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

Alpha-dystroglycan (alpha-DG) has been identified as a major receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa virus, two Old World arenaviruses. The situation with New World arenaviruses is less clear: previous studies demonstrated that Oliveros virus also exhibited high-affinity binding to alpha-DG but that Guanarito virus did not. To extend these initial studies, several additional Old and New World arenaviruses were screened for entry into mouse embryonic stem cells possessing or lacking alpha-DG. In addition, representative viruses were further analyzed for direct binding to alpha-DG by means of a virus overlay protein blot assay technique. These studies indicate that Old World arenaviruses use alpha-DG as a major receptor, whereas, of the New World arenaviruses, only clade C viruses (i.e., Oliveros and Latino viruses) use alpha-DG as a major receptor. New World clade A and B arenaviruses, which include the highly pathogenic Machupo, Guanarito, Junin, and Sabia viruses, appear to use a different receptor or coreceptor for binding. Previous studies with LCMV have suggested the need for a small aliphatic amino acid at LCMV GP1 glycoprotein amino acid position 260 to allow high-affinity binding to alpha-DG. As reported herein, this requirement appears to be broadly applicable to the arenaviruses as determined by more extensive analysis of alpha-DG receptor usage and GP1 sequences of Old and New World arenaviruses. In addition, GP1 amino acid position 259 also appears to be important, since all arenaviruses showing high-affinity alpha-DG binding possess a bulky aromatic amino acid (tyrosine or phenylalanine) at this position.     

Unique small molecule entry inhibitors of hemorrhagic fever arenaviruses

Viral hemorrhagic fevers caused by the arenaviruses Lassa virus in Africa and Machupo, Guanarito, Junin, and Sabia virus in South America are among the most devastating emerging human diseases with fatality rates of 15-35% and a limited antiviral therapeutic repertoire available. Here we used high throughput screening of synthetic combinatorial small molecule libraries to identify inhibitors of arenavirus infection using pseudotyped virion particles bearing the glycoproteins (GPs) of highly pathogenic arenaviruses. Our screening efforts resulted in the discovery of a series of novel small molecule inhibitors of viral entry that are highly active against both Old World and New World hemorrhagic arenaviruses. We observed potent inhibition of infection of human and primate cells with live hemorrhagic arenaviruses (IC(50)=500-800 nm). Investigations of the mechanism of action revealed that the candidate compounds efficiently block pH-dependent fusion by the arenavirus GPs (IC(50) of 200-350 nm). Although our lead compounds were potent against phylogenetically distant arenaviruses, they did not show activity against other enveloped viruses with class I viral fusion proteins, indicating specificity for arenavirus GP-mediated membrane fusion.     

Characterization of the interaction of lassa fever virus with its cellular receptor alpha-dystroglycan

The cellular receptor for the Old World arenaviruses Lassa fever virus (LFV) and lymphocytic choriomeningitis virus (LCMV) has recently been identified as alpha-dystroglycan (alpha-DG), a cell surface receptor that provides a molecular link between the extracellular matrix and the actin-based cytoskeleton. In the present study, we show that LFV binds to alpha-DG with high affinity in the low-nanomolar range. Recombinant vesicular stomatitis virus pseudotyped with LFV glycoprotein (GP) adopted the receptor binding characteristics of LFV and depended on alpha-DG for infection of cells. Mapping of the binding site of LFV on alpha-DG revealed that LFV binding required the same domains of alpha-DG that are involved in the binding of LCMV. Further, LFV was found to efficiently compete with laminin alpha1 and alpha2 chains for alpha-DG binding. Together with our previous studies on receptor binding of the prototypic immunosuppressive LCMV isolate LCMV clone 13, these findings indicate a high degree of conservation in the receptor binding characteristics between the highly human-pathogenic LFV and murine-immunosuppressive LCMV isolates.     

Cell entry by human pathogenic arenaviruses

The arenaviruses Lassa virus (LASV) in Africa and Machupo (MACV), Guanarito (GTOV) and Junin viruses (JUNV) in South America cause severe haemorrhagic fevers in humans with fatality rates of 15–35%. The present review focuses on the first steps of infection with human pathogenic arenaviruses, the interaction with their cellular receptor molecules and subsequent entry into the host cell. While similarities exist in genomic organization, structure and clinical disease caused by pathogenic Old World and New World arenaviruses these pathogens use different primary receptors. The Old World arenaviruses employ α-dystroglycan, a cellular receptor for proteins of the extracellular matrix, and the human pathogenic New World arenaviruses use the cellular cargo receptor transferrin receptor 1. While the New World arenavirus JUNV enters cells via clathrin-dependent endocytosis, evidence occurred for clathrin-independent entry of the prototypic Old World arenavirus lymphocytic choriomeningitis virus. Upon internalization, arenaviruses are delivered to the endosome, where pH-dependent membrane fusion is mediated by the envelope glycoprotein (GP). While arenavirus GPs share characteristics with class I fusion GPs of other enveloped viruses, unusual mechanistic features of GP-mediated membrane fusion have recently been discovered for arenaviruses with important implications for viral entry.     

Old World and clade C New World arenaviruses mimic the molecular mechanism of receptor recognition used by alpha-dystroglycan's host-derived ligands

alpha-Dystroglycan (DG) is an important cellular receptor for extracellular matrix (ECM) proteins and also serves as the receptor for Old World arenaviruses Lassa fever virus (LFV) and lymphocytic choriomeningitis virus (LCMV) and clade C New World arenaviruses. In the host cell, alpha-DG is subject to a remarkably complex pattern of O glycosylation that is crucial for its interactions with ECM proteins. Two of these unusual sugar modifications, protein O mannosylation and glycan modifications involving the putative glycosyltransferase LARGE, have recently been implicated in arenavirus binding. Considering the complexity of alpha-DG O glycosylation, our present study was aimed at the identification of the specific O-linked glycans on alpha-DG that are recognized by arenaviruses. As previously shown for LCMV, we found that protein O mannosylation of alpha-DG is crucial for the binding of arenaviruses of distinct phylogenetic origins, including LFV, Mobala virus, and clade C New World arenaviruses. In contrast to the highly conserved requirement for O mannosylation, more generic O glycans present on alpha-DG are dispensable for arenavirus binding. Despite the critical role of O-mannosyl glycans for arenavirus binding under normal conditions, the overexpression of LARGE in cells deficient in O mannosylation resulted in highly glycosylated alpha-DG that was functional as a receptor for arenaviruses. Thus, modifications by LARGE but not O-mannosyl glycans themselves are most likely the crucial structures recognized by arenaviruses. Together, the data demonstrate that arenaviruses recognize the same highly conserved O-glycan structures on alpha-DG involved in ECM protein binding, indicating a strikingly similar mechanism of receptor recognition by pathogen- and host-derived ligands.     

Site 1 protease is required for proteolytic processing of the glycoproteins of the South American hemorrhagic fever viruses Junin, Machupo, and Guanarito

The cellular proprotein convertase site 1 protease (S1P) has been implicated in the proteolytic processing of the glycoproteins (GPs) of Old World arenaviruses. Here we report that S1P is also involved in the processing of the GPs of the genetically more-distant South American hemorrhagic fever viruses Guanarito, Machupo, and Junin. Efficient cleavage of Guanarito virus GP, whose protease recognition sites deviate from the reported S1P consensus sequence, indicates a broader specificity of S1P than anticipated. Lack of GP processing of Junin virus dramatically reduced production of infectious virus and prevented cell-to-cell propagation. Infection of S1P-deficient cells resulted in viral persistence over several weeks without the emergence of escape variants able to use other cellular proteases for GP processing.     

SAR studies of 4-acyl-1,6-dialkylpiperazin-2-one arenavirus cell entry inhibitors

Old World (Africa) and New World (South America) arenaviruses are associated with human hemorrhagic fevers. Efforts to develop small molecule therapeutics have yielded several chemical series including the 4-acyl-1,6-dialkylpiperazin-2-ones. Herein, we describe an extensive exploration of this chemotype. In initial Phase I studies, R¹ and R⁴ scanning libraries were assayed to identify potent substituents against Old World (Lassa) virus. In subsequent Phase II studies, R⁶ substituents and iterative R¹, R⁴ and R⁶ substituent combinations were evaluated to obtain compounds with improved Lassa and New World (Machupo, Junin, and Tacaribe) arenavirus inhibitory activity, in vitro human liver microsome metabolic stability and aqueous solubility.     

Old World arenavirus infection interferes with the expression of functional alpha-dystroglycan in the host cell

alpha-Dystroglycan (alpha-DG) is an important cellular receptor for extracellular matrix (ECM) proteins as well as the Old World arenaviruses lymphocytic choriomeningitis virus (LCMV) and the human pathogenic Lassa fever virus (LFV). Specific O-glycosylation of alpha-DG is critical for its function as receptor for ECM proteins and arenaviruses. Here, we investigated the impact of arenavirus infection on alpha-DG expression. Infection with an immunosuppressive LCMV isolate caused a marked reduction in expression of functional alpha-DG without affecting biosynthesis of DG core protein or global cell surface glycoprotein expression. The effect was caused by the viral glycoprotein (GP), and it critically depended on alpha-DG binding affinity and GP maturation. An equivalent effect was observed with LFVGP. Viral GP was found to associate with a complex between DG and the glycosyltransferase LARGE in the Golgi. Overexpression of LARGE restored functional alpha-DG expression in infected cells. We provide evidence that virus-induced down-modulation of functional alpha-DG perturbs DG-mediated assembly of laminin at the cell surface, affecting normal cell-matrix interactions.     

Posttranslational modification of alpha-dystroglycan, the cellular receptor for arenaviruses, by the glycosyltransferase LARGE is critical for virus binding

The receptor for lymphocytic choriomeningitis virus (LCMV), the human pathogenic Lassa fever virus (LFV), and clade C New World arenaviruses is α-dystroglycan (α-DG), a cell surface receptor for proteins of the extracellular matrix (ECM). Specific posttranslational modification of α-DG by the glycosyltransferase LARGE is critical for its function as an ECM receptor. In the present study, we show that LARGE-dependent modification is also crucial for α-DG's function as a cellular receptor for arenaviruses. Virus binding involves the mucin-type domain of α-DG and depends on modification by LARGE. A crucial role of the LARGE-dependent glycosylation of α-DG for virus binding is found for several isolates of LCMV, LFV, and the arenaviruses Mobala and Oliveros. Since the posttranslational modification by LARGE is crucial for α-DG recognition by both arenaviruses and the host-derived ligand laminin, it also influences competition between virus and laminin for α-DG. Hence, LARGE-dependent glycosylation of α-DG has important implications for the virus-host cell interaction and the pathogenesis of LFV in humans.     

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

The Old World arenavirus Lassa virus (LASV) is the causative agent of severe viral hemorrhagic fever (VHF) in humans and is the most prevalent human pathogen among arenaviruses. The present study investigated the largely unknown mechanisms of cell entry of LASV, a process know to be mediated solely by the virus envelope glycoprotein (GP). To circumvent biosafety restrictions associated with the use of live LASV, we used reverse genetics to generate a recombinant variant of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV GP (rLCMV-LASVGP). The rescued rLCMV-LASVGP grew to titers comparable to that of LCMV and showed the receptor binding characteristics of LASV. We used rLCMV-LASVGP to characterize the cellular mechanisms of LASV entry in the context of a productive arenavirus infection. The kinetics of pH-dependent membrane fusion of rLCMV-LASVGP resembled those of the human-pathogenic New World arenavirus Junin virus (JUNV) and other enveloped viruses that use clathrin-mediated endocytosis for entry. However, rLCMV-LASVGP entered cells predominantly via a clathrin-, caveolin-, and dynamin-independent endocytotic pathway similar to the one recently described for LCMV. Productive infection of rLCMV-LASVGP was only mildly affected by a dominant negative mutant of Rab5 and was independent of Rab7, suggesting an unusual mechanism of delivery to endosomes. In addition, rLCMV-LASVGP infection was independent of actin but required intact microtubules. Our data indicate that LASV enters cells via a pathway distinct from the one used by human-pathogenic New World arenaviruses.     

A Multivalent and Cross-Protective Vaccine Strategy against Arenaviruses Associated with Human Disease

Arenaviruses are the causative pathogens of severe hemorrhagic fever and aseptic meningitis in humans, for which no licensed vaccines are currently available. Pathogen heterogeneity within the Arenaviridae family poses a significant challenge for vaccine development. The main hypothesis we tested in the present study was whether it is possible to design a universal vaccine strategy capable of inducing simultaneous HLA-restricted CD8⁺ T cell responses against 7 pathogenic arenaviruses (including the lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses), either through the identification of widely conserved epitopes, or by the identification of a collection of epitopes derived from multiple arenavirus species. By inoculating HLA transgenic mice with a panel of recombinant vaccinia viruses (rVACVs) expressing the different arenavirus proteins, we identified 10 HLA-A02 and 10 HLA-A03-restricted epitopes that are naturally processed in human antigen-presenting cells. For some of these epitopes we were able to demonstrate cross-reactive CD8⁺ T cell responses, further increasing the coverage afforded by the epitope set against each different arenavirus species. Importantly, we showed that immunization of HLA transgenic mice with an epitope cocktail generated simultaneous CD8⁺ T cell responses against all 7 arenaviruses, and protected mice against challenge with rVACVs expressing either Old or New World arenavirus glycoproteins. In conclusion, the set of identified epitopes allows broad, non-ethnically biased coverage of all 7 viral species targeted by our studies.     

DNA vaccination against persistent viral infection.

This study shows that DNA vaccination can confer protection against a persistent viral infection by priming CD8+ cytotoxic T lymphocytes (CTL). Adult BALB/c (H-2d) mice were injected intramuscularly with a plasmid expressing the nucleoprotein (NP) gene of lymphocytic choriomeningitis virus (LCMV) under the control of the cytomegalovirus promoter. The LCMV NP contains the immunodominant CTL epitope (amino acids 118 to 126) recognized by mice of the H-2d haplotype. After three injections with 200 micrograms of NP DNA, the vaccinated mice were challenged with LCMV variants (clones 13 and 28b) that establish persistent infection in naive adult mice. Fifty percent of the DNA-vaccinated mice were protected, as evidenced by decreased levels of infectious virus in the blood and tissues, eventual clearance of viral antigen from all organs tested, the presence of an enhanced LCMV-specific CD8+ CTL response, and maintenance of memory CTL after clearance of virus infection. However, it should be noted that protection was seen in only half of the vaccinated mice, and we were unable to directly measure virus-specific immune responses in any of the DNA-vaccinated mice prior to LCMV challenge. Thus, at least in the system that we have used, gene immunization was a suboptimal method of inducing protective immunity and was several orders of magnitude less efficient than vaccination with live virus. In conclusion, our results show that DNA immunization works against a persistent viral infection but that efforts should be directed towards improving this novel method of vaccination.     

Infection of Type I Interferon Receptor-Deficient Mice with Various Old World Arenaviruses: A Model for Studying Virulence and Host Species Barriers

Lassa virus causes hemorrhagic Lassa fever in humans, while the related Old World arenaviruses Mopeia, Morogoro, and Mobala are supposedly apathogenic to humans and cause only inapparent infection in non-human primates. Here, we studied whether the virulence of Old World arenaviruses in humans and non-human primates is reflected in type I interferon receptor deficient (IFNAR^-/-) mice by testing several strains of Lassa virus vs. the apathogenic viruses Mopeia, Morogoro, and Mobala. All Lassa virus strains tested—Josiah, AV, BA366, and Nig04-10—replicated to high titers in blood, lung, kidney, heart, spleen, brain, and liver and caused disease as evidenced by weight loss and elevation of aspartate and alanine aminotransferase (AST and ALT) levels with a high AST/ALT ratio. Lassa fever-like pathology included acute hepatitis, interstitial pneumonia, and pronounced disturbance of splenic cytoarchitecture. Infiltrations of activated monocytes/macrophages expressing inducible nitric oxide synthase and T cells were found in liver and lung. In contrast, Mopeia, Morogoro, and Mobala virus replicated poorly in the animals and acute inflammatory alterations were not noted. Depletion of CD4⁺ and CD8⁺ T cells strongly enhanced susceptibility of IFNAR^-/- mice to the apathogenic viruses. In conclusion, the virulence of Old World arenaviruses in IFNAR^-/- mice correlates with their virulence in humans and non-human primates. In addition to the type I interferon system, T cells seem to regulate whether or not an arenavirus can productively infect non-host rodent species. The observation that Lassa virus overcomes the species barrier without artificial depletion of T cells suggests it is able to impair T cell functionality in a way that corresponds to depletion.     

In silico characterization of immunogenic epitopes presented by HLA-Cw*0401

Background

Arenaviruses are a family of rodent-borne viruses that cause several hemorrhagic fevers. These diseases can be devastating and are often lethal. Herein, to aid in the design and development of diagnostics, treatments and vaccines for arenavirus infections, we have developed a database containing protein sequences from the seven pathogenic arenaviruses (Junin, Guanarito, Sabia, Machupo, Whitewater Arroyo, Lassa and LCMV).

Results

The database currently contains a non-redundant set of 333 protein sequences which were manually annotated. All entries were linked to NCBI and cited PubMed references. The database has a convenient query interface including BLAST search. Sequence variability analyses were also performed and the results are hosted in the database.

Conclusion

The database is available at http://epitope.liai.org:8080/projects/arena and can be used to aid in studies that require proteomic information from pathogenic arenaviruses.     

Cellular entry of lymphocytic choriomeningitis virus

In contrast to most enveloped viruses that enter the host cell via clathrin-dependent endocytosis, the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) enters cells via noncoated vesicles that deliver the virus to endosomes, where pH-dependent membrane fusion occurs. Here, we investigated the initial steps of LCMV infection. We found that the attachment of LCMV to its cellular receptor α-dystroglycan occurs rapidly and is not dependent on membrane cholesterol. However, subsequent virus internalization is sensitive to cholesterol depletion, indicating the involvement of a cholesterol-dependent pathway. We provide evidence that LCMV entry involves an endocytotic pathway that is independent of clathrin and caveolin and that does not require the GTPase dynamin. In addition, neither the structural integrity nor the dynamics of the actin cytoskeleton are required for infection. These findings indicate that the prototypic Old World arenavirus LCMV uses a mechanism of entry that is different from clathrin-mediated endocytosis, which is used by the New World arenavirus Junin virus, and pathways used by other enveloped viruses.     

Vaccination with a synthetic peptide modulates lymphocytic choriomeningitis virus-mediated immunopathology.

Vaccination with a nucleopeptide (NP 118; amino acids 118 to 132) representing a cytotoxic T-cell epitope of lymphocytic choriomeningitis virus (LCMV) can modulate immunopathology. Immunization with NP 118 protected H-2d mice against intracerebral infection with the LCMV-ARMSTRONG isolate. However, when NP 118-primed H-2d mice were challenged intracerebrally with an intermediate dose (5 x 10(4) PFU) of the LCMV-DOCILE strain, all mice primed with NP 118 emulsified in incomplete Freund's adjuvant died, whereas unprimed mice survived. Correspondingly, peptide vaccination enhanced specifically the cytotoxic T-cell response, influencing the critical balance between T-cell response and virus spread.     

Signal-regulatory protein alpha is an anti-viral entry factor targeting viruses using endocytic pathways

Signal-regulatory protein alpha (SIRPA) is a well-known inhibitor of phagocytosis when it complexes with CD47 expressed on target cells. Here we show that SIRPA decreased in vitro infection by a number of pathogenic viruses, including New World and Old World arenaviruses, Zika virus, vesicular stomatitis virus and pseudoviruses bearing the Machupo virus, Ebola virus and SARS-CoV-2 glycoproteins, but not HSV-1, MLV or mNoV. Moreover, mice with targeted mutation of the Sirpa gene that renders it non-functional were more susceptible to infection with the New World arenaviruses Junín virus vaccine strain Candid 1 and Tacaribe virus, but not MLV or mNoV. All SIRPA-inhibited viruses have in common the requirement for trafficking to a low pH endosomal compartment. This was clearly demonstrated with SARS-CoV-2 pseudovirus, which was only inhibited by SIRPA in cells in which it required trafficking to the endosome. Similar to its role in phagocytosis inhibition, SIRPA decreased virus internalization but not binding to cell surface receptors. We also found that increasing SIRPA levels via treatment with IL-4 led to even greater anti-viral activity. These data suggest that enhancing SIRPA’s activity could be a target for anti-viral therapies.