Helical Domain Changes between hGBP3 and hGBP3ΔC Result in Distinct Oligomers and Anti-HCV Activity.

Human guanylate binding proteins (hGBPs), which are large GTPases, are crucial for cell-autonomous immunity, including antiviral activity. hGBPs contain two domains: an N-terminal catalytic domain and a C-terminal helical domain. hGBP3 and its splice variant hGBP3ΔC have been shown to possess anti-influenza activity in lung epithelial cells. These two proteins have identical catalytic domains but different helical domains. It is unclear whether this difference affects GTPase activity or protein oligomerization. Using combined approaches, we show that both proteins hydrolyze GTP to GDP and further to GMP. However, they form different oligomers. hGBP3 exists as a hexamer in the free form, whereas hGBP3ΔC forms large oligomers, indicating that helical domain modifications of the splice variant result in distinct oligomers. Furthermore, unlike other homologues, neither protein changes its oligomeric state upon substrate binding or hydrolysis. Deleting the helical domain of hGBP3 (hGBP3 ^1-309 ) yields a monomer, suggesting that the helical domain promotes the hexamerization of hGBP3. We overexpressed hGBP3 and hGBP3ΔC to test their efficacy against HCV growth and found that hGBP3 inhibits HCV multiplication, while the splice variant has little effect. Our mutational studies on hGBP3 show that substrate hydrolysis, rather than substrate binding, is required for inhibiting HCV growth. This suggests that substrate hydrolysis generates a protein conformation essential for anti-HCV activity. Additionally, truncated hGBP3 ^1-309 does not exhibit anti-HCV activity. Altogether, these findings suggest that the helical domain of hGBP3 is crucial for reducing HCV growth through hexamer formation and that its variations result in different oligomers and antiviral activities.