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.

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.

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.

Breaking the Barrier: Host Cell Invasion by Lujo Virus.

Lujo virus (LUJV) is an arenavirus that emerged in 2008 associated with a cluster of human cases of severe hemorrhagic fever. In this issue of Cell Host & Microbe, Raaben et al. (2017) identify neuropilin (NRP)-2 as cell surface receptor and the tetraspannin protein CD63 as intracellular entry factor for LUJV.

NRP2 and CD63 Are Host Factors for Lujo Virus Cell Entry.

Arenaviruses cause fatal hemorrhagic disease in humans. Old World arenavirus glycoproteins (GPs) mainly engage α-dystroglycan as a cell-surface receptor, while New World arenaviruses hijack transferrin receptor. However, the Lujo virus (LUJV) GP does not cluster with New or Old World arenaviruses. Using a recombinant vesicular stomatitis virus containing LUJV GP as its sole attachment and fusion protein (VSV-LUJV), we demonstrate that infection is independent of known arenavirus receptor genes. A genome-wide haploid genetic screen identified the transmembrane protein neuropilin 2 (NRP2) and tetraspanin CD63 as factors for LUJV GP-mediated infection. LUJV GP binds the N-terminal domain of NRP2, while CD63 stimulates pH-activated LUJV GP-mediated membrane fusion. Overexpression of NRP2 or its N-terminal domain enhances VSV-LUJV infection, and cells lacking NRP2 are deficient in wild-type LUJV infection. These findings uncover this distinct set of host cell entry factors in LUJV infection and are attractive focus points for therapeutic intervention.

[Lujo hemorrhagic fever].

Lujo hemorrhagic fever (LHF) is a viral disease accompanied with fever, headache, vomiting, diarrhea, arthralgia, myalgia and numerous signs of hemorrhagic syndrome. LHF causes a clinical syndrome remarkably similar to Lassa hemorrhagic fever. The first case of LHF occurred in Johannesburg, South Africa, in 2008. There was a secondary transmission from the index patient to four healthcare workers. Four of the five patients died. The etiologic agent of LHF is Lujo virus (LUJV) belonging to Arenavirus genus of the Arenaviridae Family. Virus Lujo is the second pathogenic arenavirus, after Lassa virus, to be recognized in Africa during the last 40 years. Data about epidemiology, clinical characteristics and diagnostics of LHF, properties of Lujo virus (according to phylogenetic analysis), and recommended precautions for preventing secondary transmission are considered in this paper.

Lujo viral hemorrhagic fever: considering diagnostic capacity and preparedness in the wake of recent Ebola and Zika virus outbreaks.

Lujo virus is a novel Old World arenavirus identified in Southern Africa in 2008 as the cause of a viral hemorrhagic fever (VHF) characterized by nosocomial transmission with a high case fatality rate of 80% (4/5 cases). Whereas this outbreak was limited, the unprecedented Ebola virus disease outbreak in West Africa, and recent Zika virus disease epidemic in the Americas, has brought into acute focus the need for preparedness to respond to rare but potentially highly pathogenic outbreaks of zoonotic or arthropod-borne viral infections. A key determinant for effective control of a VHF outbreak is the time between primary infection and diagnosis of the index case. Here, we review the Lujo VHF outbreak of 2008 and discuss how preparatory measures with respect to developing diagnostic capacity might be effectively embedded into existing national disease control networks, such as those for human immunodeficiency virus, tuberculosis, and malaria.

A Molecular Sensor To Characterize Arenavirus Envelope Glycoprotein Cleavage by Subtilisin Kexin Isozyme 1/Site 1 Protease.

UNLABELLED: Arenaviruses are emerging viruses including several causative agents of severe hemorrhagic fevers in humans. The advent of next-generation sequencing technology has greatly accelerated the discovery of novel arenavirus species. However, for many of these viruses, only genetic information is available, and their zoonotic disease potential remains unknown. During the arenavirus life cycle, processing of the viral envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) is crucial for productive infection. The ability of newly emerging arenaviruses to hijack human SKI-1/S1P appears, therefore, to be a requirement for efficient zoonotic transmission and human disease potential. Here we implement a newly developed cell-based molecular sensor for SKI-1/S1P to characterize the processing of arenavirus GPC-derived target sequences by human SKI-1/S1P in a quantitative manner. We show that only nine amino acids flanking the putative cleavage site are necessary and sufficient to accurately recapitulate the efficiency and subcellular location of arenavirus GPC processing. In a proof of concept, our sensor correctly predicts efficient processing of the GPC of the newly emergent pathogenic Lujo virus by human SKI-1/S1P and defines the exact cleavage site. Lastly, we employed our sensor to show efficient GPC processing of a panel of pathogenic and nonpathogenic New World arenaviruses, suggesting that GPC cleavage represents no barrier for zoonotic transmission of these pathogens. Our SKI-1/S1P sensor thus represents a rapid and robust test system for assessment of the processing of putative cleavage sites derived from the GPCs of newly discovered arenavirus by the SKI-1/S1P of humans or any other species, based solely on sequence information. IMPORTANCE: Arenaviruses are important emerging human pathogens that can cause severe hemorrhagic fevers with high mortality in humans. A crucial step in productive arenavirus infection of human cells is the processing of the viral envelope glycoprotein by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P). In order to break the species barrier during zoonotic transmission and cause severe disease in humans, newly emerging arenaviruses must be able to hijack human SKI-1/S1P efficiently. Here we implement a newly developed cell-based molecular sensor for human SKI-1/S1P to characterize the processing of arenavirus glycoproteins in a quantitative manner. We further use our sensor to correctly predict efficient processing of the glycoprotein of the newly emergent pathogenic Lujo virus by human SKI-1/S1P. Our sensor thus represents a rapid and robust test system with which to assess whether the glycoprotein of any newly emerging arenavirus can be efficiently processed by human SKI-1/S1P, based solely on sequence information.

Analysis of Assembly and Budding of Lujo Virus.

The recently identified arenavirus Lujo virus (LUJV) causes fatal hemorrhagic fever in humans. We analyzed its mechanism of viral release driven by matrix protein Z and the cell surface glycoprotein precursor GPC. The L domains in Z are required for efficient virus-like particle release, but Tsg101, ALIX/AIP1, and Vps4A/B are unnecessary for budding. LUJV GPC is cleaved by site 1 protease (S1P) at the RKLM motif, and treatment with the S1P inhibitor PF-429242 reduced LUJV production.

Detecting the emergence of novel, zoonotic viruses pathogenic to humans.

RNA viruses, with their high potential for mutation and epidemic spread, are the most common class of pathogens found as new causes of human illness. Despite great advances made in diagnostic technology since the 1950s, the annual rate at which novel virulent viruses have been found has remained at 2-3. Most emerging viruses are zoonoses; they have jumped from mammal or bird hosts to humans. An analysis of virus discovery indicates that the small number of novel viruses discovered annually is an artifact of inadequate surveillance in tropical and subtropical countries, where even established endemic pathogens are often misdiagnosed. Many of the emerging viruses of the future are already infecting humans but remain to be uncovered by a strategy of disease surveillance in selected populations.

Delayed inflammatory and cell death responses are associated with reduced pathogenicity in Lujo virus-infected cynomolgus macaques.

UNLABELLED: To identify host factors associated with arenavirus virulence, we used a cynomolgus macaque model to evaluate the pathogenesis of Lujo virus (LUJV), a recently emerged arenavirus that caused an outbreak of severe viral hemorrhagic fever in southern Africa. In contrast to human cases, LUJV caused mild, nonlethal illness in macaques. We then compared this to contrasting clinical outcomes during arenavirus infection, specifically to samples obtained from macaques infected with three highly pathogenic lines of Lassa virus (LASV), the causative agent of Lassa fever (LF). We assessed gene expression in peripheral blood mononuclear cells (PBMC) and determined genes that significantly changed expression relative to that in uninfected animals over the course of infection. We detected a 72-h delay in the induction of host responses to infection during LUJV infection compared to that of the animals infected with LASV. This included genes associated with inflammatory and antiviral responses and was particularly apparent among groups of genes promoting cell death. We also observed early differential expression of a subset of genes specific to LUJV infection that accounts for the delayed inflammatory response. Cell type enrichment analysis suggested that host response induction delay and an LUJV-specific profile are due to a different proportion of natural killer cells responding in LUJV infection than that in the LASV-infected animals. Together, these data indicate that delayed proinflammatory and proapoptotic host responses to arenavirus infection could ameliorate disease severity. This conclusion provides insight into the cellular and molecular mechanisms of arenaviral hemorrhagic fever and suggests potential strategies for therapeutic development. IMPORTANCE: Old World arenaviruses are significant human pathogens that often are associated with high mortality. However, mechanisms underlying disease severity and virulence in arenavirus hemorrhagic fever are largely unknown, particularly regarding host responses that contribute to pathogenicity. This study describes a comparison between Lujo and Lassa virus infection in cynomolgus macaques. Lujo virus-infected macaques developed only mild illness, while Lassa virus-infected macaques developed severe illness consistent with Lassa fever. We determined that mild disease is associated with a delay in host expression of genes linked to virulence, such as those causing inflammation and cell death, and with distinct cell types that may mediate this delay. This is the first study to associate the timing and directionality of gene expression with arenaviral pathogenicity and disease outcome and evokes new potential approaches for developing effective therapeutics for treating these deadly emerging pathogens.

Clinical features and patient management of Lujo hemorrhagic fever.

BACKGROUND: In 2008 a nosocomial outbreak of five cases of viral hemorrhagic fever due to a novel arenavirus, Lujo virus, occurred in Johannesburg, South Africa. Lujo virus is only the second pathogenic arenavirus, after Lassa virus, to be recognized in Africa and the first in over 40 years. Because of the remote, resource-poor, and often politically unstable regions where Lassa fever and other viral hemorrhagic fevers typically occur, there have been few opportunities to undertake in-depth study of their clinical manifestations, transmission dynamics, pathogenesis, or response to treatment options typically available in industrialized countries. METHODS AND FINDINGS: We describe the clinical features of five cases of Lujo hemorrhagic fever and summarize their clinical management, as well as providing additional epidemiologic detail regarding the 2008 outbreak. Illness typically began with the abrupt onset of fever, malaise, headache, and myalgias followed successively by sore throat, chest pain, gastrointestinal symptoms, rash, minor hemorrhage, subconjunctival injection, and neck and facial swelling over the first week of illness. No major hemorrhage was noted. Neurological signs were sometimes seen in the late stages. Shock and multi-organ system failure, often with evidence of disseminated intravascular coagulopathy, ensued in the second week, with death in four of the five cases. Distinctive treatment components of the one surviving patient included rapid commencement of the antiviral drug ribavirin and administration of HMG-CoA reductase inhibitors (statins), N-acetylcysteine, and recombinant factor VIIa. CONCLUSIONS: Lujo virus causes a clinical syndrome remarkably similar to Lassa fever. Considering the high case-fatality and significant logistical impediments to controlled treatment efficacy trials for viral hemorrhagic fever, it is both logical and ethical to explore the use of the various compounds used in the treatment of the surviving case reported here in future outbreaks. Clinical observations should be systematically recorded to facilitate objective evaluation of treatment efficacy. Due to the risk of secondary transmission, viral hemorrhagic fever precautions should be implemented for all cases of Lujo virus infection, with specialized precautions to protect against aerosols when performing enhanced-risk procedures such as endotracheal intubation.

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.

Rapid molecular detection of Lujo virus RNA.

Lujo virus is an emerging arenavirus circulating in Southern Africa. Although to date there has only been a single outbreak of the novel haemorrhagic disease resulting from human infection with this virus, the case-fatality rate of exposed individuals, including nosocomial transmission, was 80%. The ability to identify viral haemorrhagic fevers accurately, especially those capable of nosocomial transmission, is of critical importance. Timely identification of these diseases allow medical professionals to isolate patients and implement barrier nursing techniques in order to prevent onward transmission of the virus. While rapid diagnostic methods are published for most viral haemorrhagic fevers, at present there are no such virus specific protocols for Lujo haemorrhagic fever. This report details the first set of diagnostic molecular assays designed to identify Lujo viral RNA rapidly, and demonstrates the potential functionality of these assays for use in the clinical setting. Although these assays have been designed and validated against a solitary isolate of Lujo virus, this represents the entirety of strains detected to date, and offer quick, cheap and easy methods for use in diagnostic laboratories.