Helical Domain\nChanges between hGBP3 and hGBP3ΔC\nResult in Distinct Oligomers and Anti-HCV Activity

Human\nguanylate binding proteins (hGBPs), which are large GTPases,\nare crucial for cell-autonomous immunity, including antiviral activity.\nhGBPs contain two domains: an N-terminal catalytic domain and a C-terminal\nhelical domain. hGBP3 and its splice variant hGBP3ΔC have been\nshown to possess anti-influenza activity in lung epithelial cells.\nThese two proteins have identical catalytic domains but different\nhelical domains. It is unclear whether this difference affects GTPase\nactivity or protein oligomerization. Using combined approaches, we\nshow that both proteins hydrolyze GTP to GDP and further to GMP. However,\nthey form different oligomers. hGBP3 exists as a hexamer in the free\nform, whereas hGBP3ΔC forms large oligomers, indicating that\nhelical domain modifications of the splice variant result in distinct\noligomers. Furthermore, unlike other homologues, neither protein changes\nits oligomeric state upon substrate binding or hydrolysis. Deleting\nthe helical domain of hGBP3 (hGBP3<sup>1−309</sup>) yields\na monomer, suggesting that the helical domain promotes the hexamerization\nof hGBP3. We overexpressed hGBP3 and hGBP3ΔC to test their efficacy\nagainst HCV growth and found that hGBP3 inhibits HCV multiplication,\nwhile the splice variant has little effect. Our mutational studies\non hGBP3 show that substrate hydrolysis, rather than substrate binding,\nis required for inhibiting HCV growth. This suggests that substrate\nhydrolysis generates a protein conformation essential for anti-HCV\nactivity. Additionally, truncated hGBP3<sup>1−309</sup> does\nnot exhibit anti-HCV activity. Altogether, these findings suggest\nthat the helical domain of hGBP3 is crucial for reducing HCV growth\nthrough hexamer formation and that its variations result in different\noligomers and antiviral activities.