A Single Point Mutation in the Rhinovirus 2B Protein Reduces the Requirement for Phosphatidylinositol 4-Kinase Class III Beta in Viral Replication

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 phosphatidylinositol 4 (PI4)-kinase 3beta (PI4K3b), which boosts PI4-phosphate (PI4P) levels and drives lipid countercurrent exchange of PI4P against cholesterol at endoplasmic reticulum-Golgi membrane contact sites through the lipid shuttling protein oxysterol binding protein 1 (OSBP1). 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 (termed 2B[I92T]). The mutation did not confer resistance to cholesterol-sequestering compounds or OSBP1 inhibition, suggesting invariant dependency on the PI4P/cholesterol lipid countercurrents. In the presence of PI4K3b inhibitor, Golgi reorganization and PI4P lipid induction occurred in RV-A16 2B[I92] but not in wild-type infection. The knockout of PI4K3b abolished the replication of both the 2B[I92T] mutant and the wild type. Doxycycline-inducible expression of PI4K3b in PI4K3b knockout 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 that of 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 while still maintaining the PI4P/cholesterol countercurrent for establishing Golgi-derived RV replication membranes. Human rhinoviruses (RVs) are the major cause of the 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 phosphatidylinositol 4 phosphate for RV replication.

Label-Free Digital Holo-tomographic Microscopy Reveals Virus-Induced Cytopathic Effects in Live Cells

“…7 ). The partial effect of PIK-93 on RIG was, however, consistent with the partial reduction of PI4P levels and the incomplete inhibition of RV-A1a replication ( 24 , 29 ). These results support the notion that an increase in RIG in virus-infected host cells occurs in a virus type-specific manner and that RIG changes are a reliable indicator of early and late CPE.…”

Label-free digital holo-tomographic microscopy reveals virus-induced cytopathic effects in live cells

“…6). The partial effect of PIK-93 on RIG was, however, consistent with the partial reduction of PI4P levels and the incomplete inhibition of RV-A1a replication (24,29). These results support the notion that an increase of RIG in virus-infected host cells occurs in a virus type-specific manner, and that RIG changes are a reliable indicator of early and late CPE.…”

Host Lipids in Positive-Strand RNA Virus Genome Replication

“…The OSBP/PI4P-dependent cholesterol enrichment pathway is known to be exploited by several diverse (+)RNA viruses, including poliovirus (Arita, 2014), HCV (Wang et al, 2014), HRV (Roulin et al, 2014), AiV (Ishikawa-Sasaki et al, 2018), and encephalomyocarditis virus (EMCV) (Dorobantu et al, 2015). Specifically, PI4KIIIβ is recruited by poliovirus protein 2BC (Arita, 2014) or HRV 2B or 2BC (Roulin et al, 2014, 2018) to promote the biosynthesis of PI4P and the recruitment of OSBP to virus-induced membranous structures or VRCs. These recruitments promote the accumulation of free cholesterol.…”