Abstract No virus encodes a ribosome, thus all viruses require the host translation machinery for the synthesis of viral proteins. This fundamental host:pathogen interface creates competition for the host translation machinery, which is a critical barrier to human cytomegalovirus (HCMV) mRNA translation. Many viruses prevent competition for the translation machinery by inhibiting host translation, however host translation is maintained during HCMV infection. How HCMV mRNAs successfully compete for access to host translation machinery to ensure efficient viral protein synthesis is poorly understood, highlighting a critical gap in our knowledge of the mechanisms controlling HCMV mRNA translation. How might HCMV mRNAs compete with host mRNAs for access to ribosomes? Based on our data we hypothesize that HCMV avoids competition by using a unique complement of host and viral translation initiation factors to facilitate viral mRNA translation. Formation of the eIF4F translation initiation complex is the limiting step in host mRNA translation, thus competition between host and viral mRNAs would be most intense for eIF4F components. We showed that the eIF4F complex is necessary for the continued translation of host mRNAs during infection; however HCMV mRNAs translate efficiently when the eIF4F complex is disrupted. We found that translation of the HCMV IE1 and IE2 mRNAs, which encode critical regulators of virus replication, is resistant to eIF4F disruption, depletion or inhibition. How HCMV mRNAs translate in the absence of eIF4F is unknown. As translation initiation factors assemble on the mRNA 5' untranslated region (5'UTR), in Aim1 we determine how RNA sequence and structure in the shared 5'UTR of the IE1 and IE2 mRNAs recruit cellular factors to allow for their eIF4F-independent translation. We also determine how these factors contribute to the widespread eIF4F-independent translation of HCMV mRNAs as a whole. Aim 2 builds on our published results suggesting that the HCMV TRS1 protein (pTRS1) acts as a viral translation initiation factor to facilitate viral protein synthesis. While pTRS1 antagonizes the antiviral kinase PKR, which inhibits protein synthesis, we showed that pTRS1 stimulates translation in PKR deficient cells. Further, we found that pTRS1 binds the mRNA cap independent of the eIF4F complex and co- purifies with 40S ribosomal subunits, suggesting a role in translation initiation. We now show that pTRS1 interacts with the DHX29 RNA helicase, a translation initiation factor that can recruit ribosomes to mRNAs in the absence of the eIF4F complex, and that DHX29 is necessary for efficient IE1/2 mRNA translation and virus replication. In Aim 2 we role of pTRS1 and DHX29 in translation initiation complex on HCMV mRNAs, and the role of their interaction in the ability of pTRS1 to enhance translation independent of PKR antagonism. We further determine how pTRS1 binding to DHX29 impacts virus replication and the translation of HCMV mRNAs in the...