Mehmet Sahin

Arenaviruses such as Lassa virus cause arenavirus hemorrhagic fever (AVHF), but protective vaccines and effective antiviral therapy remain unmet medical needs. Our prior work has revealed that inducible nitric oxide synthase (iNOS) induction by IFN-γ represents a key pathway to microvascular leak and terminal shock in AVHF. Here we hypothesized that Ruxolitinib, an FDA-approved JAK inhibitor known to prevent IFN-γ signaling, could be repurposed for host-directed therapy in AVHF. We tested the efficacy of Ruxolitinib in MHC-humanized (HHD) mice, which develop Lassa fever-like disease upon infection with the monkey-pathogenic lymphocytic choriomeningitis virus strain WE. Anti-TNF antibody therapy was tested as an alternative strategy owing to its expected effect on macrophage activation. Ruxolitinib but not anti-TNF antibody prevented hypothermia and terminal disease as well as pleural effusions and skin edema, which served as readouts of microvascular leak. As expected, neither treatment influenced viral loads. Intriguingly, however, and despite its potent disease-modifying activity, Ruxolitinib did not measurably interfere with iNOS expression or systemic NO metabolite levels. These findings suggest that the FDA-approved JAK-inhibitor Ruxolitinib has potential in the treatment of AVHF. Moreover, our observations indicate that besides IFN-γ-induced iNOS additional druggable pathways contribute essentially to AVHF and are amenable to host-directed therapy.

RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected Trim21^-/- mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host. RNA-dependent RNA-polymerases (RdRps) play a key role in the life cycle of RNA viruses. They interact with cellular proteins during replication and transcription processes and impact the immunobiology of viral infections. This study characterized the host protein interactome of the RdRp-containing L protein of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). Several L protein interactors with proviral and antiviral effects were identified in vitro, and mice lacking the identified L protein interactor TRIM21 exhibited impaired control of chronic LCMV infection. Integration of the L protein interactomes with known RdRp interactomes from other RNA viruses highlighted common and virus-specific strategies to interact with the host proteome, which may indicate novel avenues for antiviral interventions.

Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein’s globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy. Neutralizing antibodies (nAbs) represent a key principle of antiviral immunity. Protective vaccines aim at inducing nAbs to prevent viral infection, and infusion of nAbs in convalescent patient serum can offer a potent antiviral therapy. Certain viruses, however, have found ways to evade nAb control. Amongst them are high-risk pathogens of the arenavirus family such as Lassa virus (LASV), which is a frequent cause of hemorrhagic fever in West Africa. Here we unveil the molecular strategy by which arenaviruses escape antibody neutralization and avoid efficient immune control. We show that their surface is decorated with sugar moieties, serving to shield the virus against the neutralizing effect of the host’s antibodies. This immune evasion strategy differs from those described for other viruses, in which sugars impair primarily the induction of antibodies or allow for viral mutational escape. The arenavirus sugar coat renders the host nAb response inefficient and as a consequence thereof, the host fails to promptly control the infection. Our results offer a compelling explanation for the long history of failures in trying to make a nAb-based vaccine against LASV or in using convalescent serum for therapy. These mechanistic insights will support vaccine development efforts against arenaviruses such as LASV.