Abstract
Rhinoviruses (RVs) replicate on cytoplasmic membranes derived from the Golgi apparatus. They encode membrane-targeted proteins 2B, 2C and 3A, which control trafficking and lipid composition of the replication membrane. The virus recruits host factors for replication, such as the phosphatidylinositol 4 (PI4)-kinase 3beta (PI4K3b), which boosts PI4-phosphate (PI4P) levels, and drives lipid counter-current exchange of PI4P against cholesterol at endoplasmic reticulum-Golgi membrane contact sites through the lipid shuttling protein oxysterol binding protein (OSBP) 1. We identified a PI4K3b-inhibitor resistant RV-A16 variant with a single point mutation in the conserved 2B protein near the cytosolic carboxy-terminus, isoleucine 92 to threonine [I92T]. The mutation did not confer resistance to cholesterol sequestering compounds or OSBP1 inhibition, suggesting invariant dependency on the PI4P/cholesterol lipid counter-currents. In presence of PI4K3b-inhibitor, Golgi reorganization and PI4P lipid induction occurred in RV-A16 2B[I92], but not wild-type infection. The knock-out of PI4K3b abolished the replication of both 2B[I92T] mutant and wild-type. Doxycyclin-inducible expression of PI4K3b in PI4K3b knock-out cells efficiently rescued the 2B[I92T] mutant, and less effectively wild-type virus infection. Ectopic expression of 2B[I92T] or 2B was less efficient than 3A in recruiting PI4K3b to perinuclear membranes, suggesting a supportive rather than decisive role of 2B in recruiting PI4K3b. The data suggest that 2B tunes the recruitment of PI4K3b to the replication membrane, and allows the virus to adapt to cells with low levels of PI4K3b, yet maintaining the PI4P/cholesterol counter-current for establishing Golgi-derived RV replication membranes.Human rhinoviruses (RVs) are the major cause of common cold worldwide. They cause asthmatic exacerbations and chronic obstructive pulmonary disease. Despite recent advances, the development of antivirals and vaccines has proven difficult due to the high number and variability of RV types. The identification of critical host factors and their interactions with viral proteins and membrane lipids for the establishment of viral replication is a basis for drug development strategies. Our findings here shed new light on the interactions between nonstructural viral membrane proteins and class III phosphatidylinositol 4 kinases from the host, and highlight the importance of phosphatidyl-inositol 4 phosphate for RV replication.
Roulin, Pascal S