Inhibitors of cannabinoid receptor 1 suppress the cellular entry of Lujo virus.

Lujo virus (LUJV), which belongs to Mammarenavirus, family Arenaviridae, has emerged as a pathogen causing severe hemorrhagic fever with high mortality. Currently, there are no effective treatments for arenaviruses, including LUJV. Here, we screened chemical compound libraries of Food and Drug Administration (FDA)-approved drugs and G protein-coupled receptor-associated drugs to identify effective antivirals against LUJV targeting cell entry using a vesicular stomatitis virus-based pseudotyped virus bearing the LUJV envelope glycoprotein (GP). Cannabinoid receptor 1 (CB1) antagonists, such as rimonabant, AM251 and AM281, have been identified as robust inhibitors of LUJV entry. The IC50 of rimonabant was 0.26 and 0.53 µM in Vero and Huh7 cells, respectively. Analysis of the cell fusion activity of the LUJV GP in the presence of CB1 inhibitors revealed that these inhibitors suppressed the fusion activity of the LUJV GP. Moreover, rimonabant, AM251 and AM281 reduced the infectivity of authentic LUJV in vitro, suggesting that the antiviral activity of CB1 antagonists against LUJV is mediated, at least in part, by inhibition of the viral entry, especially, membrane fusion. These findings suggest promising candidates for developing new therapies against LUJV infections.

Molecular Mechanisms Underlying the Cellular Entry and Host Range Restriction of Lujo Virus.

Like other human-pathogenic arenaviruses, Lujo virus (LUJV) is a causative agent of viral hemorrhagic fever in humans. LUJV infects humans with high mortality rates, but the susceptibilities of other animal species and the molecular determinants of its host specificity remain unknown. We found that mouse- and hamster-derived cell lines (NIH 3T3 and BHK, respectively) were less susceptible to a replication-incompetent recombinant vesicular stomatitis virus (Indiana) pseudotyped with the LUJV glycoprotein (GP) (VSVΔG*-LUJV/GP) than were human-derived cell lines (HEK293T and Huh7). To determine the cellular factors involved in the differential susceptibilities between the human and mouse cell lines, we focused on the CD63 molecule, which is required for pH-activated GP-mediated membrane fusion during LUJV entry into host cells. The exogenous introduction of human CD63, but not mouse or hamster CD63, into BHK cells significantly increased susceptibility to VSVΔG*-LUJV/GP. Using chimeric human-mouse CD63 proteins, we found that the amino acid residues at positions 141 to 150 in the large extracellular loop (LEL) region of CD63 were important for the cellular entry of VSVΔG*-LUJV/GP. By site-directed mutagenesis, we further determined that a phenylalanine at position 143 in human CD63 was the key residue for efficient membrane fusion and VSVΔG*-LUJV/GP infection. Our data suggest that the interaction of LUJV GP with the LEL region of CD63 is essential for cell susceptibility to LUJV, thus providing new insights into the molecular mechanisms underlying the cellular entry of LUJV and the host range restriction of this virus. IMPORTANCE Lujo virus (LUJV) infects humans with high mortality rates, but the host range of LUJV remains unknown. We found that rodent-derived cell lines were less susceptible to LUJV infection than were human-derived cell lines, and the differential susceptibilities were determined by the difference of CD63, the intercellular receptor of LUJV. We further identified an amino acid residue on human CD63 important for efficient LUJV infection. These results suggest that the interaction between LUJV glycoprotein and CD63 is one of the important factors determining the host range of LUJV. Our findings on the CD63-regulated susceptibilities of the cell lines to LUJV infection provide important information for the development of anti-LUJV drugs as well as the identification of natural hosts of LUJV. Importantly, our data support a concept explaining the molecular mechanism underlying viral tropisms controlled by endosomal receptors.

Structural basis for receptor recognition by Lujo virus.

Lujo virus (LUJV) has emerged as a highly fatal human pathogen. Despite its membership among the Arenaviridae, LUJV does not classify with the known Old and New World groups of that viral family. Likewise, LUJV was recently found to use neuropilin-2 (NRP2) as a cellular receptor instead of the canonical receptors used by Old World and New World arenaviruses. The emergence of a deadly pathogen into human populations using an unprecedented entry route raises many questions regarding the mechanism of cell recognition. To provide the basis for combating LUJV in particular, and to increase our general understanding of the molecular changes that accompany an evolutionary switch to a new receptor for arenaviruses, we used X-ray crystallography to reveal how the GP1 receptor-binding domain of LUJV (LUJVGP1) recognizes NRP2. Structural data show that LUJVGP1 is more similar to Old World than to New World arenaviruses. Structural analysis supported by experimental validation further suggests that NRP2 recognition is metal-ion dependent and that the complete NRP2 binding site is formed in the context of the trimeric spike. Taken together, our data provide the mechanism for the cell attachment step of LUJV and present indispensable information for combating this phatogen.

Analysis of Lujo virus cell entry using pseudotype vesicular stomatitis virus.

UNLABELLED: Several arenaviruses are known to cause viral hemorrhagic fever (VHF) in sub-Saharan Africa and South America, where VHF is a major public health and medical concern. The biosafety level 4 categorization of these arenaviruses restricts their use and has impeded biological studies, including therapeutic drug and/or vaccine development. Due to difficulties associated with handling live viruses, pseudotype viruses, which transiently bear arenavirus envelope proteins based on vesicular stomatitis virus (VSV) or retrovirus, have been developed as surrogate virus systems. Here, we report the development of a pseudotype VSV bearing each envelope protein of various species of arenaviruses (AREpv), including the newly identified Lujo virus (LUJV) and Chapare virus. Pseudotype arenaviruses generated in 293T cells exhibited high infectivity in various mammalian cell lines. The infections by New World and Old World AREpv were dependent on their receptors (human transferrin receptor 1 [hTfR1] and α-dystroglycan [αDG], respectively). However, infection by pseudotype VSV bearing the LUJV envelope protein (LUJpv) occurred independently of hTfR1 and αDG, indicating that LUJpv utilizes an unidentified receptor. The pH-dependent endocytosis of AREpv was confirmed by the use of lysosomotropic agents. The fusion of cells expressing these envelope proteins, except for those expressing the LUJV envelope protein, was induced by transient treatment at low pH values. LUJpv infectivity was inhibited by U18666A, a cholesterol transport inhibitor. Furthermore, the infectivity of LUJpv was significantly decreased in the Niemann-Pick C1 (NPC1)-deficient cell line, suggesting the necessity for NPC1 activity for efficient LUJpv infection. IMPORTANCE: LUJV is a newly identified arenavirus associated with a VHF outbreak in southern Africa. Although cell entry for many arenaviruses has been studied, cell entry for LUJV has not been characterized. In this study, we found that LUJpv utilizes neither αDG nor hTfR1 as a receptor and found unique characteristics of LUJV glycoprotein in membrane fusion and cell entry. Proper exclusion of cholesterol or some kinds of lipids may play important roles in LUJpv cell entry.