Inhibition of Hepadnavirus Reverse Transcriptase-ε RNA Interaction by Porphyrin Compounds

Abstract

The hepatitis B virus (HBV) reverse transcriptase (RT) plays a multitude of fundamental roles in the viral life cycle and is the key target in the development of anti-HBV chemotherapy. We report here that the endogenous small molecule iron protoporphyrin IX (hemin) and several related porphyrin compounds potently blocked a critical RT interaction with the viral RNA packaging signal/origin of replication, called . As RTinteraction is essential for the initiation of viral reverse transcription, which is primed by RT itself (protein priming), the porphyrin compounds dramatically suppressed the protein-priming reaction. Further studies demonstrated that these compounds could target the unique N-terminal domain of the RT protein, the so-called terminal protein. Hemin and related porphyrin compounds thus represent a novel class of agents that can block HBV RT functions through a mechanism and target that are completely distinct from those of existing anti-HBV drugs.Hepatitis B virus (HBV) belongs to the family Hepadnaviridae, a group of small, hepatotropic DNA viruses that also includes related animal viruses, such as the duck HBV (DHBV) and the woodchuck hepatitis virus (31). Chronic HBV infection remains a major public health problem worldwide, with over 350 million chronic HBV carriers who are at serious risk of developing liver cirrhosis and hepatocellular carcinoma (5,7,12). At present, the immunomodulatory and antiviral cytokine alpha interferon and several nucleoside analogs represent two distinct classes of therapies for chronic HBV infections (14). Unfortunately, alpha interferon therapy induces a sustained antiviral response in only 20 to 30% of patients and is fraught with adverse reactions. In the short term, nucleoside analogs exhibit a potent inhibitory effect on HBV DNA synthesis. However, long-term treatment with nucleoside analogs is required to maintain viral suppression, which leads to the development of drug-resistant HBV variants (13,26). Therefore, the current inventory of therapeutics against HBV infections is inadequate, and novel anti-HBV therapy needs to be developed.All hepadnaviruses replicate the DNA genome through an RNA intermediate (the pregenomic RNA [pgRNA]) by reverse transcription (31, 33). The virally encoded reverse transcriptase (RT) protein carries the two essential enzymatic activities required for the conversion of pgRNA to the DNA copy, i.e., DNA polymerase activity and RNase H activity (18). In contrast to retroviral RTs, the hepadnavirus RT initiates viral DNA synthesis via a novel protein-priming mechanism, whereby the RT protein itself serves a dual role as a protein primer and as the polymerase. As an obligate template for the protein-priming action, a short (ca. 60-nucleotide) viral RNA structure termed ε, located at the 5Ј end of pgRNA, is specifically recognized by the RT protein to form a stable ribonucleoprotein (RNP) complex (28, 38). Using an internal bulge located on the ε stem-loop structure as a template and a specific tyrosine residue of the RT protein as a pr...

Carbonyl J Acid Derivatives Block Protein Priming of Hepadnaviral P Protein and DNA-Dependent DNA Synthesis Activity of Hepadnaviral Nucleocapsids

“…Several of our inhibition data for KM-1 resemble those reported for selected porphine derivatives (28). Hence, the tetrapyrrole and naphthylurea derivatives may have similar inhibition mechanisms.…”

“…4A, bottom). A reduced inhibitory activity in RRL has also been reported for hemin (28). Possibly, the high concentration of proteins and other factors in RRL reduced Grp-DP, a GrpE fusion protein lacking just the spacer and the C-terminal 25 aa of DHBV P protein, was activated by using Hsc70/Hsp40 and ATP as described previously (46).…”

Functional and Structural Dynamics of Hepadnavirus Reverse Transcriptase during Protein-Primed Initiation of Reverse Transcription: Effects of Metal Ions

“…pSP64pA-DP expresses the full-length DHBV RT from the SP6 promoter in the pSP64pA vector. pGEX-TP and pGEX-RT express the DHBV TP (residues 75 to 220) and DHBV RT (residues 349 to 575) domain sequences, respectively, fused to GST, and were derived from pGEX-MiniRT2 by removing the RT and TP sequences, respectively (33).…”

“…The protein priming assay was carried out as previously described (20,24,33,55) with minor modifications. Briefly, the following components were added to a 10-l reaction mixture: GST-MiniRT2 (ca.…”

Analysis of hepatitis B virus drug-resistant mutation (M204V) using molecular dynamics simulation techniques

“…The virally encoded reverse transcriptase (RT) contains initiation and elongation activity, helps in the reverse transcription of the double-stranded DNA genome of hepatitis B from the viral pregenome (Seifer & Standring 1993). Since the HBV RT plays an important role in the viral life cycle, antiviral drugs are developed to target the RT activity of the HDP (Lin & Hu 2008).…”