The C-Terminal 42 Residues of the Tula Virus Gn Protein Regulate Interferon Induction

Pathogenic hantaviruses delay the type I interferon response during early stages of viral infection. However, the robust interferon response and induction of interferon-stimulated genes observed during later stages of hantavirus infection fail to combat the virus replication in infected cells. Protein kinase R (PKR), a classical interferon-stimulated gene product, phosphorylates the eukaryotic translation initiation factor eIF2␣ and causes translational shutdown to create roadblocks for the synthesis of viral proteins. The PKR-induced translational shutdown helps host cells to establish an antiviral state to interrupt virus replication. However, hantavirus-infected cells do not undergo translational shutdown and fail to establish an antiviral state during the course of viral infection. In this study, we showed for the first time that Andes virus infection induced PKR overexpression. However, the overexpressed PKR was not active due to a significant inhibition of autophosphorylation. Further studies revealed that Andes virus nucleocapsid protein inhibited PKR dimerization, a critical step required for PKR autophosphorylation to attain activity. The studies reported here establish a hantavirus nucleocapsid protein as a new PKR inhibitor. These studies provide mechanistic insights into hantavirus resistance to the host interferon response and solve the puzzle of the lack of translational shutdown observed in hantavirus-infected cells. The sensitivity of hantavirus replication to PKR has likely imposed a selective evolutionary pressure on hantaviruses to evade the PKR antiviral response for survival. We envision that evasion of the PKR antiviral response by NP has likely helped hantaviruses to exist during evolution and to survive in infected hosts with a multifaceted antiviral defense. IMPORTANCEProtein kinase R (PKR), a versatile antiviral host factor, shuts down the translation machinery upon activation in virus-infected cells to create hurdles for the manufacture of viral proteins. The studies reported here reveal that the hantavirus nucleocapsid protein counteracts the PKR antiviral response by inhibiting PKR dimerization, which is required for its activation. We report the discovery of a new PKR inhibitor whose expression in hantavirus-infected cells prevents the PKR-induced host translational shutdown to ensure the continuous synthesis of viral proteins required for efficient virus replication.

Section: Discussionmentioning

confidence: 53%

Andes Virus Nucleocapsid Protein Interrupts Protein Kinase R Dimerization To Counteract Host Interference in Viral Protein Synthesis

Wang

Mir

2015

“…Gc contains a short (Ͻ10-residue) cytoplasmic tail, while Gn contains a 142-residue cytoplasmic tail (GnT) with several potential functions in the viral life cycle (5). The GnT directs binding of viral nucleocapsid complexes, has matrix protein functions that nucleate viral assembly and budding (23), and with the exception of the PHV GnT, regulates RIG-I-directed beta interferon (IFN-␤) induction (19,20,(24)(25)(26).…”

Section: Hfrs Results From Infection By Eurasian Hantaviruses (Hantaamentioning

confidence: 99%

Hantavirus GnT Elements Mediate TRAF3 Binding and Inhibit RIG-I/TBK1-Directed Beta Interferon Transcription by Blocking IRF3 Phosphorylation

Matthys

Cimica

Dalrymple

et al. 2014

Self Cite

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.

“…At 12 or 24 h posttransfection GFP signal was examined by FACS analysis, and fold change related to the cells lacking the drug was calculated and plotted. response (1,2,22,26). The cytoplasmic tail domain of hantavirus glycoprotein Gn has been reported to inhibit RIG-I-and TBK-1-directed interferon responses by disrupting the formation of TBK1-TRF3 complex (1, 2).…”

Section: Discussionmentioning

confidence: 99%

Autophagic Clearance of Sin Nombre Hantavirus Glycoprotein Gn Promotes Virus Replication in Cells

Hussein

Cheng

Ganaie

et al. 2012

Hantavirus glycoprotein precursor (GPC) is posttranslationally cleaved into two glycoproteins, Gn and Gc. Cells transfected with plasmids expressing either GPC or both Gn and Gc revealed that Gn is posttranslationally degraded. Treatment of cells with the autophagy inhibitors 3-methyladenine, LY-294002, or Wortmanin rescued Gn degradation, suggesting that Gn is degraded by the host autophagy machinery. Confocal microscopic imaging showed that Gn is targeted to autophagosomes for degradation by an unknown mechanism. Examination of autophagy markers LC3-I and LC3-II demonstrated that both Gn expression and Sin Nombre hantavirus (SNV) infection induce autophagy in cells. To delineate whether induction of autophagy and clearance of Gn play a role in the virus replication cycle, we downregulated autophagy genes BCLN-1 and ATG7 using small interfering RNA (siRNA) and monitored virus replication over time. These studies revealed that inhibition of host autophagy machinery inhibits Sin Nombre virus replication in cells, suggesting that autophagic clearance of Gn is required for efficient virus replication. Our studies provide mechanistic insights into viral pathogenesis and reveal that SNV exploits the host autophagy machinery to decrease the intrinsic steady-state levels of an important viral component for efficient replication in host cells.