Polioviruses, Coxsackieviruses, and Echoviruses belonging to the Picornaviridae family of positive-stranded, non-enveloped viruses, are highly infectious and associated with a range of illnesses in children from minor febrile illness to severe, potentially fatal conditions (eg, aseptic meningitis, encephalitis, paralysis and myocarditis). The viruses encodes 11 viral proteins along with the transient set of intermediates unique to viral propagation. 3A, one of the non-structural proteins, plays a crucial role in viral replication by anchoring the replication complex to the membrane vesicle and by recruiting essential cellular factors to the site of replication. It is an 89 amino-acid longprotein, and consists of a soluble N-terminal region and a hydrophobicC-terminal region. The soluble region contains two amphipathic alphahelices that form a hairpin, which are flanked by unstructured regions.Since, Enteroviruses have limited coding capacity,viral protein interactions with cellular proteins and lipids are essentialin viral replication, translation, polyprotein processing andpathogenesis. Understanding these interactions is essential inunderstanding the molecular mechanisms associated pathogenesis, andidentifying drug targets. Our studies are aimed at identifying hostfactors interacting with 3A protein and their functional significance invirus replication.We have identified thepotential 3A-interacting cellular candidate proteins using pull-down followed by liquid chromatography associated mass spectrometry. Gene ontology analysis revealed asignificant enrichment in cellular pathways, functions, and proteindomains in comparison with the control. Further studies were focused on Sec12 (guanine nucleotideexchange factor), ACBD3 (acyl-CoA binding domain containing 3) andPhosphatidylinositol 4-kinase beta (PI4KIIIß) interactions with the 3Aprotein, and their significance in viral replication. Sec12 (GEF) initiates the COPII vesicle-mediated ER-to-Golgi membrane trafficking by recruiting and activating the small GTP binding protein Sar1A to the membrane, which further recruits Sec23/24, cargo and Sec13/31 coat proteins to form functional COPII vesicles.We demonstrated that Sec12 and 3A interact directly in the ER through their C-terminal hydrophobic regions in oligomerization independent manner, leading toreduced the level of recruitment of individual COPII components such as Sar1A, Sec24A, and Sec31A to the membranes, thereby inhibiting virus replication. But in infected cells, other viral proteins such as 2B and 2BC likely stabilize the membrane-recruited Sar1A to support the viral replication. The viral proteins, ACBD3, PI4KIIIß interacted and co-localized with the Echovirus 3A protein.Knockdown of Sec12 or PI4KIIIß and expression of 3A or DN-Sar1A inhibited Echovirus replication, unlike proteins which support the COPII vesicle mediated ER-to-Golgi trafficking.Our results collectively indicate Sec12 is a crucial component in the anterograde membrane trafficking and is a novel host factor in Echovirus replication.