Despite replicating to very high titers, coxsackieviruses do not elicit strong CD8 T-cell responses, perhaps because antigen presentation is inhibited by virus-induced disruption of host protein trafficking. Herein, we evaluated the effects of three viral nonstructural proteins (2B, 2BC, and 3A) on intracellular trafficking. All three of these proteins inhibited secretion, to various degrees, and directly associated with the Golgi complex, causing trafficking proteins to accumulate in this compartment. The 3A protein almost completely ablated trafficking and secretion, by moving rapidly to the Golgi, and causing its disruption. Using an alanine-scanning 3A mutant, we show that Golgi targeting and disruption can be uncoupled. Thus, coxsackieviruses rely on the combined effects of several gene products that target a single cellular organelle to successfully block protein secretion during an infection. These findings have implications for viral pathogenesis.Following their entry into a permissive cell, viruses quickly initiate a cascade of events that modify the intracellular space, making it an environment conducive to viral gene expression, genome replication, and, ultimately, the rapid spread of infectious particles to other susceptible cells within the host. To accomplish this, viral genomes often encode polypeptides that have very specific, often nonoverlapping functions ranging from the shutoff of host cell transcription and translation to inhibiting the antiviral immune response by curbing cytokine secretion and antigen presentation (9). While the often very large genomes of DNA and RNA viruses can encode a small army of gene products to accomplish these tasks (2), smaller viruses must rely on a more limited number of multifunctional polypeptides.RNA viruses in the Picornaviridae family fall into this latter category, possessing very small genomes (ϳ7.5 kilobases) that encode no more than a dozen gene products. Despite having such a limited number of genes, these viruses are remarkably efficient at causing a wide range of diseases, including (but not limited to) the common cold (human rhinovirus), foot-andmouth disease (foot-and-mouth-disease virus), poliomyelitis (poliovirus), hepatitis (hepatitis A virus), and myocarditis, pancreatitis, and meningitis (coxsackievirus) (38). How do these viruses, with their limited coding capacities, achieve the rapid and complete subversion of multiple cell types within the host? The answer lies in the expression of a large ϳ250 kDa viral polyprotein (from a single long open reading frame) (Fig. 1A) that is processed by virally encoded proteinases (33) to yield several precursors and mature cleavage products, each with highly specific functions during the viral life cycle (17). Interestingly, many of the nonstructural proteins have membraneassociative properties (e.g., 2B, 2BC, 2C) and act, individually or in concert with each other, to form cytoplasmic vesicles (5,27,29,32) that compartmentalize the host cell and provide an environment suitable for RNA replication. Concur...