Unique Interferon Pathway Regulation by the Andes Virus Nucleocapsid Protein Is Conferred by Phosphorylation of Serine 386

Andes virus (ANDV) causes hantavirus pulmonary syndrome (HPS) and is the only hantavirus shown to spread person to person and cause a highly lethal HPS-like disease in Syrian hamsters. The unique ability of ANDV N protein to inhibit beta interferon (IFNβ) induction may contribute to its virulence and spread. Here we analyzed IFNβ regulation by ANDV N protein substituted with divergent residues from the nearly identical Maporal virus (MAPV) N protein. We found that MAPV N fails to inhibit IFNβ signaling and that replacing ANDV residues 252 to 296 with a hypervariable domain (HVD) from MAPV N prevents IFNβ regulation. In addition, changing ANDV residue S386 to the histidine present in MAPV N or the alanine present in other hantaviruses prevented ANDV N from regulating IFNβ induction. In contrast, replacing serine with phosphoserine-mimetic aspartic acid (S386D) in ANDV N robustly inhibited interferon regulatory factor 3 (IRF3) phosphorylation and IFNβ induction. Additionally, the MAPV N protein gained the ability to inhibit IRF3 phosphorylation and IFNβ induction when ANDV HVD and H386D replaced MAPV residues. Mass spectroscopy analysis of N protein from ANDV-infected cells revealed that S386 is phosphorylated, newly classifying ANDV N as a phosphoprotein and phosphorylated S386 as a unique determinant of IFN regulation. In this context, the finding that the ANDV HVD is required for IFN regulation by S386 but dispensable for IFN regulation by D386 suggests a role for HVD in kinase recruitment and S386 phosphorylation. These findings delineate elements within the ANDV N protein that can be targeted to attenuate ANDV and suggest targeting cellular kinases as potential ANDV therapeutics. IMPORTANCE ANDV contains virulence determinants that uniquely permit it to spread person to person and cause highly lethal HPS in immunocompetent hamsters. We discovered that ANDV S386 and an ANDV-specific hypervariable domain permit ANDV N to inhibit IFN induction and that IFN regulation is directed by phosphomimetic S386D substitutions in ANDV N. In addition, MAPV N proteins containing D386 and ANDV HVD gained the ability to inhibit IFN induction. Validating these findings, mass spectroscopy analysis revealed that S386 of ANDV N protein is uniquely phosphorylated during ANDV infection. Collectively, these findings reveal new paradigms for ANDV N protein as a phosphoprotein and IFN pathway regulator and suggest new mechanisms for hantavirus regulation of cellular kinases and signaling pathways. Our findings define novel IFN-regulating virulence determinants of ANDV, identify residues that can be modified to attenuate ANDV for vaccine development, and suggest the potential for kinase inhibitors to therapeutically restrict ANDV replication.