AV Congress – Boston USA, November 2012
CADA, a Specific Down-Modulator of the Primary HIV Receptor, Acts as a Small-Molecule
Inhibitor of CD4 Protein Translocation into the Endoplasmic Reticulum
Anne Giraut1, Dominique Schols1, Thomas W. Bell2 and Kurt Vermeire1
1
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium and
Chemistry, University of Nevada, Reno, USA.
2
Department of
Introduction : CD4 is a crucial component of the cellular multi-receptor complex for HIV-entry. Thus,
regulating CD4 expression can be an important target for antiviral intervention. Previously, we
identified cyclotriazadisulfonamide (CADA) as a small-molecule that down-modulates CD4 expression
in immune cells and inhibits HIV entry. Recently, we have discovered that CADA inhibits the synthesis
of human CD4 protein by disrupting translocation across the ER membrane in a signal peptidedependent manner. Signal peptides (SP) consist in 20 to 40 amino acid residues located at the Nterminal extremity of nascent polypeptides and are crucial for proper translocation and posttranslational modifications of proteins and receptors.
Methods: A yellow-fluorescent protein (YFP)-coding construct containing the signal sequence of
human CD4 was generated, allowing easy flow cytometric quantification of protein expression after
transfection. Via site-directed mutagenesis, single residues of the SP were evaluated in an alaninescan.
Results: Three characteristic domains of the SP of CD4 were investigated by exchanging the domains
between CADA-susceptible human CD4 and non-susceptible mouse CD4. Our data demonstrate that
the hydrophobic alpha-helix of the SP of CD4 is the primary domain for CADA-interaction. Next, we
uncovered several critical residues within this hydrophobic mid-region of the SP that are crucial for
the drug-induced inhibition of co-translational translocation of CD4. In addition to the SP, a few
residues in the NH2-terminal domain of the mature protein also affect the potency of CADA.
Discussion: We gained significant information on the interaction of CADA with specific residues in the
SP of CD4. Further study of SP is essential to understand the regulation of the biosynthesis of
proteins which may help in the development of new antiviral strategies that act (i) on the viral level
by inhibiting the synthesis of viral proteins and (ii) on the host level by down-modulation of receptor
proteins utilized during viral entry.