ABSTRACT
The 2013-2015 West African epidemic of Ebola virus disease (EVD) reminds us of how little is known about biosafety level 4 viruses. Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever with high case fatality rates. We generated a genomic catalog of almost 200 LASV sequences from clinical and rodent reservoir samples. We show that whereas the 2013-2015 EVD epidemic is fueled by human-to-human transmissions, LASV infections mainly result from reservoir-to-human infections. We elucidated the spread of LASV across West Africa and show that this migration was accompanied by changes in LASV genome abundance, fatality rates, codon adaptation, and translational efficiency. By investigating intrahost evolution, we found that mutations accumulate in epitopes of viral surface proteins, suggesting selection for immune escape. This catalog will serve as a foundation for the development of vaccines and diagnostics. VIDEO ABSTRACT.
Subject(s)
Genome, Viral , Lassa Fever/virology , Lassa virus/genetics , RNA, Viral/genetics , Africa, Western/epidemiology , Animals , Biological Evolution , Disease Reservoirs , Ebolavirus/genetics , Genetic Variation , Glycoproteins/genetics , Hemorrhagic Fever, Ebola/virology , Humans , Lassa Fever/epidemiology , Lassa Fever/transmission , Lassa virus/classification , Lassa virus/physiology , Murinae/genetics , Mutation , Nigeria/epidemiology , Viral Proteins/genetics , Zoonoses/epidemiology , Zoonoses/virologyABSTRACT
Lassa virus (LASV) is a mammarenavirus that can cause lethal Lassa fever disease with no FDA-approved vaccine and limited treatment options. Fatal LASV infections are associated with innate immune suppression. We have previously shown that the small matrix Z protein of LASV, but not of a nonpathogenic arenavirus Pichinde virus (PICV), can inhibit the cellular RIG-I-like receptors (RLRs), but its biological significance has not been evaluated in an infectious virus due to the multiple essential functions of the Z protein required for the viral life cycle. In this study, we developed a stable HeLa cell line (HeLa-iRIGN) that could be rapidly and robustly induced by doxycycline (Dox) treatment to express RIG-I N-terminal effector, with concomitant production of type I interferons (IFN-Is). We also generated recombinant tri-segmented PICVs, rP18tri-LZ, and rP18tri-PZ, which encode LASV Z and PICV Z, respectively, as an extra mScarlet fusion protein that is nonessential for the viral life cycle. Upon infection, rP18tri-LZ consistently expressed viral genes at a higher level than rP18tri-PZ. rP18tri-LZ also showed a higher level of a viral infection than rP18tri-PZ did in HeLa-iRIGN cells, especially upon Dox induction. The heterologous Z gene did not alter viral growth in Vero and A549 cells by growth curve analysis, while LASV Z strongly increased and prolonged viral gene expression, especially in IFN-competent A549 cells. Our study provides important insights into the biological role of LASV Z-mediated RIG-I inhibition and implicates LASV Z as a potential virulence factor. IMPORTANCE Lassa virus (LASV) can cause lethal hemorrhagic fever disease in humans but other arenaviruses, such as Pichinde virus (PICV), do not cause obvious disease. We have previously shown that the Z protein of LASV but not of PICV can inhibit RIG-I, a cytosolic innate immune receptor. In this study, we developed a stable HeLa cell line that can be induced to express the RIG-I N-terminal effector domain, which allows for timely control of RIG-I activation. We also generated recombinant PICVs encoding LASV Z or PICV Z as an extra gene that is nonessential for the viral life cycle. Compared to PICV Z, LASV Z could increase viral gene expression and viral infection in an infectious arenavirus system, especially when RIG-I signaling is activated. Our study presented a convenient cell system to characterize RIG-I signaling and its antagonists and revealed LASV Z as a possible virulence factor and a potential antiviral target.
Subject(s)
Lassa virus , Viral Proteins/metabolism , HeLa Cells , Humans , Lassa Fever/virology , Lassa virus/pathogenicity , Lassa virus/physiology , Pichinde virus/genetics , Virulence FactorsABSTRACT
Arenavirus entry into host cells occurs through a low pH-dependent fusion with late endosomes that is mediated by the viral glycoprotein complex (GPC). The mechanisms of GPC-mediated membrane fusion and of virus targeting to late endosomes are not well understood. To gain insights into arenavirus fusion, we examined cell-cell fusion induced by the Old World Lassa virus (LASV) GPC complex. LASV GPC-mediated cell fusion is more efficient and occurs at higher pH with target cells expressing human LAMP1 compared to cells lacking this cognate receptor. However, human LAMP1 is not absolutely required for cell-cell fusion or LASV entry. We found that GPC-induced fusion progresses through the same lipid intermediates as fusion mediated by other viral glycoproteins-a lipid curvature-sensitive intermediate upstream of hemifusion and a hemifusion intermediate downstream of acid-dependent steps that can be arrested in the cold. Importantly, GPC-mediated fusion and LASV pseudovirus entry are specifically augmented by an anionic lipid, bis(monoacylglycero)phosphate (BMP), which is highly enriched in late endosomes. This lipid also specifically promotes cell fusion mediated by Junin virus GPC, an unrelated New World arenavirus. We show that BMP promotes late steps of LASV fusion downstream of hemifusion-the formation and enlargement of fusion pores. The BMP-dependence of post-hemifusion stages of arenavirus fusion suggests that these viruses evolved to use this lipid as a cofactor to selectively fuse with late endosomes.
Subject(s)
Endosomes/metabolism , Lassa Fever/metabolism , Lassa virus/physiology , Lysophospholipids/metabolism , Monoglycerides/metabolism , Virus Internalization , Animals , COS Cells , Chlorocebus aethiops , HEK293 Cells , Humans , Viral Envelope Proteins/metabolismABSTRACT
Lassa fever is an haemorrhagic fever caused by Lassa virus (LASV). There is no vaccine approved against LASV and the only recommended antiviral treatment relies on ribavirin, despite limited evidence of efficacy. Recently, the nucleotide analogue favipiravir showed a high antiviral efficacy, with 100% survival obtained in an otherwise fully lethal non-human primate (NHP) model of Lassa fever. However the mechanism of action of the drug is not known and the absence of pharmacokinetic data limits the translation of these results to the human setting. Here we aimed to better understand the antiviral effect of favipiravir by developping the first mathematical model recapitulating Lassa viral dynamics and treatment. We analyzed the viral dynamics in 24 NHPs left untreated or treated with ribavirin or favipiravir, and we put the results in perspective with those obtained with the same drugs in the context of Ebola infection. Our model estimates favipiravir EC50 in vivo to 2.89 µg.mL-1, which is much lower than what was found against Ebola virus. The main mechanism of action of favipiravir was to decrease virus infectivity, with an efficacy of 91% at the highest dose. Based on our knowledge acquired on the drug pharmacokinetics in humans, our model predicts that favipiravir doses larger than 1200 mg twice a day should have the capability to strongly reduce the production infectious virus and provide a milestone towards a future use in humans.
Subject(s)
Amides , Antiviral Agents , Lassa Fever/virology , Lassa virus , Pyrazines , Ribavirin , Amides/pharmacokinetics , Amides/pharmacology , Amides/therapeutic use , Animals , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Female , Host-Pathogen Interactions/drug effects , Lassa Fever/drug therapy , Lassa virus/drug effects , Lassa virus/pathogenicity , Lassa virus/physiology , Macaca fascicularis , Models, Biological , Pyrazines/pharmacokinetics , Pyrazines/pharmacology , Pyrazines/therapeutic use , Ribavirin/pharmacokinetics , Ribavirin/pharmacology , Ribavirin/therapeutic use , Viral Load/drug effectsABSTRACT
Lassa fever virus (LASV) is endemic in West Africa and causes severe hemorrhagic fever and sensorineural hearing loss. We identified a small molecule inhibitor of LASV and used it to analyze the mechanism of entry. Using a photo-reactive analog that retains antiviral activity as a probe, we identified the inhibitor target as lysosome-associated membrane protein 1 (LAMP1), a host factor that binds to the LASV glycoprotein (GP) during infection. We found that LAMP1 binding to LASV GP is cholesterol-dependent, and that the inhibitor blocks infection by competing with cholesterol in LAMP1. Mutational analysis of a docking-based model identified a putative inhibitor binding site in the cholesterol-binding pocket within the LAMP1 domain that binds GP. These findings identify a critical role for cholesterol in LASV entry and a potential target for therapeutic intervention.
Subject(s)
Cholesterol/metabolism , Lassa virus/physiology , Lassa virus/pathogenicity , Lysosomal Membrane Proteins/physiology , Receptors, Virus/physiology , Adamantane/analogs & derivatives , Adamantane/chemistry , Adamantane/pharmacology , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Cell Line , Chlorocebus aethiops , HEK293 Cells , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/physiology , Humans , Lassa Fever/etiology , Lassa virus/drug effects , Lysosomal Membrane Proteins/antagonists & inhibitors , Lysosomal Membrane Proteins/genetics , Models, Molecular , Mutation , Protein Stability , Protein Structure, Tertiary , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/genetics , Vero Cells , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Envelope Proteins/physiology , Virus Internalization/drug effectsABSTRACT
Lassa virus (LASV), a hemorrhagic fever virus endemic to West Africa, causes conjunctivitis in patients with acute disease. To examine ocular manifestations of LASV, we histologically examined eyes from infected guinea pigs. In fatal disease, LASV immunostaining was most prominent in the anterior uvea, especially in the filtration angle, ciliary body, and iris and in and around vessels in the bulbar conjunctiva and peripheral cornea, where it co-localized with an endothelial marker (platelet endothelial cell adhesion molecule). Antigen was primarily associated with infiltration of T-lymphocytes around vessels in the anterior uvea and with new vessel formation at the peripheral cornea. In animals that exhibited clinical signs but survived infection, eyes had little to no inflammation and no LASV immunostaining 6 weeks after infection. Overall, in this model, LASV antigen was restricted to the anterior uvea and was associated with mild chronic inflammation in animals with severe disease but was not detected in survivors.
Subject(s)
Conjunctivitis/virology , Endothelium, Corneal/virology , Iritis/virology , Keratitis/virology , Lassa virus/physiology , Animals , Biopsy , Conjunctivitis/pathology , Disease Models, Animal , Endothelium, Corneal/pathology , Female , Guinea Pigs , Immunohistochemistry , Iritis/pathology , Keratitis/pathology , Male , Polymerase Chain Reaction , RNA, ViralABSTRACT
Lassa virus (LASV) is an Old World arenavirus responsible for hundreds of thousands of infections in West Africa every year. LASV entry into a variety of cell types is mediated by interactions with glycosyltransferase LARGE-modified O-linked glycans present on the ubiquitous receptor α-dystroglycan (αDG). However, cells lacking αDG are permissive to LASV infection, suggesting that alternative receptors exist. Previous studies demonstrated that the phosphatidylserine (PtdSer)-binding receptors Axl and Tyro3 along with C-type lectin receptors mediate αDG-independent entry. Here, we demonstrate that another PtdSer receptor, TIM-1, mediates LASV glycoprotein (GP)-pseudotyped virion entry into αDG-knocked-out HEK 293T and wild-type (WT) Vero cells, which express αDG lacking appropriate glycosylation. To investigate the mechanism by which TIM-1 mediates enhancement of entry, we demonstrate that mutagenesis of the TIM-1 IgV domain PtdSer-binding pocket abrogated transduction. Furthermore, the human TIM-1 IgV domain-binding monoclonal antibody ARD5 blocked transduction of pseudovirions bearing LASV GP in a dose-dependent manner. Finally, as we showed previously for other viruses that use TIM-1 for entry, a chimeric TIM-1 protein that substitutes the proline-rich region (PRR) from murine leukemia virus envelope (Env) for the mucin-like domain served as a competent receptor. These studies provide evidence that, in the absence of a functional αDG, TIM-1 mediates the entry of LASV pseudoviral particles through interactions of virions with the IgV PtdSer-binding pocket of TIM-1.IMPORTANCE PtdSer receptors, such as TIM-1, are emerging as critical entry factors for many enveloped viruses. Most recently, hepatitis C virus and Zika virus have been added to a growing list. PtdSer receptors engage with enveloped viruses through the binding of PtdSer embedded in the viral envelope, defining them as GP-independent receptors. This GP-independent entry mechanism should effectively mediate the entry of all enveloped viruses, yet LASV GP-pseudotyped viruses were previously found to be unresponsive to PtdSer receptor enhancement in HEK 293T cells. Here, we demonstrate that LASV pseudovirions can utilize the PtdSer receptor TIM-1 but only in the absence of appropriately glycosylated α-dystroglycan (αDG), the high-affinity cell surface receptor for LASV. Our studies shed light on LASV receptor utilization and explain why previous studies performed with α-DG-expressing cells did not find that LASV pseudovirions utilize PtdSer receptors for virus uptake.
Subject(s)
Dystroglycans/deficiency , Hepatitis A Virus Cellular Receptor 1/metabolism , Host-Pathogen Interactions , Lassa virus/physiology , Receptors, Virus/metabolism , Virus Internalization , Animals , Chlorocebus aethiops , DNA Mutational Analysis , HEK293 Cells , Hepatitis A Virus Cellular Receptor 1/genetics , Humans , Receptors, Virus/genetics , Vero CellsABSTRACT
Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae) and causes severe hemorrhagic fever in humans. At present, there are no Food and Drug Administration (FDA)-approved drugs or vaccines specific for LASV. Here, high-throughput screening of an FDA-approved drug library was performed against LASV entry by using pseudotype virus bearing LASV envelope glycoprotein (GPC). Two hit compounds, lacidipine and phenothrin, were identified as LASV entry inhibitors in the micromolar range. A mechanistic study revealed that both compounds inhibited LASV entry by blocking low-pH-induced membrane fusion. Accordingly, lacidipine showed virucidal effects on the pseudotype virus of LASV. Adaptive mutant analyses demonstrated that replacement of T40, located in the ectodomain of the stable-signal peptide (SSP), with lysine (K) conferred LASV resistance to lacidipine. Furthermore, lacidipine showed antiviral activity against LASV, the closely related Mopeia virus (MOPV), and the New World arenavirus Guanarito virus (GTOV). Drug-resistant variants indicated that V36M in the ectodomain of the SSP mutant and V436A in the transmembrane domain of the GP2 mutant conferred GTOV resistance to lacidipine, suggesting the interface between SSP and GP2 is the target of lacidipine. This study shows that lacidipine is a candidate for LASV therapy, reinforcing the notion that the SSP-GP2 interface provides an entry-targeted platform for arenavirus inhibitor design.IMPORTANCE Currently, there is no approved therapy to treat Lassa fever; therefore, repurposing of approved drugs will accelerate the development of a therapeutic stratagem. In this study, we screened an FDA-approved library of drugs and identified two compounds, lacidipine and phenothrin, which inhibited Lassa virus entry by blocking low-pH-induced membrane fusion. Additionally, both compounds extended their inhibition against the entry of Guanarito virus, and the viral targets were identified as the SSP-GP2 interface.
Subject(s)
Antiviral Agents/pharmacology , Dihydropyridines/pharmacology , Drug Evaluation, Preclinical/methods , High-Throughput Screening Assays/methods , Lassa virus/drug effects , Pyrethrins/pharmacology , Virus Internalization/drug effects , Arenaviridae/drug effects , Arenaviruses, New World/drug effects , DNA Mutational Analysis , Drug Resistance, Viral , Lassa virus/physiologyABSTRACT
Fatal infection with the highly pathogenic Lassa virus (LASV) is characterized by extensive viral dissemination, indicating broad tissue tropism. The major cellular receptor for LASV is the highly conserved extracellular matrix receptor dystroglycan (DG). Binding of LASV depends on DG's tissue-specific posttranslational modification with the unusual O-linked polysaccharide matriglycan. Interestingly, functional glycosylation of DG does not always correlate with viral tropism observed in vivo The broadly expressed phosphatidylserine (PS) receptors Axl and Tyro3 were recently identified as alternative LASV receptor candidates. However, their role in LASV entry is not entirely understood. Here, we examine LASV receptor candidates in primary human cells and found coexpression of Axl with differentially glycosylated DG. To study LASV receptor use in the context of productive arenavirus infection, we employed recombinant lymphocytic choriomeningitis virus expressing LASV glycoprotein (rLCMV-LASV GP) as a validated biosafety level 2 (BSL2) model. We confirm and extend previous work showing that Axl can contribute to LASV entry in the absence of functional DG using "apoptotic mimicry" in a way similar to that of other enveloped viruses. We further show that Axl-dependent LASV entry requires receptor activation and involves a pathway resembling macropinocytosis. Axl-mediated LASV entry is facilitated by heparan sulfate and critically depends on the late endosomal protein LAMP-1 as an intracellular entry factor. In endothelial cells expressing low levels of functional DG, both receptors are engaged by the virus and can contribute to productive entry. In sum, we characterize the role of Axl in LASV entry and provide a rationale for targeting Axl in antiviral therapy.IMPORTANCE The highly pathogenic arenavirus Lassa virus (LASV) represents a serious public health problem in Africa. Although the principal LASV receptor, dystroglycan (DG), is ubiquitously expressed, virus binding critically depends on DG's posttranslational modification, which does not always correlate with tissue tropism. The broadly expressed phosphatidylserine receptor Axl was recently identified as an alternative LASV receptor candidate, but its role in LASV entry is unclear. Here, we investigate the exact role of Axl in LASV entry as a function of DG's posttranslational modification. We found that in the absence of functional DG, Axl can mediate LASV entry via apoptotic mimicry. Productive entry requires virus-induced receptor activation, involves macropinocytosis, and critically depends on LAMP-1. In endothelial cells that express low levels of glycosylated DG, both receptors can promote LASV entry. In sum, our study defines the roles of Axl in LASV entry and provides a rationale for targeting Axl in antiviral therapy.
Subject(s)
Dystroglycans/metabolism , Lassa virus/physiology , Proto-Oncogene Proteins/physiology , Receptor Protein-Tyrosine Kinases/physiology , Receptors, Virus/metabolism , Virus Attachment , Virus Internalization , A549 Cells , Antiviral Agents/pharmacology , Arenaviridae Infections/metabolism , Cell Line, Tumor , Dystroglycans/genetics , Endosomes/metabolism , Gene Expression , Glycosylation , HEK293 Cells , HeLa Cells , Heparitin Sulfate/pharmacology , Humans , Lassa virus/drug effects , Lassa virus/pathogenicity , Lymphocytic choriomeningitis virus/genetics , Lymphocytic choriomeningitis virus/metabolism , Lysosomal Membrane Proteins/metabolism , Pinocytosis/physiology , Protein Processing, Post-Translational , Proto-Oncogene Proteins/drug effects , Proto-Oncogene Proteins/genetics , RNA Interference , Receptor Protein-Tyrosine Kinases/drug effects , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction , Tropism , Axl Receptor Tyrosine KinaseABSTRACT
Many emerging infections are RNA virus spillovers from animal reservoirs. Reservoir identification is necessary for predicting the geographic extent of infection risk, but rarely are taxonomic levels below the animal species considered as reservoir, and only key circumstances in nature and methodology allow intrinsic virus-host associations to be distinguished from simple geographic (co-)isolation. We sampled and genetically characterized in detail a contact zone of two subtaxa of the rodent Mastomys natalensis in Tanzania. We find two distinct arenaviruses, Gairo and Morogoro virus, each spatially confined to a single M. natalensis subtaxon, only co-occurring at the contact zone's centre. Inter-subtaxon hybridization at this centre and a continuum of quality habitat for M. natalensis show that both viruses have the ecological opportunity to spread into the other substaxon's range, but do not, strongly suggesting host-intrinsic barriers. Such barriers could explain why human cases of another M. natalensis-borne arenavirus, Lassa virus, are limited to West Africa.
Subject(s)
Arenavirus/classification , Arenavirus/metabolism , Disease Reservoirs/virology , Murinae/virology , Rodent Diseases/virology , Animals , Arenavirus/physiology , Humans , Lassa Fever/virology , Lassa virus/physiology , Phylogeography , Species Specificity , TanzaniaABSTRACT
BACKGROUND: Infectious disease prevention and control strategies require a coordinated, transnational approach. To establish core capacities of the International Health Regulations (IHR), the World Health Organization (WHO) developed the Integrated Diseases Surveillance and Response (IDSR) strategy. Epidemic-prone Lassa fever, caused by Lassa virus, is an endemic disease in the West African countries of Ghana, Guinea, Mali, Benin, Liberia, Sierra Leone, Togo and Nigeria. It's one of the major public health threats in these countries. Here it is reported an epidemiological investigation of a cross-border case of Lassa fever, which demonstrated the importance of strengthened capacities of IHR and IDSR. CASE PRESENTATION: On January 9th, 2018 a 35-year-old Guinean woman with fever, neck pain, body pain, and vomiting went to a hospital in Ganta, Liberia. Over the course of her illness, the case visited various health care facilities in both Liberia and Guinea. A sample collected on January 10th was tested positive for Lassa virus by RT-PCR in a Liberian laboratory. The Guinean Ministry of Health (MoH) was officially informed by WHO Country Office for Guinea and for Liberia. CONCLUSION: This case report revealed how an epidemic-prone disease such as Lassa fever can rapidly spread across land borders and how such threat can be quickly controlled with communication and collaboration within the IHR framework.
Subject(s)
Emigration and Immigration , Lassa Fever/diagnosis , Lassa virus/physiology , Adult , Africa, Western/epidemiology , Epidemiological Monitoring , Female , Humans , International Health Regulations/standards , Lassa Fever/epidemiology , Lassa Fever/pathology , Lassa virus/genetics , World Health OrganizationABSTRACT
Viral entry represents the first step of every viral infection and is a determinant for the host range and disease potential of a virus. Here, we review the latest developments on cell entry of the highly pathogenic Old World arenavirus Lassa virus, providing novel insights into the complex virus-host cell interaction of this important human pathogen. We will cover new discoveries on the molecular mechanisms of receptor recognition, endocytosis, and the use of late endosomal entry factors.
Subject(s)
Host-Pathogen Interactions , Lassa Fever/virology , Lassa virus/physiology , Virus Internalization , Animals , Dystroglycans/metabolism , Endocytosis , Endosomes/metabolism , Endosomes/virology , Humans , Lassa Fever/metabolism , Lysosomal-Associated Membrane Protein 1/metabolism , Pinocytosis , Receptors, Virus/metabolism , Viral TropismABSTRACT
Lassa virus (LASV) small zinc-finger protein (Z), which contains two L-domain motifs, plays a central role in virus budding. Here, we report that co-expression of glycoprotein (GPC) altered the requirements for cholesterol but not the L-domains and host factor, Tsg101, for Z-induced virus-like particle (VLP) production. In particular, the cholesterol requirement for VLP production was cell-type-dependent. In addition, GPC was found to be important for co-localization of Z with CD63, a late endosomal marker. We also found that the N-terminal region (aa 3-10) of Z was critical for its myristoylation and VLP production. These findings will contribute to our understanding of LASV assembly and budding.
Subject(s)
Lassa virus/physiology , Viral Proteins/genetics , Viral Proteins/metabolism , Virosomes/genetics , Virosomes/metabolism , Virus Assembly , Virus Release , Animals , Cell Line , Cholesterol/metabolism , Humans , Lassa virus/geneticsABSTRACT
The N terminus of arenavirus L protein contains an endonuclease presumably involved in "cap snatching." Here, we employed the Lassa virus replicon system to map other L protein sites that might be involved in this mechanism. Residues Phe-1979, Arg-2018, Phe-2071, Asp-2106, Trp-2173, Tyr-2179, Arg-2200, and Arg-2204 were important for viral mRNA synthesis but dispensable for genome replication. Thus, the C terminus of L protein is involved in the mRNA synthesis process, potentially by mediating cap binding.
Subject(s)
Lassa virus/physiology , RNA, Messenger/genetics , RNA, Viral/genetics , Viral Proteins/metabolism , Cell Line , Humans , Lassa virus/genetics , Viral Proteins/genetics , Virus ReplicationABSTRACT
Lassa virus (LASV), which causes a viral hemorrhagic fever, inhibits the innate immune response. The exonuclease (ExoN) domain of its nucleoprotein (NP) is implicated in the suppression of retinoic acid-inducible gene I (RIG-I) signaling. We show here that a LASV in which ExoN function has been abolished strongly activates innate immunity and that this effect is dependent on RIG-I signaling. These results highlight the key role of NP ExoN function in the immune evasion that occurs during LASV infection.
Subject(s)
Exonucleases/immunology , Immune Tolerance , Immunity, Innate , Lassa virus/immunology , Lassa virus/physiology , Nucleoproteins/immunology , Signal Transduction , Cells, Cultured , DEAD Box Protein 58 , DEAD-box RNA Helicases/metabolism , Exonucleases/metabolism , Humans , Nucleoproteins/metabolism , Receptors, ImmunologicABSTRACT
The arenavirus Lassa virus (LASV) causes a severe hemorrhagic fever with high mortality in humans. Antigen-presenting cells, in particular dendritic cells (DCs), are early and preferred targets of LASV, and their productive infection contributes to the virus-induced immunosuppression observed in fatal disease. Here, we characterized the role of the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) in LASV entry into primary human DCs using a chimera of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV glycoprotein (rLCMV-LASVGP). We found that differentiation of human primary monocytes into DCs enhanced virus attachment and entry, concomitant with the upregulation of DC-SIGN. LASV and rLCMV-LASVGP bound to DC-SIGN via mannose sugars located on the N-terminal GP1 subunit of LASVGP. We provide evidence that DC-SIGN serves as an attachment factor for rLCMV-LASVGP in monocyte-derived immature dendritic cells (MDDC) and can accelerate the capture of free virus. However, in contrast to the phlebovirus Uukuniemi virus (UUKV), which uses DC-SIGN as an authentic entry receptor, productive infection with rLCMV-LASVGP was less dependent on DC-SIGN. In contrast to the DC-SIGN-mediated cell entry of UUKV, entry of rLCMV-LASVGP in MDDC was remarkably slow and depended on actin, indicating the use of different endocytotic pathways. In sum, our data reveal that DC-SIGN can facilitate cell entry of LASV in human MDDC but that its role seems distinct from the function as an authentic entry receptor reported for phleboviruses.
Subject(s)
Cell Adhesion Molecules/metabolism , Dendritic Cells/virology , Host-Pathogen Interactions , Lassa virus/physiology , Lectins, C-Type/metabolism , Receptors, Cell Surface/metabolism , Virus Internalization , Cells, Cultured , Humans , Lassa virus/genetics , Lymphocytic choriomeningitis virus/genetics , Receptors, Virus/metabolismABSTRACT
The small matrix protein Z of arenaviruses has been identified as the main driving force to promote viral particle production at the plasma membrane. Although multiple functions of Z in the arenaviral life cycle have been uncovered, the mechanism of intracellular transport of Z to the site of virus budding is poorly understood and cellular motor proteins that mediate Z trafficking remain to be identified. In the present study, we report that the Z protein of the Old World arenavirus Lassa virus (LASV) interacts with the kinesin family member 13A (KIF13A), a plus-end-directed microtubule-dependent motor protein. Plasmid-driven overexpression of KIF13A results in relocalization of Z to the cell periphery, while functional blockage of endogenous KIF13A by overexpression of a dominant-negative mutant or KIF13A-specific siRNA causes a perinuclearaccumulation and decreased production of both Z-induced virus-like particles and infectious LASV. The interaction of KIF13A with Z proteins from both Old and New World arenaviruses suggests a conserved intracellular transport mechanism. In contrast, the intracellular distribution of the matrix proteins of prototypic members of the paramyxo- and rhabdovirus family is independent of KIF13A. In summary, our studies identify for the first time a molecular motor protein as a critical mediator for intracellular microtubule-dependent transport of arenavirus matrix proteins.
Subject(s)
Carrier Proteins/metabolism , Kinesins/metabolism , Lassa virus/physiology , Microtubules/metabolism , Viral Matrix Proteins/metabolism , Virus Release/physiology , Animals , Carrier Proteins/genetics , Cell Line , Cell Membrane/metabolism , Cell Membrane/virology , Chlorocebus aethiops , Gene Expression , Host-Pathogen Interactions , Humans , Kidney/pathology , Kidney/virology , Kinesins/antagonists & inhibitors , Kinesins/genetics , Liver/pathology , Liver/virology , Microtubules/virology , Protein Binding , Protein Transport , RNA, Small Interfering/genetics , RNA-Binding Proteins , Vero Cells , Viral Matrix Proteins/geneticsABSTRACT
This study aims to formulate a mathematical framework to examine how the Lassa virus spreads in humans of opposite genders. The stability of the model is analyzed at an equilibrium point in the absence of the Lassa fever. The model's effectiveness is evaluated using real-life data, and all the parameters needed to determine the basic reproduction number are estimated. Sensitivity analysis is performed to pinpoint the crucial parameters significantly influencing the spread of the infection. The interaction between threshold parameters and the basic reproduction number is simulated. Control theory is employed to devise and evaluate strategies, such as awareness campaigns, advocating condom usage, and deploying rodenticides to reduce the possibility of virus transmission efficiently.
Subject(s)
Lassa Fever , Lassa virus , Humans , Lassa Fever/transmission , Lassa Fever/prevention & control , Lassa Fever/epidemiology , Lassa virus/physiology , Female , Male , Basic Reproduction Number , Epidemics/prevention & control , Models, TheoreticalABSTRACT
Lassa fever is a West African rodent-borne viral haemorrhagic fever that kills thousands of people a year, with 100 000 to 300 000 people a year probably infected by Lassa virus (LASV). The main reservoir of LASV is the Natal multimammate mouse, Mastomys natalensis. There is reported asynchrony between peak infection in the rodent population and peak Lassa fever risk among people, probably owing to differing seasonal contact rates. Here, we developed a susceptible-infected-recovered ([Formula: see text])-based model of LASV dynamics in its rodent host, M. natalensis, with a persistently infected class and seasonal birthing to test the impact of changes to seasonal birthing in the future owing to climate and land use change. Our simulations suggest shifting rodent birthing timing and synchrony will alter the peak of viral prevalence, changing risk to people, with viral dynamics mainly stable in adults and varying in the young, but with more infected individuals. We calculate the time-average basic reproductive number, [Formula: see text], for this infectious disease system with periodic changes to population sizes owing to birthing using a time-average method and with a sensitivity analysis show four key parameters: carrying capacity, adult mortality, the transmission parameter among adults and additional disease-induced mortality impact the maintenance of LASV in M. natalensis most, with carrying capacity and adult mortality potentially changeable owing to human activities and interventions.
Subject(s)
Lassa Fever , Lassa virus , Murinae , Animals , Lassa Fever/epidemiology , Lassa Fever/transmission , Lassa Fever/virology , Lassa virus/physiology , Murinae/virology , Humans , Models, Biological , Disease Reservoirs/virology , Africa, Western/epidemiology , Seasons , FemaleABSTRACT
Lassa virus (LASV), a risk-group 4 pathogen, must be handled in biosafety level-4 (BSL-4) conditions, thereby limiting its research and antiviral development. Here, we developed a novel LASV reverse genetics system which, to our knowledge, is the first to study the complete LASV life cycle under BSL-2 conditions. Viral particles can be produced efficiently when LASV minigenomic RNA harbouring minimal viral cis-elements and reporter genes is transfected into a helper cell line stably expressing viral NP, GP, Z and L proteins. The resulting defective virions, named LASVmg, can propagate only in the helper cell line, providing a BSL-2 model to study the complete LASV life cycle. Using this model, we found that a previously reported cellular receptor α-dystroglycan is dispensable for LASVmg infection. Furthermore, we showed that ribavirin can inhibit LASVmg infection by inducing viral mutations. This new BSL-2 system should facilitate studying the LASV life cycle and screening antivirals.