Structural features in common of HBV and HIV-1 resistance against chirally-distinct nucleoside analogues entecavir and lamivudine

Abstract

Chronic hepatitis B virus (HBV) infection is a major public health problem that affects millions of people worldwide. Nucleoside analogue reverse transcriptase (RT) inhibitors, such as entecavir (ETV) and lamivudine (3TC), serve as crucial anti-HBV drugs. However, structural studies of HBV RT have been hampered due to its unexpectedly poor solubility. Here, we show that human immunodeficiency virus type-1 (HIV-1) with HBV-associated amino acid substitutions Y115F/F116Y/Q151M in its RT (HiV Y115F/F116Y/Q151M) is highly susceptible to ETV and 3TC. Additionally, we experimentally simulated previously reported ETV/3TC resistance for HBV using HIV Y115F/F116Y/Q151M with F160M/M184V (L180M/ M204V in HBV RT) substituted. We determined crystal structures for HIV-1 RT Y115F/F116Y/Q151M :DNA complexed with 3TC-triphosphate (3TC-TP)/ETV-triphosphate (ETV-TP)/dCTP/dGTP. These structures revealed an atypically tight binding conformation of 3TC-TP, where the Met184 side-chain is pushed away by the oxathiolane of 3TC-TP and exocyclic methylene of ETV-TP. Structural analysis of Rt Y115F/F116Y/Q151M/F160M/M184V :DNA:3TC-TP also demonstrated that the loosely bound 3TC-TP is misaligned at the active site to prevent a steric clash with the side chain γ-methyl of Val184. These findings shed light on the common structural mechanism of HBV and HIV-1 resistance to 3TC and ETV and should aid in the design of new agents to overcome drug resistance to 3TC and ETV. Hepatitis B virus (HBV) is a major pathogen causing human liver diseases such as chronic hepatitis B, liver cirrhosis, and hepatocellular carcinoma 1 , and affects approximately 250 million worldwide, resulting in nearly 1 million deaths per year 2. Although HBV is a DNA virus comprised of a compact 3.2 kb genome, the polymerase (Pol) gene that spans three-quarters of the HBV genome contains reverse transcriptase (RT) and is essential for viral replication, as HBV replicates via a pre-genomic RNA intermediate 3,4. HBV Pol is a multifunctional protein with molecular weight of ~90 kDa, and is composed of four distinct domains: terminal protein (TP), spacer, RT, and ribonuclease H (RH). Duck HBV Pol has provided a model to study a unique reverse transcription initiation mechanism whereby HBV Pol binds to a signal sequence termed ε on the template pre-genomic RNA. Reverse transcription is then initiated through a conserved Tyr residue in the TP domain acting as the sole protein primer 5,6. The resultant covalently-linked ribonucleoprotein is simultaneously packaged with HBV core protein to form nucleocapsid core particles. Recombinant expression attempts to obtain large amounts of HBV Pol protein for structural studies have not been successful due to the insolubility of HBV Pol 7. In fact, HBV Pol is considered to be stably folded and enzymatically active only within the nucleocapsid core particle 8. Fortunately, the RT/RH domains of HBV Pol exhibit weak sequence homology with human immunodeficiency virus type-1 (HIV-1) RT. In particular, a consensus sequence present across multiple ...

Selection of Primer–Template Sequences That Bind with Enhanced Affinity to Vaccinia Virus E9 DNA Polymerase

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