Phenotypic Divergence of P Proteins of Australian Bat Lyssavirus Lineages Circulating in Microbats and Flying Foxes

Abstract

Bats are reservoirs of many pathogenic viruses, including the lyssaviruses rabies virus (RABV) and Australian bat lyssavirus (ABLV). Lyssavirus strains are closely associated with particular host reservoir species, with evidence of specific adaptation. Associated phenotypic changes remain poorly understood but are likely to involve phosphoprotein (P protein), a key mediator of the intracellular virus–host interface. Here, we examine the phenotype of P protein of ABLV, which circulates as two defined lineages associated with frugivorous and insectivorous bats, providing the opportunity to compare proteins of viruses adapted to divergent bat species. We report that key functions of P protein in the antagonism of interferon/signal transducers and activators of transcription 1 (STAT1) signaling and the capacity of P protein to undergo nuclear trafficking differ between lineages. Molecular mapping indicates that these differences are functionally distinct and appear to involve modulatory effects on regulatory regions or structural impact rather than changes to defined interaction sequences. This results in partial but significant phenotypic divergence, consistent with “fine-tuning” to host biology, and with potentially distinct properties in the virus–host interface between bat families that represent key zoonotic reservoirs.

The Ebola Virus Interferon Antagonist VP24 Undergoes Active Nucleocytoplasmic Trafficking

“…Some viral IFN antagonists use NESs for immune evasion, including through mislocalization of associated STATs [ 39 ]. We thus assessed the effect of NES mutations in VP24 on antagonism of IFN/STAT1 using CLSM and an IFN-α/STAT1-dependent luciferase reporter gene assay ( Figure 3 ) [ 11 , 21 , 24 , 25 ]. Rabies virus N-protein fused to GFP (GFP-N), which localizes to the cytoplasm and does not affect STAT signaling [ 29 ], was included as a standard negative control, as used previously [ 11 , 21 , 25 ].…”

“…We thus assessed the effect of NES mutations in VP24 on antagonism of IFN/STAT1 using CLSM and an IFN-α/STAT1-dependent luciferase reporter gene assay ( Figure 3 ) [ 11 , 21 , 24 , 25 ]. Rabies virus N-protein fused to GFP (GFP-N), which localizes to the cytoplasm and does not affect STAT signaling [ 29 ], was included as a standard negative control, as used previously [ 11 , 21 , 25 ]. GFP-VP24 NM clearly inhibited IFN-α-dependent STAT1 nuclear localization ( Figure 3 a,b) and reporter gene activity ( Figure 3 c), to an extent similar to that observed for WT VP24, indicating that the NES mutations do not disrupt VP24 IFN antagonist function.…”

“…Cells were treated 8 h post-transfection with or without IFN-α (1000 U/mL) before lysis 16 h later using Passive Lysis Buffer (Promega, Madison, WI, USA). Firefly and Renilla luciferase activity was determined in a dual luciferase assay [ 11 , 21 , 24 , 25 ]. The ratio of Firefly to Renilla luciferase activity was determined for each condition, and calculated relative to that for control cells treated with IFN-α (relative luciferase activity).…”

Structural Determination of the Australian Bat Lyssavirus Nucleoprotein and Phosphoprotein Complex

“…Furthermore, RABV phosphoprotein CTD inhibits downstream IFN signalling by interacting with phosphorylated STAT-1, STAT-2, and STAT-3. This interaction causes the accumulation of STATS in the cytoplasm, thereby blocking IFN signalling and failing to induce a robust host antiviral response [ 58 , 59 , 60 , 61 ]. Binding sites for host proteins, including PML, microtubules, STATs, and nuclear import/export machinery, have all been attributed to the phosphoprotein CTD [ 62 , 63 , 64 , 65 ].…”