Identification of cellular deoxyhypusine synthase as a novel target for antiretroviral therapy
Ilona Hauber, Dorian Bevec, Jochen Heukeshoven, Friedrich Krätzer, Florian Horn, Axel Choidas, Thomas Harrer, Joachim Hauber
Ilona Hauber, Dorian Bevec, Jochen Heukeshoven, Friedrich Krätzer, Florian Horn, Axel Choidas, Thomas Harrer, Joachim Hauber
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Article AIDS/HIV

Identification of cellular deoxyhypusine synthase as a novel target for antiretroviral therapy

Abstract

The introduction of highly active antiretroviral therapy (HAART) has significantly decreased morbidity and mortality among patients infected with HIV-1. However, HIV-1 can acquire resistance against all currently available antiretroviral drugs targeting viral reverse transcriptase, protease, and gp41. Moreover, in a growing number of patients, the development of multidrug-resistant viruses compromises HAART efficacy and limits therapeutic options. Therefore, it is an ongoing task to develop new drugs and to identify new targets for antiretroviral therapy. Here, we identified the guanylhydrazone CNI-1493 as an efficient inhibitor of human deoxyhypusine synthase (DHS). By inhibiting DHS, this compound suppresses hypusine formation and, thereby, activation of eukaryotic initiation factor 5A (eIF-5A), a cellular cofactor of the HIV-1 Rev regulatory protein. We demonstrate that inhibition of DHS by CNI-1493 or RNA interference efficiently suppressed the retroviral replication cycle in cell culture and primary cells. We show that CNI-1493 inhibits replication of macrophage- and T cell–tropic laboratory strains, clinical isolates, and viral strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Moreover, no measurable drug-induced adverse effects on cell cycle transition, apoptosis, and general cytotoxicity were observed. Therefore, human DHS represents a novel and promising drug target for the development of advanced antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.

Authors

Ilona Hauber, Dorian Bevec, Jochen Heukeshoven, Friedrich Krätzer, Florian Horn, Axel Choidas, Thomas Harrer, Joachim Hauber

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CNI-1493 negatively affects HIV-1 Rev trans-activation. (A) Inhibition o...
CNI-1493 negatively affects HIV-1 Rev trans-activation. (A) Inhibition of cytoplasmic accumulation of incompletely spliced viral mRNAs. PM1 cells were infected with HIV-1 BaL and subsequently cultured in the presence of either CNI-1493 (0.8 μM) or DMSO. Cytoplasmic (C) or total (T) RNA was isolated at day 7 after infection and subjected to HIV-1 mRNA–specific or U6 snRNA–specific Northern analysis. (B) Inhibition of Rev-dependent virus production. HeLa cells were cultured for 7 days in 1.0 μM CNI-1493 or DMSO and subsequently cotransfected with a rev-defective proviral DNA, a Rev-expressing vector, or the respective parental plasmid (Rev-deficient control) and a construct expressing constitutively secreted alkaline phosphatase (SEAP). The transfected cell monolayers were further maintained in 1.0 μM CNI-1493 or DMSO and analyzed at 60 hours after transfection for levels of p24 antigen. The respective values were adjusted for transfection efficiency by determination of the level of SEAP (upper panel). In addition, total cell protein extracts were prepared and subjected to p55Gag-specific or α-tubulin–specific (protein loading control) Western analysis (lower panel). (C) Analysis of Rev trans-activation. HeLa cells were cultured in CNI-1493 or DMSO as before and subsequently cotransfected with the Rev-responsive reporter plasmid pCMVgagLucRRE, a Rev-expressing vector, or the respective parental plasmid (Rev-deficient control) and the constitutive internal control vector pBC12/CMV/βGal. (D) Analysis of Tat trans-activation. HeLa cells were cultured in CNI-1493 or DMSO as before and subsequently cotransfected with the Tat-responsive reporter plasmid pBC12/HIV/CAT, a Tat-expressing vector, or the respective parental plasmid (Tat-deficient control) and the constitutive internal control vector pBC12/CMV/βGal.