Background:  Phosphorodiamidate morpholino oligomers (PMO) are nuclease-resistant, water-soluble antisense oligonucleotide analogs that act by sterically blocking complementary RNA sequences. PMO have recently been reported to be effective against several families of RNA viruses and therefore represent a prospective new class of anti-HIV compounds. To evaluate their potential 2 targets were selected:  the highly conserved start codon region of the HIV-1 Vif gene and; the Tar stem-loop. Knockdown of Vif gene expression is expected to produce virions that incorporate APOBEC3. Steric blocking of the Tar stem-loop is predicted to inhibit both transcription and translation of viral mRNA. Both classes of PMO were evaluated using cell reporter genes and/or viral replication in tissue culture.

Methods:  A series of overlapping PMO that target the HIV and feline immunodeficiency virus (FIV) Vif start codon regions were synthesized and evaluated in cell-free translation (CFT) assays. A rank order of efficacy was determined and the most potent PMO were synthesized as peptide-conjugated PMO (P-PMO) for cell culture studies. For HIV experiments, H9 cells were infected with pNL4-3 cloned viral stock at an multiplicity of infection of 0.001. Two hours post-infection, P-PMO were added at concentrations from 10 to 5000 nM. Viral replication was determined 5 and 7 days post-infection by p24 antigen capture ELISA. FIV experiments were performed similarly using MCH5-4 cells and pPPR-derived viral stocks. Inhibition of HIV transcription using P-PMO targeted to the HIV Tar stem-loop was measured using the HeLa-Tat-III/LTR/d1EGFP cell line.

Results:  CFT assays of HIV Vif-luc reporter constructs identified an efficacious P-PMO that selectively inhibits viral replication in H9 cells with an EC₅₀ of 260±40 nM. Inhibition of CFT of FIV Vif-luc constructs has produced similar candidate PMO. Tat-induced reporter gene expression in cell culture was inhibited by P-PMO targeting the HIV Tar stem-loop with an EC₅₀ of 3.6 mM. P-PMO targeted to the 5’terminal stem-loop of FIV inhibited viral replication in MCH5-4 cells with an EC₅₀ of 1.3 mM.

Conclusions:  The highly conserved region surrounding the HIV Vif start codon and the importance of Vif to viral infectivity suggest an excellent PMO target. PMO compounds that target Vif and the Tar stem-loop may be candidates for further investigation in animal model systems such as FIV. Studies with P-PMO designed against these targets have shown promise as effective antiretroviral compounds.