xiaohao; Zhang, Yueli; Liu, Chenchen; Xiao, Gengfu; Wang, Wei title: Functional Characterization of a Lassa Virus Fusion Inhibitors Adaptive Mutant date: 2020-12-24 journal: bioRxiv DOI: 10.1101/2020.12.23.424274 sha: 1cae6a032e9120dc17a036ba3b968d9cbfc6c224 doc_id: 262021 cord_uid: 1lvagm7c Lassa virus (LASV) glycoprotein complex (GPC) contains retained stable-signal peptide (SSP), GP1, and GP2. SSP interacts with GP2 and provides an interface targeted by numerous fusion inhibitors. Serially passaging of LASV with inhibitors allowed some adaptive mutants to be obtained of which most had mutations located in the transmembrane (TM) domain of GP2. In the current study, we focused on the F446L mutant, which is reported to confer resistance to ST-series inhibitors. We found that F446L conferred cross-resistance to structurally distinct inhibitors. Furthermore, F446L increased the fusion activities of LASV and Mopeia virus GPC, elevating the pH threshold for fusion of LASV and promoting fusion of MOPV at neutral pH. F446L exerted little effect on the pseudotype viral growth profile or thermostability. By introducing other residues to the conserved F446 locus, it was found that this site was less compatible with a similar tyrosine residue and was intolerable to charged residues. These results help characterize the fusion inhibitor target located in the TM domain of GP2, which should be useful for drug and vaccine design. IMPORTANCE The LASV SSP-GP2 interface provides an Achilles heel that is targeted by numerous inhibitors. However, the emergence of resistant viruses is a major concern for direct antiviral drugs. In this study, we investigated the F446L mutant located in the GPC TM domain to determine the relationship between drug resistance, membrane fusion activity, viral growth kinetics, and thermostability. These results will be helpful in monitoring drug-resistant variants, as well as the advancement of drug and vaccine design. F446L mutant, which is reported to confer resistance to ST-series inhibitors. We found 23 that F446L conferred cross-resistance to structurally distinct inhibitors. Furthermore, 24 F446L increased the fusion activities of LASV and Mopeia virus GPC, elevating the 25 pH threshold for fusion of LASV and promoting fusion of MOPV at neutral pH. F446L exerted little effect on the pseudotype viral growth profile or thermostability. By introducing other residues to the conserved F446 locus, it was found that this site 28 was less compatible with a similar tyrosine residue and was intolerable to charged 29 residues. These results help characterize the fusion inhibitor target located in the TM 30 domain of GP2, which should be useful for drug and vaccine design. IMPORTANCE The LASV SSP-GP2 interface provides an Achilles heel that is 32 targeted by numerous inhibitors. However, the emergence of resistant viruses is a 33 major concern for direct antiviral drugs. In this study, we investigated the F446L 34 mutant located in the GPC TM domain to determine the relationship between drug 35 resistance, membrane fusion activity, viral growth kinetics, and thermostability. These 36 results will be helpful in monitoring drug-resistant variants, as well as the 37 advancement of drug and vaccine design. The bi-segmented RNA genomes contain the small (S) and large (L) segments. 48 The S segment encodes the glycoprotein complex (GPC) and nucleoprotein, while the 49 L segment encodes the matrix protein (Z) and viral polymerase. The mature GPC is 50 present on the surface of the virion and consists of three subunits, the stable-signal 51 peptide (SSP), receptor-binding subunit GP1, and membrane fusion subunit GP2 (5-7). The three subunits are non-covalently bound and form a (SSP/GP1/GP2) 3 trimeric 53 complex ( Fig. 1A and 1B) . Notably, the unusual retained SSP plays an essential role 54 in GPC-mediated membrane fusion and cell entry. SSP anchors to the viral membrane 55 by interacting with the membrane proximal external region, the transmembrane 56 domain (TM), and the cytoplasmic tail of GP2, stabilizing the pre-fusion 57 conformation of GPC and providing an interface that is targeted by fusion inhibitors 58 (5-7). It has been widely reported that mammarenaviruses generate adaptive mutants 59 against entry inhibitors mostly through mutations located at the SSP-GP2 interface. Intriguingly, some adaptive mutants show cross-resistance to other entry inhibitors (8). The GPC F446L mutant of LASV and corresponding mutants in other 62 mammarenaviruses, such as JUNV F438L, have been identified among these adaptive 63 mutants to be resistant to multiple small-molecule inhibitors (8-11). To date, insight 64 into the mechanisms underlying resistance is limited. Whether the adaptive mutant 65 de-stabilizes the prefusion conformation of GPC or if the native residue of the 66 adaptive mutant serves as a viral target that interacts directly with the inhibitors is 67 unclear. In the current work, we aimed to characterize the effects of the F446L mutant on LASV GPC F446L exhibited resistance to structurally distinct inhibitors. 76 As noted, the F446L mutation is reported to confer resistance to numerous inhibitors. Passaging LASV with ST-161 derivative led to the isolation of the F446L mutant, 78 while passaging Tacaribe virus (TCRV) in the presence of the F436I mutant, corresponding to LASV GPC F446 mutants (9-11). The F446L 80 mutation has been reported to confer resistance to these ST-series inhibitors and their 81 structurally similar inhibitors ( Fig. 1C-1F ) (8, 9) . This raises the question whether 82 5 F446L confers resistance to other mammarenavirus entry inhibitors that caused 83 adaptive mutants with changes at loci other than the F446L change itself. To this end, 84 we investigated the sensitivity of LASV F446Lrv to lacidipine, in accordance with our 85 previous reported that lacidipine caused the adaptive T40K mutant in LASV GPC SSP 86 (6). As shown in Fig. 1H which was significantly higher than that of wild-type (WT) LASV (P = 0.00359). F446 is located in the transmembrane domain of GP2 (Fig 1A and 1B) . As GP2 is the 95 fusion subunit of the glycoprotein and TM plays an essential role in the 96 GPC-mediated fusion activity (9), we further evaluated the effect of F446L on 97 GPC-mediated fusion. We transfected 293t cells with WT or F446L GPC and 24 h 98 later treated the cells with buffers of various pH for 15 min. Syncytium formation was 99 then observed 4 h later. As shown in Fig. 2A , both WT and F446L GP2 could lead to a 100 complete fusion when treated with buffer at either pH 4.5 or 5.0. In comparison, WT 101 GP2 was able to induce only partial syncytium formation after exposure to pH 5.5, 102 while F446L still demonstrated complete fusion. To confirm this finding, a 103 quantitative evaluation was performed using a dual-luciferase assay. Consistent with 104 6 the results from the qualitative assay, the quantitative assay indicated that F446L was 105 able to reach a much greater degree of fusion activity compared to that of WT when 106 treated with either pH 5.5 or pH 6.0 buffer (Fig. 2B ). As pH 6.0 and pH 7.0 were 107 barely able to trigger GPC-mediated fusion for either WT or F446L, the qualitative 108 assay was not conducted at these two pH values. Based on our findings that F446L 109 raised the pH threshold for GPC-mediated fusion, we investigated whether F446L 110 entered the cells through the pH-dependent endocytic pathway. It was found that 111 LASV F446Lrv remained sensitive to NH 4 Cl treatment, similar to that of LASV WTrv and 112 VSV rv , which indicated the entry of LASV F446Lrv remained pH-dependent (Fig. 2C ). 114 Alfa-DG is the primary receptor of LASV (12, 13). It was expected that a TM mutant infected with the MOPV GPC WT virus treated with pH 5.5 (Fig. 6B) . We also 169 introduced the corresponding mutation into other mammarenaviruses and evaluated The unique retention of SSP and its interaction with GP2 provides an interface target 179 for numerous structurally distinct inhibitors (5-7). However, considering the high 180 mutation rate of arenaviruses, drug resistance is a constant major concern in the 181 context of direct antiviral agents. Among the reported drug-resistant variants, LASV 182 F446L and its corresponding mutants in other arenaviruses have been extensively 183 recorded. In the current study, we found that F446L conferred cross-resistance, not 184 only to the ST series of inhibitors, but also to lacidipine, which we previously 185 reported resulted in the emergence of an adaptive mutant with a T40K change in SSP 186 (6). The detailed molecular mechanism underlying drug resistance is unknown. Being Operetta High-Content Imaging System. Data are presented as means ± SDs from 4-6 423 independent experiments. *P < 0.05, ** P < 0.01, *** P < 0.001. Sequence ( TAGATGCTAATAAGATAGAAACTTGTACTGAA TCTAGTCAGCATCTTACTGCACCTTG TAAGATGCTGACTAGATATGCAGATG TTTGATCTCCGTGTTACTCCACCTAA TAACACGGAGATCAAATAAAAGCTAG CACCACAAGTCTCTTATTACACCTGG TAAGAGACTTGTGGTGAAAAAGATTG TACCGCCAGCCTGTTACTGCACCTCG TAACAGGCTGGCGGTAAAGAACACGG CACCGCCTCCCTCTTACTGCACCTGG TAAGAGGGAGGCGGTGAAGAAAATGG CACAACAACACTGTTACTTCACCTGG TAACAGTGTTGTTGTGAAAAACAAAG CACAACAACATTGTTACTCCATCTAG TAACAATGTTGTTGTGAAGAACAGTG Taxonomy of the family Arenaviridae and the order Bunyavirales: 302 update Structure-function relationship of the 304 mammarenavirus envelope glycoprotein Fields Virology Animal models, prophylaxis, and therapeutics for arenavirus infections Fusion Inhibitors Bind the pH-Sensing Stable Signal Peptide-GP2 Subunit Interface of the Lassa Virus Envelope Glycoprotein Screening 313 and Identification of Lassa Virus Entry Inhibitors from an FDA-Approved Drugs Library Identification of the dietary supplement capsaicin as an 316 inhibitor of Lassa virus entry pH-induced activation of arenavirus membrane 318 fusion is antagonized by small-molecule inhibitors Structure-activity relationship optimization for lassa virus fusion inhibitors targeting the 321 transmembrane domain of GP2 Identification and characterization of potent small molecule inhibitor of 325 hemorrhagic fever New World arenaviruses Identification of a 327 broad-spectrum arenavirus entry inhibitor Identification of alpha-dystroglycan as a receptor for 330 lymphocytic choriomeningitis virus and Lassa fever virus Lassa virus entry requires a 333 trigger-induced receptor switch Lassa Virus Cell Entry via Dystroglycan 335 Involves an Unusual Pathway of Macropinocytosis Cell 337 entry of Lassa virus induces tyrosine phosphorylation of dystroglycan LaBranche 342 CC, Saphire EO, Montefiori DC. 2020. Tracking Changes in SARS-CoV-2 Spike: Evidence that 343 D614G Increases Infectivity of the COVID-19 Virus Spike mutation D614G alters 347 SARS-CoV-2 fitness SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and 352 transmission in vivo Ebola Virus Glycoprotein with Increased Infectivity Dominated the 2013-2016 Epidemic Biochemical Basis for Increased Activity of 358 Ebola Glycoprotein in the 2013-16 Epidemic Genomic Analysis of Lassa Virus during an Increase in 366 Cases in Nigeria The major determinant of attenuation in mice of the Candid1 vaccine for Argentine 369 hemorrhagic fever is located in the G2 glycoprotein transmembrane domain A 373 potent Lassa virus antiviral targets an arenavirus virulence determinant Substitutions in the glycoprotein (GP) of the Candid#1 vaccine strain of Junin virus 377 increase dependence on human transferrin receptor 1 for entry and destabilize the 378 metastable conformation of GP A substitution in the transmembrane region of the glycoprotein leads to an unstable 381 attenuation of Machupo virus Drugs for Inhibitors of Japanese Encephalitis Virus Infection Identification of H209 as Essential for pH 8-Triggered Receptor-Independent Syncytium Formation by S Protein of 386 Mouse Hepatitis Virus A59 A single amino acid in the stalk region of the H1N1pdm 388 influenza virus HA protein affects viral fusion, stability and infectivity Modulation of the pH Stability of Influenza Virus 391 Hemagglutinin: A Host Cell Adaptation Strategy Stability of the Hemagglutinin of H5N1 Influenza A Viruses Influences Susceptibility to 394 Broadly Neutralizing Stem Antibodies Development of a new vaccine for the prevention of Lassa fever A 400 recombinant vesicular stomatitis virus-based Lassa fever vaccine protects guinea pigs and 401 macaques against challenge with geographically and genetically distinct Lassa viruses Cell fusion 404 activity of hepatitis C virus envelope proteins A specific interaction of small molecule entry 407 inhibitors with the envelope glycoprotein complex of the Junin hemorrhagic fever arenavirus Characterizing the Lassa Virus Envelope 410 Glycoprotein Membrane Proximal External Region for Its Role in Fusogenicity