Background:  Entecavir (ETV) is a potent antiviral drug that is used to treat infection with the hepatitis B virus (HBV). Recent studies in humans have shown that ETV has anti-HIV activity and can select for the M184V mutation in HIV-1 reverse transcriptase (RT), which limits its clinical use in HIV/HBV co-infected individuals. However, the mechanism of drug action remains elusive. ETV is a guanosine nucleoside analogue that contains a 3’-hydroxyl group. Thus, the drug may exert its inhibitory effects at a certain point following its incorporation and/or later during synthesis of the second DNA strand when ETV-5’-monophpsphate (ETV-MP) is part of the template. 

Methods:  We utilized site-specific footprinting tools in combination with enzyme kinetics and binding studies to elucidate the anti-HIV mechanism of ETV.

Results:  Incorporation of ETV-MP at position n causes strong pausing at positions n and n+3. Increasing concentrations of natural dNTP pools at positions n+1 and n+4 can overcome pausing, which points to an immediate effect of ETV-MP on rates of incorporation of the next nucleotide and later at n+4. Steady-state kinetic measurements revealed an 8-fold decrease in efficiency of nucleotide incorporation at position n+1 when ETV-terminated primers are compared with the natural counterpart. Site-specific footprinting experiments show that the incorporation of ETV-MP prevents RT translocation. As a result, the complex remains trapped in its pre-translocational conformation in which the nucleotide binding site is occluded. A steric clash with the highly conserved Y183 residue in the post-translocational state helps to explain these findings. Despite the clear bias toward pre-translocation, excision of ETV-MP in the presence of PPi or ATP is insignificant with wild type RT. In addition to the specific inhibitory effects of ETV during synthesis of the first DNA strand, this drug also diminishes rates of incorporation at position n+1 when present in the template. Steric clashes between the exocyclic double bond of ETV and the sugar moiety of the adjacent nucleotide provide a plausible explanation.  

Conclusions:  The results of this study delineate 3 interlinked mechanisms of inhibition of HIV-1 RT by ETV. DNA synthesis is compromised at positions n+1 and n+4, and, during synthesis of the second DNA strand, at position n+1. The combined data provide a rational for mechanism-based approaches in the development of more potent non-obligate chain-terminators.