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A dynamic actin cytoskeleton is necessary for viral entry, intracellular migration, and virion release. For HIV-1 infection, during entry, the virus triggers early actin activity by hijacking chemokine coreceptor signaling, which activates a host dependency factor, cofilin, and its kinase, the LIM domain kinase (LIMK). Although knockdown of human LIM domain kinase 1 (LIMK1) with short hairpin RNA (shRNA) inhibits HIV infection, no specific small-molecule inhibitor of LIMK has been available. Here, we describe the design and discovery of novel classes of small-molecule inhibitors of LIMK for inhibiting HIV infection. We identified R10015 as a lead compound that blocks LIMK activity by binding to the ATP-binding pocket. R10015 specifically blocks viral DNA synthesis, nuclear migration, and virion release. In addition, R10015 inhibits multiple viruses, including Zaire ebolavirus (EBOV), Rift Valley fever virus (RVFV), Venezuelan equine encephalitis virus (VEEV), and herpes simplex virus 1 (HSV-1), suggesting that LIMK inhibitors could be developed as a new class of broad-spectrum antiviral drugs. IMPORTANCE The actin cytoskeleton is a structure that gives the cell shape and the ability to migrate. Viruses frequently rely on actin dynamics for entry and intracellular migration. In cells, actin dynamics are regulated by kinases, such as the LIM domain kinase (LIMK), which regulates actin activity through phosphorylation of cofilin, an actin-depolymerizing factor. Recent studies have found that LIMK/cofilin are targeted by viruses such as HIV-1 for propelling viral intracellular migration. Although inhibiting LIMK1 expression blocks HIV-1 infection, no highly specific LIMK inhibitor is available. This study describes the design, medicinal synthesis, and discovery of small-molecule LIMK inhibitors for blocking HIV-1 and several other viruses and emphasizes the feasibility of developing LIMK inhibitors as broad-spectrum antiviral drugs.

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. IMPORTANCE 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.

We show that interferon-induced transmembrane protein 1 (IFITM-1), IFITM-2, and IFITM-3 exhibit a broad spectrum of antiviral activity against several members of the Bunyaviridae family, including Rift Valley fever virus (RVFV), La Crosse virus, Andes virus, and Hantaan virus, all of which can cause severe disease in humans and animals. We found that RVFV was restricted by IFITM-2 and -3 but not by IFITM-1, whereas the remaining viruses were equally restricted by all IFITMs. Indeed, at low doses of alpha interferon (IFN-α), IFITM-2 and -3 mediated more than half of the antiviral activity of IFN-α against RVFV. IFITM-2 and -3 restricted RVFV infection mostly by preventing virus membrane fusion with endosomes, while they had no effect on virion attachment to cells, endocytosis, or viral replication kinetics. We found that large fractions of IFITM-2 and IFITM-3 occupy vesicular compartments that are distinct from the vesicles coated by IFITM-1. In addition, although overexpression of all IFITMs expanded vesicular and acidified compartments within cells, there were marked phenotypic differences among the vesicular compartments occupied by IFITMs. Collectively, our data provide new insights into the possible mechanisms by which the IFITM family members restrict distinct viruses.

With the exception of Reston and Lloviu viruses, filoviruses (marburgviruses, ebolaviruses, and “cuevaviruses”) cause severe viral hemorrhagic fevers in humans. Filoviruses use a class I fusion protein, GP 1,2 , to bind to an unknown, but shared, cell surface receptor to initiate virus-cell fusion. In addition to GP 1,2 , ebolaviruses and cuevaviruses, but not marburgviruses, express two secreted glycoproteins, soluble GP (sGP) and small soluble GP (ssGP). All three glycoproteins have identical N termini that include the receptor-binding region (RBR) but differ in their C termini. We evaluated the effect of the secreted ebolavirus glycoproteins on marburgvirus and ebolavirus cell entry, using Fc-tagged recombinant proteins. Neither sGP-Fc nor ssGP-Fc bound to filovirus-permissive cells or inhibited GP 1,2 -mediated cell entry of pseudotyped retroviruses. Surprisingly, several Fc-tagged Δ-peptides, which are small C-terminal cleavage products of sGP secreted by ebolavirus-infected cells, inhibited entry of retroviruses pseudotyped with Marburg virus GP 1,2 , as well as Marburg virus and Ebola virus infection in a dose-dependent manner and at low molarity despite absence of sequence similarity to filovirus RBRs. Fc-tagged Δ-peptides from three ebolaviruses (Ebola virus, Sudan virus, and Taï Forest virus) inhibited GP 1,2 -mediated entry and infection of viruses comparably to or better than the Fc-tagged RBRs, whereas the Δ-peptide-Fc of an ebolavirus nonpathogenic for humans (Reston virus) and that of an ebolavirus with lower lethality for humans (Bundibugyo virus) had little effect. These data indicate that Δ-peptides are functional components of ebolavirus proteomes. They join cathepsins and integrins as novel modulators of filovirus cell entry, might play important roles in pathogenesis, and could be exploited for the synthesis of powerful new antivirals.

Marburgvirus (MARV) infections are generally lethal in humans and nonhuman primates but require in vivo lethal mouse variant selection by the serial transfer (passage) of the nonlethal virus into naïve mice to propagate a lethal infection. The passage of progenitor (wild-type) MARV or Ravn virus (RAVV) from infected scid BALB/c mouse liver homogenates into immunocompetent BALB/c mice results in the selection of lethal mouse viruses from within the quasispecies sufficient to establish lethality in immunocompetent mice. Genomic analysis in conjunction with the passage history of each mutation detailed the altered primary and secondary structures of the viral genomic RNA throughout the process. Key findings included the following: (i) a VP40:D184N mutation previously identified in the lethal guinea pig MARV genome was the first mutation to occur during the passage of both the MARV and RAVV variants; (ii) there was biased hypermutagenesis in the RAVV variant genome; (iii) there were two identical mutations in lethal mouse MARV and RAVV variants, VP40:Y19H in the PPPY motif and VP40:D184N in a loop structure between the two VP40 domains; (iv) the passage of wild-type MARV and RAVV in mice resulted in the selection of viral variants from among the quasispecies with different genotypes than those of the wild-type viruses; and (v) a lethal mouse RAVV variant had different tissue tropisms distinct from those of its wild-type virus. These studies provide insights into how marburgviruses manipulate the host for enzymes, metabolites, translation regulators, and effectors of the innate immune response to serve as potential viral countermeasures.

The lack of a mouse model has hampered an understanding of the pathogenesis and immunity of Marburg hemorrhagic fever (MHF), the disease caused by marburgvirus (MARV), and has created a bottleneck in the development of antiviral therapeutics. Primary isolates of the filoviruses, i.e., ebolavirus (EBOV) and MARV, are not lethal to immunocompetent adult mice. Previously, pathological, virologic, and immunologic evaluation of a mouse-adapted EBOV, developed by sequential passages in suckling mice, identified many similarities between this model and EBOV infections in nonhuman primates. We recently demonstrated that serially passaging virus recovered from the liver homogenates of MARV-infected immunodeficient (SCID) mice was highly successful in reducing the time to death in these mice from 50 to 70 days to 7 to 10 days after challenge with the isolate MARV-Ci67, -Musoke, or -Ravn. In this study, we extended our findings to show that further sequential passages of MARV-Ravn in immunocompetent mice caused the MARV to kill BALB/c mice. Serial sampling studies to characterize the pathology of mouse-adapted MARV-Ravn revealed that this model is similar to the guinea pig and nonhuman primate MHF models. Infection of BALB/c mice with mouse-adapted MARV-Ravn caused uncontrolled viremia and high viral titers in the liver, spleen, lymph node, and other organs; profound lymphopenia; destruction of lymphocytes within the spleen and lymph nodes; and marked liver damage and thrombocytopenia. Sequencing the mouse-adapted MARV-Ravn strain revealed differences in 16 predicted amino acids from the progenitor virus, although the exact changes required for adaptation are unclear at this time. This mouse-adapted MARV strain can now be used to develop and evaluate novel vaccines and therapeutics and may also help to provide a better understanding of the virulence factors associated with MARV.

Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.

Ocozocoautla de Espinosa virus (OCEV) is a novel, uncultured arenavirus. We found that the OCEV glycoprotein mediates entry into grivet and bat cells through transferrin receptor 1 (TfR1) binding but that OCEV glycoprotein precursor (GPC)-pseudotyped retroviruses poorly entered 53 human cancer cell lines. Interestingly, OCEV and Tacaribe virus could use bat, but not human, TfR1. Replacing three human TfR1 amino acids with their bat ortholog counterparts transformed human TfR1 into an efficient OCEV and Tacaribe virus receptor.