Viruses often hijack cellular pathways to facilitate infection and replication. Kaposi's sarcoma-associated herpesvirus (KSHV) is IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus associated with several cancers. Through genome-wide kinase screening, we found that KSHV activates the MSK1/2-CREB1 pathway during primary infection and that it depends on this pathway for viral lytic replication. Inhibition of this pathway blocks KSHV lytic replication. These results illustrate a mechanism by which KSHV hijacks a cellular pathway for its replication, and they identify a potential therapeutic target. V iruses depend on cell signaling pathways for successful infection and replication. Identification of pathways hijacked by viruses not only reveals the mechanisms of infection and replication of these viruses but also provides novel therapeutic targets. Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus etiologically associated with Kaposi's sarcoma (KS), a vascular tumor of endothelial cells commonly found in AIDS patients, and with two B-cell lymphoproliferative diseases, namely, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD) (1-3). The early phase of primary KSHV infection is a highly regulated multistep event consisting of virion attachment and binding, membrane fusion, internalization, intracellular trafficking, and early viral gene expression (4). KSHV infection induces phosphorylation of cellular proteins, leading to the activation of signal transduction pathways. A number of these pathways regulate KSHV entry, trafficking, and viral gene expression (4). Binding of KSHV glycoproteins to cellular receptors activates focal adhesion kinase (FAK), Src, phosphatidylinositol-3-kinases (PI3Ks), and mitogen-activated protein kinases (MAPKs), including MEK/extracellular signal-regulated kinase (MEK/ERK), p38, and Jun N-terminal kinase (JNK), facilitating KSHV internalization and trafficking (5-10). This process depends on rearrangements of the actin and microtubule cytoskeletons and on factors, such as Rho-GTPases and diaphanous-2 (Dia-2), that regulate their dynamics (8, 11). KSHV entry and trafficking rely on the dynamics of the ubiquitin/proteasome system and on activation of the E3 ligase c-Cbl to maintain the endosomal activities and cellular signaling (12, 13).Successful KSHV infection requires the coordinated expres-