Hantaviruses cause two diseases with vascular permeability defects, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Non-pathogenic PHV elicits early interferon responses in human, suggesting that hantavirus pathogenesis may in part be determined by viral regulation of cellular interferon responses. In contrast to pathogenic NY-1V and HTNV, PHV replication is blocked in IFN competent human endothelial cells, further suggesting NY-1V might regulate early IFN responses. These findings led us to investigate the mechanism of interferon regulation by pathogenic hantaviruses and what permits pathogenic hantavirus replication in human endothelial cells.

Expression of the NY-1V Gn cytoplasmic tail inhibited RIG-I and TBK1 directed transcription from interferon stimulated response elements (ISRE) or β-interferon promoters. In contrast, expression of the NY-1V nucleocapsid or PHV Gn-tail had no effect on RIG-I or TBK1 directed transcription. Further, neither the NY-1V nor PHV G1-tails inhibited transcriptional responses directed by a constitutively active IRF-3 protein. These findings indicate that the pathogenic NY-1V Gn protein regulates cellular IFN responses upstream of IRF-3 phosphorylation at the level of TBK1 signaling complexes.

TBK1 phosphorylates IRF-3, and forms a signaling complex with TRAF3, which is required for IFN transcription directed by a variety of upstream stimuli. TBK1 also forms a signaling complex with TRAF2, which directs NF-κB activation. Here we report that the NY-1V Gn-tail co-immunoprecipitates TRAF3, but not TBK1, from cellular lysates. Analysis of TRAF3 deletion mutants demonstrated that the Gn-tail bound the N-terminus of TRAF3, as well as the corresponding N-terminal domain of TRAF2. Furthermore, the NY-1V Gn-tail also inhibits TBK1 and TRAF2 directed NF-κB activation, indicating that the Gn-tail regulates the activation of two factors required for IFN-β transcription. In contrast, the Gn-tail of the non-pathogenic hantavirus PHV failed to bind TRAF3 or TRAF2 proteins or inhibit NF-κB or IFN-β transcriptional responses. Expression of the NY-1V blocked TBK1 co-precipitation of TRAF3 and similarly, infection by NY-1V, but not PHV blocked the formation of TBK1-TRAF3 complexes. These findings suggest that both the NY-1V Gn-tail and infection by NY-1V virus disrupt the formation of TBK1-TRAF3 signaling complexes required for IFN-β induction.

Collectively, these findings indicate that the ability to inhibit interferon-β induction at early times post-infection is critical for hantavirus infection of human endothelial cells and suggest that the pathogenic hantavirus Gn cytoplasmic tail is the primary determinate of hantavirus pathogenic potential in humans.