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

Abstract

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/IMPβ1-dependent nuclear import by conferring direct binding to the IMPα2/IMPβ1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPβ-binding autoinhibitory domain (ΔIBB-IMPα2), and IMPβ1 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.

Roles of nuclear trafficking in infection by cytoplasmic negative-strand RNA viruses: paramyxoviruses and beyond

“…the NLSs of the nucleoproteins of influenza virus (TKGTKRSYEQM) and canine distemper virus (CDV) (TGILISIL), respectively] (Sato et al, 2006;Wang et al, 1997). Conformational NLSs that function only in the context of the native protein/ domain have also been described, such as the predicted NLS of the globular C-terminal domain (CTD) of RABV P protein, comprising basic residues distantly localized in the primary sequence but closely aligned within the domain fold (Moseley et al, 2007a;Pasdeloup et al, 2005;Rowe et al, 2016). Several IMP-independent modes of nuclear import have also been reported, including Ca 2+ -dependent transport of sex-determining region Y protein, that is mediated by calmodulin and heat shock cognate protein 70 (Kaur & Jans, 2011;Kaur et al, 2013), as well as carrier-independent transport of b-catenin and human T-cell lymphotropic virus 1 Tax, which interact directly with Nups (Fagotto et al, 1998;Yokoya et al, 1999).…”

“…Notably, several key trafficking sequences show broad conservation in the lyssavirus genus, indicative of important roles in viral biology (Jacob et al, 2000;Pasdeloup et al, 2005;Wiltzer et al, 2012). RABV P protein contains two nuclear trafficking modules/domains, located in the N-terminal region (NTR) (Oksayan et al, 2012b) and globular CTD (Pasdeloup et al, 2005;Rowe et al, 2016), both of which incorporate a CRM1-dependent NES and an IMPbinding NLS in overlapping or closely associated sequences, enabling efficient co-regulation of import and export. Truncation of the NTR to generate isoforms impacts localization both by inactivating/deleting the N-terminal NES and, in P3, by activating the N-terminal NLS de novo resulting in strong nuclear import (Oksayan et al, 2012b).…”

“…Trafficking function of the CTD, which is present in all RABV P isoforms, is proposed to be regulated by a phosphorylation-dependent mechanism that switches between conformations favouring export or import by the NES and non-conventional conformational NLS (Moseley et al, 2007a, b;Oksayan et al, 2012b;Pasdeloup et al, 2005;Rowe et al, 2016). Notably, the CTD can interact directly with IMPa/b heterodimer and IMPb alone, suggesting that the conformational NLS has non-classical function in coupling to multiple transport pathways (Rowe et al, 2016), which parallels the broad IMP-binding specificity of some paramyxovirus M proteins (Pentecost et al, 2015).…”

“…Notably, the CTD can interact directly with IMPa/b heterodimer and IMPb alone, suggesting that the conformational NLS has non-classical function in coupling to multiple transport pathways (Rowe et al, 2016), which parallels the broad IMP-binding specificity of some paramyxovirus M proteins (Pentecost et al, 2015). Regulation of P protein trafficking is also likely to derive from its role as an interaction hub at the virus-host interface , such that NLSs/NESs may be regulated by competing intermolecular interactions.…”

“…Regulation of P protein trafficking is also likely to derive from its role as an interaction hub at the virus-host interface , such that NLSs/NESs may be regulated by competing intermolecular interactions. In particular, the binding of IMPs and RABV N protein to overlapping sequences in the P protein CTD (Moseley et al, 2007a, b;Pasdeloup et al, 2005;Rowe et al, 2016) might coordinate trafficking by a mechanism similar to that suggested for morbillivirus N-P protein interaction (Sato et al, 2006). P protein additionally contains two sequences for association with the cytoskeleton, which modulate nuclear localization by either inhibiting nuclear import through sequestration to microtubules (as reported for P3) or facilitating nuclear import through interaction with a component of the microtubule-associated dynein motor.…”