Hantaviruses successfully replicate in primary human endothelial cells by restricting the early induction of beta interferon (IFN-␤) and interferon-stimulated genes (ISGs). Gn proteins from NY-1V, ANDV, and TULV, but not PHV, harbor elements in their 142-residue cytoplasmic tails (GnTs) that inhibit RIG-I/MAVS/TBK1-TRAF3-directed IFN-␤ induction. Here, we define GnT interactions and residues required to inhibit TRAF3-TBK1-directed IFN-␤ induction and IRF3 phosphorylation. We observed that GnTs bind TRAF3 via residues within the TRAF-N domain (residues 392 to 415) and that binding is independent of the MAVS-interactive TRAF-C domain (residues 415 to 568). We determined that GnT binding to TRAF3 is mediated by C-terminal degrons within NY-1V or ANDV GnTs and that mutations that add degrons to TULV or PHV GnTs confer TRAF3 binding. Further analysis of GnT domains revealed that TRAF3 binding is a discrete GnT function, independent of IFN regulation, and that residues 15 to 42 from the NY-1V GnT C terminus are required for inhibiting TBK1-directed IFN-␤ transcription.

In contrast, expressing the PHV Gn-T had no effect on TBK1-induced transcriptional responses. Analysis of Gn-T truncations demonstrated that the C-terminal 42 residues of the Gn-T (Gn-T-C42) from TULV, but not PHV, inhibited IFN induction >70%. These findings demonstrate that the TULV Gn-T inhibits IFN-and ISRE-directed responses upstream of IRF3 at the level of the TBK1 complex and further define a 42-residue domain of the TULV Gn-T that inhibits IFN induction. In contrast to pathogenic hantavirusGn-Ts, the TULV Gn-T lacks a C-terminal degron domain and failed to bind tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3), a TBK1 complex component required for IRF3 activation. These findings indicate that the nonpathogenic TULV Gn-T regulates IFN induction but accomplishes this via unique interactions with cellular TBK1 complexes. These findings fundamentally distinguish nonpathogenic hantaviruses, PHV and TULV, and demonstrate that IFN regulation alone is insufficient for hantaviruses to cause disease. Yet regulating the early IFN response is necessary for hantaviruses to replicate within human endothelial cells and to be pathogenic. Thus, in addition to IFN regulation, hantaviruses contain discrete virulence determinants which permit them to be human pathogens.

Hantaviruses primarily infect human endothelial cells (ECs) and cause two highly lethal human diseases. Early addition of Type I interferon (IFN) to ECs blocks hantavirus replication and thus for hantaviruses to be pathogenic they need to prevent early interferon induction. PHV replication is blocked in human ECs, but not inhibited in IFN deficient VeroE6 cells and consistent with this, infecting ECs with PHV results in the early induction of IFNβ and an array of interferon stimulated genes (ISGs). In contrast, ANDV, HTNV, NY-1V and TULV hantaviruses, inhibit early ISG induction and successfully replicate within human ECs. Hantavirus inhibition of IFN responses has been attributed to several viral proteins including regulation by the Gn proteins cytoplasmic tail (Gn-T). The Gn-T interferes with the formation of STING-TBK1-TRAF3 complexes required for IRF3 activation and IFN induction, while the PHV Gn-T fails to alter this complex or regulate IFN induction. These findings indicate that interfering with early IFN induction is necessary for hantaviruses to replicate in human ECs, and suggest that additional determinants are required for hantaviruses to be pathogenic. The mechanism by which Gn-Ts disrupt IFN signaling is likely to reveal potential therapeutic interventions and suggest protein targets for attenuating hantaviruses.

Andes virus (ANDV) causes a fatal hantavirus pulmonary syndrome (HPS) in humans and Syrian hamsters.Human ␣ v ␤ 3 integrins are receptors for several pathogenic hantaviruses, and the function of ␣ v ␤ 3 integrins on endothelial cells suggests a role for ␣ v ␤ 3 in hantavirus directed vascular permeability. We determined here that ANDV infection of human endothelial cells or Syrian hamster-derived BHK-21 cells was selectively inhibited by the high-affinity ␣ v ␤ 3 integrin ligand vitronectin and by antibodies to ␣ v ␤ 3 integrins. Further, antibodies to the ␤ 3 integrin PSI domain, as well as PSI domain polypeptides derived from human and Syrian hamster ␤ 3 subunits, but not murine or bovine ␤ 3 , inhibited ANDV infection of both BHK-21 and human endothelial cells. These findings suggest that ANDV interacts with ␤ 3 subunits through PSI domain residues conserved in both Syrian hamster and human ␤ 3 integrins. Sequencing the Syrian hamster ␤ 3 integrin PSI domain revealed eight differences between Syrian hamster and human ␤ 3 integrins. Analysis of residues within the PSI domains of human, Syrian hamster, murine, and bovine ␤ 3 integrins identified unique proline substitutions at residues 32 and 33 of murine and bovine PSI domains that could determine ANDV recognition. Mutagenizing the human ␤ 3 PSI domain to contain the L33P substitution present in bovine ␤ 3 integrin abolished the ability of the PSI domain to inhibit ANDV infectivity. Conversely, mutagenizing either the bovine PSI domain, P33L, or the murine PSI domain, S32P, to the residue present human ␤ 3 permitted PSI mutants to inhibit ANDV infection. Similarly, CHO cells transfected with the full-length bovine ␤ 3 integrin containing the P33L mutation permitted infection by ANDV. These findings indicate that human and Syrian hamster ␣ v ␤ 3 integrins are key receptors for ANDV and that specific residues within the ␤ 3 integrin PSI domain are required for ANDV infection. Since L33P is a naturally occurring human ␤ 3 polymorphism, these findings further suggest the importance of specific ␤ 3 integrin residues in hantavirus infection. These findings rationalize determining the role of ␤ 3 integrins in hantavirus pathogenesis in the Syrian hamster model.

American hantaviruses cause a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). Hantaviruses nonlytically infect endothelial cells and cause dramatic changes in barrier functions of the endothelium without disrupting the endothelium. Instead hantaviruses cause changes in the function of infected endothelial cells that normally regulate fluid barrier functions of capillaries. The endothelium of arteries, veins, and lymphatic vessels is unique and central to the function of vast pulmonary capillary beds, which regulate pulmonary fluid accumulation. The endothelium maintains vascular barrier functions through a complex series of redundant receptors and signaling pathways that serve to both permit fluid and immune cell efflux into tissues and restrict tissue edema. Infection of the endothelium provides several mechanisms for hantaviruses to alter capillary permeability but also defines potential therapeutic targets for regulating acute pulmonary edema and HPS disease. Here we discuss interactions of HPS causing hantaviruses with the endothelium, potential endothelial cell-directed permeability mechanisms, and therapeutic targeting of the endothelium as a means of reducing the severity of HPS disease.