A novel nuclear trafficking module regulates the nucleocytoplasmic localization of the rabies virus interferon antagonist, P protein.

Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1-P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3-P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection.

Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death.

Cancer cell metabolism is increasingly recognized as providing an exciting therapeutic opportunity. However, a drug that directly couples targeting of a metabolic dependency with the induction of cell death in cancer cells has largely remained elusive. Here we report that the drug-like small-molecule ironomycin reduces the mitochondrial iron load, resulting in the potent disruption of mitochondrial metabolism. Ironomycin promotes the recruitment and activation of BAX/BAK, but the resulting mitochondrial outer membrane permeabilization (MOMP) does not lead to potent activation of the apoptotic caspases, nor is the ensuing cell death prevented by inhibiting the previously established pathways of programmed cell death. Consistent with the fact that ironomycin and BH3 mimetics induce MOMP through independent nonredundant pathways, we find that ironomycin exhibits marked in vitro and in vivo synergy with venetoclax and overcomes venetoclax resistance in primary patient samples. SIGNIFICANCE: Ironomycin couples targeting of cellular metabolism with cell death by reducing mitochondrial iron, resulting in the alteration of mitochondrial metabolism and the activation of BAX/BAK. Ironomycin induces MOMP through a different mechanism to BH3 mimetics, and consequently combination therapy has marked synergy in cancers such as acute myeloid leukemia. This article is highlighted in the In This Issue feature, p. 587.

Dual modes of rabies P-protein association with microtubules: a novel strategy to suppress the antiviral response.

Conventional nuclear import is independent of the cytoskeleton, but recent data have shown that the import of specific proteins can be either facilitated or inhibited by microtubules (MTs). Nuclear import of the P-protein from rabies virus involves a MT-facilitated mechanism, but here, we show that P-protein is unique in that it also undergoes MT-inhibited import, with the mode of MT-interaction being regulated by the oligomeric state of the P-protein. This is the first demonstration that a protein can utilise both MT-inhibited and MT-facilitated import mechanisms, and can switch between these different modes of MT interaction to regulate its nuclear trafficking. Importantly, we show that the P-protein exploits MT-dependent mechanisms to manipulate host cell processes by switching the import of the interferon-activated transcription factor STAT1 from a conventional to a MT-inhibited mechanism. This prevents STAT1 nuclear import and signalling in response to interferon, which is vital to the host innate antiviral response. This is the first report of MT involvement in the viral subversion of interferon signalling that is central to virus pathogenicity, and identifies novel targets for the development of antiviral drugs or attenuated viruses for vaccine applications.

Role of interferon antagonist activity of rabies virus phosphoprotein in viral pathogenicity.

The fixed rabies virus (RV) strain Nishigahara kills adult mice after intracerebral inoculation, whereas the chicken embryo fibroblast cell-adapted strain Ni-CE causes nonlethal infection in adult mice. We previously reported that the chimeric CE(NiP) strain, which has the phosphoprotein (P protein) gene from the Nishigahara strain in the genetic background of the Ni-CE strain, causes lethal infection in adult mice, indicating that the P gene is responsible for the different pathogenicities of the Nishigahara and Ni-CE strains. Previous studies demonstrated that RV P protein binds to the interferon (IFN)-activated transcription factor STAT1 and blocks IFN signaling by preventing its translocation to the nucleus. In this study, we examine the molecular mechanism by which RV P protein determines viral pathogenicity by comparing the IFN antagonist activities of the Nishigahara and Ni-CE P proteins. The results, obtained from both RV-infected cells and cells transfected to express P protein only, show that Ni-CE P protein is significantly impaired for its capacity to block IFN-activated STAT1 nuclear translocation and, consequently, inhibits IFN signaling less efficiently than Nishigahara P protein. Further, it was demonstrated that a defect in the nuclear export of Ni-CE P protein correlates with a defect in its ability to cause the mislocalization of STAT1. These data provide the first evidence that the capacity of the RV P protein to inhibit STAT1 nuclear translocation and IFN signaling correlates with the viral pathogenicity.

Nuclear Trafficking of the Rabies Virus Interferon Antagonist P-Protein Is Regulated by an Importin-Binding Nuclear Localization Sequence in the C-Terminal Domain.

Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP)-recognised nuclear localization sequence in the N-terminal region (N-NLS), the mechanisms underlying nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD) can result in nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro nuclear transport assays to examine in detail the nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on nuclear export, suggesting that nuclear exclusion of mutated P3 involves the P-CTD-localized nuclear export sequence (C-NES). However, assays using cells in which nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 nuclear accumulation and, importantly, prevent nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMPα2/IMPß1-dependent nuclear import by conferring direct binding to the IMPα2/IMPß1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPß-binding autoinhibitory domain (ΔIBB-IMPα2), and IMPß1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P-protein subcellular localization, consistent with important roles in infection.