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.

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.

Subcellular trafficking in rhabdovirus infection and immune evasion: a novel target for therapeutics.

Vesicular stomatitis virus (VSV) and Rabies Virus (RABV) are the prototypic members of the rhabdovirus family. These viruses have a particularly broad host range, and despite the availability of vaccines, RABV still causes more than 50,000 human deaths a year. Trafficking of the virion or viral particles is important at several stages of the replicative life cycle, including cellular entry, localization into the cytoplasmic inclusion bodies which primarily house the transcription and replication of the viral genome, and migration to the plasma membrane from whence the virus is released by budding. Intriguingly, specific viral proteins, VSV M and RABV P have also been shown to undergo intracellular trafficking independent of the other viral apparatus. These proteins are multifunctional, and play roles in antagonism of host processes, namely the IFN system, and as such enable viral evasion of the innate cellular antiviral response. A body of recent research has been aimed at characterizing the mechanisms by which these proteins are able to shuttle between and localize to various subcellular sites, including the nucleus, which is not required during the cytoplasmic replicative life cycle of the virus. This work has indicated that trafficking of these proteins plays a significant role in determining the ability of the viruses to replicate and cause infection, and as such, represents a viable target for development of a new generation of vaccines and prophylactic therapeutics which are required to battle these pathogens of human and agricultural significance.

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.