The alphaviruses Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus (EEEV), and western equine encephalitis virus (WEEV) are arthropod-borne positive-strand RNA viruses that are capable of causing acute and fatal encephalitis in many mammals, including humans. VEEV was weaponized during the Cold War and is recognized as a select agent. Currently, there are no FDAapproved vaccines or therapeutics for these viruses. The spread of VEEV and other members of this family due to climate change-mediated vector range expansion underscores the need for research aimed at developing medical countermeasures. These viruses utilize programmed Ϫ1 ribosomal frameshifting (Ϫ1 PRF) to synthesize the viral trans-frame (TF) protein, which has previously been shown to be important for neuropathogenesis in the related Sindbis virus. Here, the alphavirus Ϫ1 PRF signals were characterized, revealing novel Ϫ1 PRF stimulatory structures. Ϫ1 PRF attenuation mildly affected the kinetics of VEEV accumulation in cultured cells but strongly inhibited its pathogenesis in an aerosol infection mouse model. Importantly, the decreased viral titers in the brains of mice infected with the mutant virus suggest that the alphavirus TF protein is important for passage through the blood-brain barrier and/or for neuroinvasiveness. These findings suggest a novel approach to the development of safe and effective live attenuated vaccines directed against VEEV and perhaps other closely related Ϫ1 PRF-utilizing viruses.IMPORTANCE Venezuelan equine encephalitis virus (VEEV) is a select agent that has been weaponized. This arthropod-borne positive-strand RNA virus causes acute and fatal encephalitis in many mammals, including humans. There is no vaccine or other approved therapeutic. VEEV and related alphaviruses utilize programmed Ϫ1 ribosomal frameshifting (Ϫ1 PRF) to synthesize the viral trans-frame (TF) protein, which is important for neuropathogenesis. Ϫ1 PRF attenuation strongly inhibited VEEV pathogenesis in mice, and viral replication analyses suggest that the TF protein is critical for neurological disease. These findings suggest a new approach to the development of safe and effective live attenuated vaccines directed against VEEV and other related viruses.

The capsid structural protein of the New World alphavirus, Venezuelan equine encephalitis virus (VEEV), interacts with the host nuclear transport proteins importin α/β1 and CRM1. Novel selective inhibitor of nuclear export (SINE) compounds, KPT-185, KPT-335 (verdinexor), and KPT-350, target the host’s primary nuclear export protein, CRM1, in a manner similar to the archetypical inhibitor Leptomycin B. One major limitation of Leptomycin B is its irreversible binding to CRM1; which SINE compounds alleviate because they are slowly reversible. Chemically inhibiting CRM1 with these compounds enhanced capsid localization to the nucleus compared to the inactive compound KPT-301, as indicated by immunofluorescent confocal microscopy. Differences in extracellular versus intracellular viral RNA, as well as decreased capsid in cell free supernatants, indicated the inhibitors affected viral assembly, which led to a decrease in viral titers. The decrease in viral replication was confirmed using a luciferase-tagged virus and through plaque assays. SINE compounds had no effect on VEEV TC83_Cm, which encodes a mutated form of capsid that is unable to enter the nucleus. Serially passaging VEEV in the presence of KPT-185 resulted in mutations within the nuclear localization and nuclear export signals of capsid. Finally, SINE compound treatment also reduced the viral titers of the related eastern and western equine encephalitis viruses, suggesting that CRM1 maintains a common interaction with capsid proteins across the New World alphavirus genus.

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There is an urgent need for therapeutic development to combat infections caused by Rift Valley fever virus (RVFV), which causes devastating disease in both humans and animals. In an effort to repurpose drugs for RVFV treatment, our previous studies screened a library of FDA-approved drugs. The most promising candidate identified was the hepatocellular and renal cell carcinoma drug sorafenib. Mechanism-of-action studies indicated that sorafenib targeted a late stage in virus infection and caused a buildup of virions within cells. In addition, small interfering RNA (siRNA) knockdown studies suggested that nonclassical targets of sorafenib are important for the propagation of RVFV. Here we extend our previous findings to identify the mechanism by which sorafenib inhibits the release of RVFV virions from the cell. Confocal microscopy imaging revealed that glycoprotein Gn colocalizes and accumulates within the endoplasmic reticulum (ER) and the transport of Gn from the Golgi complex to the host cell membrane is reduced. Transmission electron microscopy demonstrated that sorafenib caused virions to be present inside large vacuoles inside the cells. p97/valosin-containing protein (VCP), which is involved in membrane remodeling in the secretory pathway and a known target of sorafenib, was found to be important for RVFV egress. Knockdown of VCP resulted in decreased RVFV replication, reduced Gn Golgi complex localization, and increased Gn ER accumulation. The intracellular accumulation of RVFV virions was also observed in cells transfected with siRNA targeting VCP. Collectively, these data indicate that sorafenib causes a disruption in viral egress by targeting VCP and the secretory pathway, resulting in a buildup of virions within dilated ER vesicles. In humans, symptoms of RVFV infection mainly include a self-limiting febrile illness. However, in some cases, infected individuals can also experience hemorrhagic fever, neurological disorders, liver failure, and blindness, which could collectively be lethal. The ability of RVFV to expand geographically outside sub-Saharan Africa is of concern, particularly to the Americas, where native mosquito species are capable of virus transmission. Currently, there are no FDA-approved therapeutics to treat RVFV infection, and thus, there is an urgent need to understand the mechanisms by which the virus hijacks the host cell machinery to replicate. The significance of our research is in identifying the cellular target of sorafenib that inhibits RVFV propagation, so that this information can be used as a tool for the further development of therapeutics used to treat RVFV infection.

Despite over 60 years of research on antiviral drugs, very few are FDA approved to treat acute viral infections. Rift Valley fever virus (RVFV), an arthropod borne virus that causes hemorrhagic fever in severe cases, currently lacks effective treatments. Existing as obligate intracellular parasites, viruses have evolved to manipulate host cell signaling pathways to meet their replication needs. Specifically, translation modulation is often necessary for viruses to establish infection in their host. Here we demonstrated phosphorylation of p70 S6 kinase, S6 ribosomal protein, and eIF4G following RVFV infection in vitro through western blot analysis and in a mouse model of infection through reverse phase protein microarrays (RPPA). Inhibition of p70 S6 kinase through rapamycin treatment reduced viral titers in vitro and increased survival and mitigated clinical disease in RVFV challenged mice. Additionally, the phosphorylation of p70 S6 kinase was decreased following rapamycin treatment in vivo. Collectively these data demonstrate modulating p70 S6 kinase can be an effective antiviral strategy.

Viruses must parasitize host cell translational machinery in order to make proteins for viral progeny. In this study, we sought to use this signal transduction conduit against them by inhibiting multiple kinases that influence translation. Previous work indicated that several kinases involved in translation, including p70 S6K, p90RSK, ERK, and p38 MAPK, are phosphorylated following Rift Valley fever virus (RVFV) infection. Furthermore, inhibiting p70 S6K through treatment with the FDA approved drug rapamycin prevents RVFV pathogenesis in a mouse model of infection. We hypothesized that inhibiting either p70 S6K, p90RSK, or p90RSK’s upstream kinases, ERK and p38 MAPK, would decrease translation and subsequent viral replication. Treatment with the p70 S6K inhibitor PF-4708671 resulted in decreased phosphorylation of translational proteins and reduced RVFV titers. In contrast, treatment with the p90RSK inhibitor BI-D1870, p38MAPK inhibitor SB203580, or the ERK inhibitor PD0325901 alone had minimal influence on RVFV titers. The combination of PF-4708671 and BI-D1870 treatment resulted in robust inhibition of RVFV replication. Likewise, a synergistic inhibition of RVFV replication was observed with p38MAPK inhibitor SB203580 or the ERK inhibitor PD0325901 combined with rapamycin treatment. These findings serve as a proof of concept regarding combination kinase inhibitor treatment for RVFV infection.