1and and paired C911 siRNAs with complementary substitutions of nucleotides 9 to 11. and replicates in vitro only in cells engineered to express a critical entry factor, cadherin-related family member 3 (CDHR3) (8). RV-C is usually strongly associated with severe respiratory tract infections in young children and is more closely related to RV-A than to RV-B (2, 7, 9, 10). Nearly all hospital visits related to RV-triggered asthma are due to infections with RV-A or RV-C viruses, with RV-C associated with more severe symptoms (10C12). The molecular mechanisms underlying replication of these RNA viruses are only partially comprehended. Enteroviral RNAs are synthesized around the cytosolic surface of membranous cytoplasmic tubulovesicular structures (13C15). These replication organelles are derived from remodeled endoplasmic reticulum (ER) or Golgi membranes and contain multiple viral nonstructural proteins, including 2B, 2C, and an RNA-dependent RNA polymerase, 3Dpol (16). The formation of replication organelles is usually associated with a striking reordering of cellular lipid metabolism, with phosphatidylinositol 4-kinase-III (PI4K) playing a Argatroban key role. PI4K is usually recruited to membranes at the site of replication by the viral 3A protein acting in concert with host acyl-CoA binding domain-containing 3 (ACBD3) (13, 17, 18). PI4K mediates the enrichment of these membranes with phosphatidylinositol 4-phosphate (PI4P), leading to subsequent recruitment of oxysterol-binding protein 1 (OSBP1), which enhances cholesterol flux into the membranes (18). Thus, ACBD3, PI4K, and OSBP1 are all crucial host factors for RV replication. The intracellular replication of poliovirus, a closely-related enterovirus, is also dependent on components of host autophagic signaling, including LC3 protein that associates with the membranes of replication organelles in a nonlipidated form (19, 20). Whether this is also true for rhinoviruses is usually uncertain. Unlike poliovirus, RV-A1a replication is not influenced by chemical compounds that promote or inhibit autophagy, rapamycin, and 3-methyadenine (3-MA) respectively, while comparable studies of RV-A2 produced conflicting results (21, 22). These latter data show that even among closely related viruses in the same picornaviral genus, host factors involved in remodeling membranes and generating replication Argatroban organelles may vary substantially. Here we describe a surprising requirement for the Stimulator of Interferon Genes (STING) protein in intracellular replication of RV-A and RV-C viruses. STING (also known as MITA, ERIS, or MPYS) is an essential adaptor protein downstream of cGMP-AMP synthase (cGAS) in the innate immune cytosolic DNA-sensing pathway, and thus is typically associated with antiviral rather than proviral effects (23C27). We show that RV-A16 replication organelles are enriched in STING, and that transfected subgenomic RV-A16 and RV-C15 RNA replicons fail to amplify in the absence of STING. Genetic evidence links Mouse monoclonal to GFP STING to the nonstructural 2C protein of RV-A, which is known to play a crucial role in the formation of replication organelles. Results Genome-Wide Screen Identifies STING as an RV-A2 Host Factor. STING was identified as a host protein required for RV-A2 replication in a genome-wide siRNA screen targeting 22,909 genes in HeLa-Ohio cells (Fig. 1 and and exceeded the C911 test for target sequence specificity (28); modified siRNAs in which bases 9 to 11 were swapped with their complement did not protect against RV-A2 CPE (Fig. 1and and paired C911 siRNAs with complementary substitutions of nucleotides 9 to 11. PPIase B (cyclophilin B) served Argatroban as a loading control. (= 0.0002 by two-way ANOVA. ( 0.001 by two-way ANOVA. (value 0.001 by two-way ANOVA with Sidaks multiple comparison test. (mRNA Argatroban and RV-A16 RNA abundance normalized to mRNA in primary SAECs transduced with value 0.001 by two-way ANOVA with Sidaks multiple comparison test. = 3. (and and and and and and and and and message were likely due to enhanced RV-A16 replication secondary to greater STING expression (Fig. 3expression also correlated positively with increased STING abundance, even though increases in STING did not enhance RV-B14 replication (Fig. 3 and.