Characterisation of the nuclear targeting mechanisms, nuclear functions, and nuclear interactors of rabies virus P3 protein

Rabies virus (RABV) causes more than 55,000 human deaths worldwide annually [1, 2]. This is largely because current inactivated vaccines for protection against RABV infection are costly and difficult to administer, and as such not available to the majority of people that are at risk of infection [3-5]. Further, there are no effective treatments for symptomatic RABV infection. Thus, there is a serious need for development of improved vaccines, including safe live vaccines, as well as antiviral drugs. This requires determining the molecular mechanisms underlying viral infection and pathogenicity in detail, and characterising viral protein interactions with the host cell. Among other recent studies that address these aims [6-9], one important publication has demonstrated that regulated nucleocytoplasmic shuttling of the RABV phosphoprotein (P) is essential for viral pathogenicity, as RABV expressing P impaired for nucleocytoplasmic trafficking becomes sensitive to interferon-mediated immunity, and thus, highly attenuated in vivo [10, 11]. This data strongly implies that regulated nucleocytoplasmic trafficking of P represents a potential target for attenuating mutations and development of antiviral therapeutics. RABV P is expressed as a full length protein, P1, and four N-terminally truncated isoforms, P2-P5 [12]. The expression of multiple isoforms from the single P gene is thought to facilitate RABV P serving its multiple functions in viral infection, including as the non-catalytic viral polymerase cofactor that is required for replication, and as an antagonist of the host antiviral immune responses [13-16]. Intriguingly, although RABV replicates in the cytoplasm and the P1 and P2 isoforms are strongly cytoplasmic, P3-P5 are reported to localise mostly in the nucleus [17]. Strongly cytoplasmic localisation of P1 and P2 is widely accepted to be due to the activity of a nuclear export signal (NES) in the N-terminal region of P, while P3-P5, which lack key residues of this NES, are reported to be driven into the nucleus by a nuclear localisation signal (NLS) within the C-terminal domain (CTD) [17]. Importantly however, much remains unresolved about these trafficking signals including mechanisms involved in their regulation and host factors involved in their recognition, and this relatively simple model of P isoform nucleocytoplasmic trafficking has been called into question by subsequent studies which have identified several additional signals that modulate P nucleocytoplasmic shuttling [18, 19]. Further, while several P isoforms are reported to localise to the nucleus, very little is known regarding their specific intranuclear functions and interactions. This PhD project addressed many of these unknowns by analysing the precise sequences and mechanisms involved in nuclear trafficking of P protein isoforms, in particular the strongly nuclear P3, and investigating the nuclear functions and binding partners of this isoform. Quantitative analysis of the subcellular distribution of the five P protein isoforms led to the identification of a novel nuclear localisation signal in the N-terminal region of P3 (the N-NLS) which is recognised by the cellular importin (IMP) α/β heterodimer and requires the presence of P residues K54 and R55 . These key residues are truncated in P4 and P5, which consequently lack N-NLS activity and are diffusely localised throughout the nucleus and cytoplasm. P3 is thus the only strongly nuclear P protein isoform. Further characterisation revealed that activation of the N-NLS also requires truncation of residues P1-52, such that it is not active in P1, but only active in the context of P3. Importantly, P residues 1-52 also contain the NES that drives P1 and P2 into the cytoplasm, which thus overlaps with the N-NLS. To our knowledge, this is the first report of a novel type of trafficking module wherein the activity of two opposing targeting signals are co-regulated through truncation. This study thus redefines the model of P protein nuclear trafficking, revealing a highly efficient regulatory mechanism that drives strong and specific nuclear targeting of P3, consistent with the idea that P3 serves key intranuclear roles in infection. The exact nuclear functions of P3 have remained unknown, however previous reports have suggested that RABV and related viruses such as Hendra virus (HeV) and Nipah virus (NiV) target proteins to the nucleus in order to inhibit IFN signalling by antagonising intranuclear STAT1 [20-23]. This was tested directly for the first time in this study using P3 hindered for nuclear trafficking by mutation of key N-NLS residues. The data indicated that P3 nuclear localisation is not critical for its antagonism of STAT1, as both wild type P3 and P3 containing mutations to key N-NLS residues can physically interact with STAT1 and inhibit STAT1 signalling to similar extents. To determine genuine reasons for the strong and specific nuclear targeting of P3, the intranuclear distribution of P3 was examined in greater detail, and this led to the finding that P3 can colocalise with nucleoli, interact specifically with the core nucleolar protein, nucleolin (NCL), and moreover, that NCL is critically required for production of infective RABV particles. To our knowledge, this is the first demonstration of a rhabdovirus targeting the nucleolus and interacting with NCL, and makes RABV one of only a handful of negative stranded RNA viruses known to interact with nucleoli and nucleolar factors. Further analysis indicated that in addition to targeting nucleoli, P3 may target distinct sub-nuclear structures, and serves additional nuclear functions including inhibition of cellular RNA synthesis. The identification if a novel host-virus interaction within the nucleus and of potentially important nuclear functions for P3 is consistent with the premise that nuclear trafficking of P is a promising target for attenuating mutations. Together, the findings of this study indicate that nuclear trafficking of P3 serves critical roles in infection, likely by enabling specific modulation of nuclear and/or nucleolar functions by the virus within infected cells. These data have thus made significant contributions to our understanding of RABV biology, and identified the nucleolus as a new host-virus interface. Identifying host virus interactions, determining where they take place, and characterising the mechanisms and sequences involved is an extremely important prerequisite to develop new targets for attenuation, generate new vaccine strains, and design antiviral drugs against the currently incurable and fatal pathogen RABV, as well as related viruses.