Replication of the human immunodeficiency virus type 1 (HIV-1) is dependent on eIF5A hypusination. Hypusine is formed post-translationally on the eIF5A precursor by two consecutive enzymatic steps; a reversible reaction involving the enzyme deoxyhypusine synthase (DHS) and an irreversible step involving the enzyme deoxyhypusine hydroxylase (DOHH). In this study we explored the effect of inhibiting DOHH activity and therefore eIF5A hypusination, on HIV-1 gene expression. Results show that the expression of proteins from an HIV-1 molecular clone is reduced when DOHH activity is inhibited by Deferiprone (DFP) or Ciclopirox (CPX). Next we evaluated the requirement of DOHH activity for internal ribosome entry site (IRES)-mediated translation initiation driven by the 5'untranslated region (5′UTR) of the full length HIV-1 mRNA. Results show that HIV-1 IRES activity relies on DOHH protein concentration and enzymatic activity. Similar results were obtained for IRES-dependent translation initiation mediated by 5′UTR of the human T-cell lymphotropic virus type 1 (HTLV-1) and the mouse mammary tumor virus (MMTV) mRNAs. Interestingly, activity of the poliovirus IRES, was less sensitive to the targeting of DOHH suggesting that not all viral IRESs are equally dependent on the cellular concentration or the activity of DOHH. In summary we present evidence indicating that the cellular concentration of DOHH and its enzymatic activity play a role in HIV-1, HTLV-1 and MMTV IRES-mediated translation initiation.