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

Abstract

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 (IFN␤) induction may contribute to its virulence and spread. Here we analyzed IFN␤ 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 IFN␤ signaling and that replacing ANDV residues 252 to 296 with a hypervariable domain (HVD) from MAPV N prevents IFN␤ 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 IFN␤ induction. In contrast, replacing serine with phosphoserine-mimetic aspartic acid (S386D) in ANDV N robustly inhibited interferon regulatory factor 3 (IRF3) phosphorylation and IFN␤ induction. Additionally, the MAPV N protein gained the ability to inhibit IRF3 phosphorylation and IFN␤ 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. Downloaded frompathogenic hantaviruses predominantly infect the endothelial cell (EC) lining of capillaries and nonlytically disrupt normal barrier functions, causing highly lethal edematous and hemorrhagic diseases (1,5,(9)(10)(11)(12)(13)(14). In Eurasia, pathogenic hantaviruses cause hemorrhagic ...

Binding of the Andes Virus Nucleocapsid Protein to RhoGDI Induces the Release and Activation of the Permeability Factor RhoA

“…Consistent with prior studies (53) , we observed a partial decline in RhoA binding to the phosphomimetic RhoGDI S34D mutant in the absence of ANDV N protein ( Fig 7A) and we found no difference in ANDV N protein binding to RhoGDI phosphorylation site mutants (Fig. 7B) (71) . However, we found no difference in the binding of RhoGDI to WT or ANDV N protein S386A/D/H mutants (Fig.…”

“…cAMP activated protein kinase A (PKA) directs RhoGDI-S174 phosphorylation which stabilizes RhoGDI binding to Rho family kinases and could impact N protein binding to RhoGDI (51,52,54,61,64,65,(74)(75)(76)(77)(78) . In addition, ANDV N protein is phosphorylated on S386 (71) , and the phosphorylation of ANDV N protein could impact its binding to RhoGDI or PKA directed phosphorylation of RhoGDI. We expressed S386A, S386D and S386H mutants of ANDV N protein and determined if mutant N proteins bind to RhoGDI or alter RhoGDI-S174 phosphorylation.…”

“…Primary human PMECs were transduced with lentiviruses expressing ANDV or TULV N proteins, puromycin selected and assayed for N protein expression by Western blot (WB). pLenti-CMV-GFP-Puro vector as previously described (70,71) . Lentiviruses were generated by cotransfecting HEK293T cells with pLenti-purovectors and 3 rd generation lentiviral packaging plasmids (130)(131)(132) Western blot of PMECs using anti-N polyclonal antibodies as previously described (29,30,70) .…”

Hantavirus infection in type I interferon receptor-deficient (A129) mice

“…It has been suggested that SEOV infection of macrophages and lymphocytes could reduce the immune response and thus contribute to persistent infection [71]. Although the A129 mice used in our study had abrogated type I IFN receptors, it is surprising that the uncoupling of the IFN response pathway was not enough to lead to disease, as hantaviruses have evolved mechanisms to inhibit production of type I IFN upon infection [72,73]. Thus our data suggest that other pathways must be disengaged in order to exert virulence.…”

Natural killer cell responses to emerging viruses of zoonotic origin

“…Syrian hamsters are tolerant to certain hantaviruses, such as Sin Nombre Virus (SNV), but succumb to the closely related Andes Virus (ANDV), with symptoms remarkably similar to the human manifestation of the disease [ 59 , 60 , 61 ]. Although comparative analyses are scarce, ANDV appears to avoid early innate immune recognition and elicit a stronger immune response later on [ 59 , 62 ]. Priming with poly I:C or SNV protects hamsters from ANDV-driven pulmonary disease, suggesting that rapid IFN responses may allow for viral control while avoiding later immunopathology [ 63 ], similar to bats.…”