INSIGHTS INTO THE IN VITRO REPLICATION OF THE LAMIVUDINE-RESISTANT MUTANT, RTM204I, USING THE HBV RECOMBINANT BACULOVIRUS SYSTEM IN HEPG2 CELLS
Open Access
- Author:
- Heipertz Jr, Richard Arthur
- Graduate Program:
- Microbiology and Immunology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 14, 2008
- Committee Members:
- Harriet C Isom, Dissertation Advisor/Co-Advisor
Harriet C Isom, Committee Chair/Co-Chair
Richard James Courtney, Committee Member
David Joseph Spector, Committee Member
Jianming Hu, Committee Member
Kristin Ann Eckert, Committee Member - Keywords:
- in vitro replication
HBV
rtM204I mutant
lamivudine-resistant - Abstract:
- Hepatitis B virus (HBV) causes acute and chronic infections of the liver and is responsible for 1.2 million deaths annually. Approximately 0.5% of acute infections terminate in fatal, fulminant hepatitis. The WHO currently estimates that 350-400 million people are chronically infected with HBV. In the United States of America, approximately 12 million people have been infected, more than 1.25 million chronically infected, with 5,000 deaths per year caused by HBV and its complications (Hepatitis B Foundation). The primary treatment for chronic HBV is lamivudine. The major clinical limitation of lamivudine is the emergence of drug-resistant mutants. The most common mutations occur in the highly conserved tyrosine-methionine-aspartate-aspartate (YMDD) motif in the catalytic domain of the HBV polymerase gene, resulting in substitutions of methionine to either valine or isoleucine at amino acid 204. Another common mutation arising during chronic HBV infection develops in the precore/core region of the HBV genome. Precore-negative chronic hepatitis B is the most prevalent form of chronic HBV in many parts of the world. Understanding the consequences of mutation in the hepatitis B virus genome on HBV replication is critical for treating chronic HBV infection. Initial studies of this Dissertation focus on the replication of the precore (PC) and lamivudine-resistant rtM204I mutant of HBV. Previously, others have performed in vitro studies on the PC mutant, reporting conflicting results with some suggesting that the PC mutant has an inherent replication advantage compared to wild-type (WT) HBV. The pattern and magnitude of HBV replication when initiated by a PC HBV recombinant baculovirus were similar to what was observed for WT HBV throughout the time course examined. Therefore, more extensive studies with the PC mutant were not carried out. During the first five days post-transduction, the rtM204I mutant replicates to levels comparable to WT HBV, in contrast to previous reports suggesting that the rtM204I mutant has a severe defect in replication compared to WT HBV. However, by day 10 post-transduction, rtM204I levels of intra- and extracellular HBV DNA were markedly reduced compared to WT HBV. Although the rtM204I mutation reduced production of HBV replicative intermediates, no effect on the levels of covalently closed circular DNA or HBV transcripts was observed at late time points. Cotransduction studies with different ratios of WT and rtM204I baculovirus show that rtM204I does not produce a product that inhibited HBV replication. However, the combination of WT and rtM204I yielded HBV DNA levels at late time points greater than WT alone, suggesting that WT polymerase may function in trans to boost rtM204I replication. Collectively, the data demonstrate that the rtM204I mutation generates a polymerase that is not only resistant to lamivudine but also replicates nucleic acids to lower levels in vitro at late time points in the recombinant baculovirus system when replication is driven primarily from CCC DNA. Recombinant baculoviruses expressing the WT hepatitis B virus polymerase were utilized to determine if the WT polymerase could correct the defect in reverse transcription at late time points post-transduction of rtM204I. Expression of a functional WT polymerase was confirmed through the restoration of replication of a polymerase-minus HBV mutant. WT polymerase can indeed restore rtM204I replicative intermediates and extracellular DNA levels without affecting the level of covalently closed circular DNA. Analysis of the genetically marked viral genomes verified that this restoration resulted from trans-complementation, rather than recombination. Furthermore, WT polymerase can function in trans to complement viral replication regardless of whether the RNA packaging signal, epsilon, which is required to activate the polymerase, was provided in cis. In contrast, the enhanced levels of total HBV RI observed when HepG2 cells were cotransduced with baculoviruses expressing the WT and rtM204I genomes was not due to trans-complementation of rtM204I by the WT polymerase. The data indicate that HBV polymerase can function in trans, without the requirement of the epsilon RNA in cis, in either RNA packaging or DNA synthesis in vivo. WT polymerase also has the capacity to trans-complement the replication defect of the polymerase mutant rtM204I; however, trans-complementation may require polymerase over-expression from a packaging-defective RNA. In contrast, when expressed from an authentic pregenomic RNA, as in a mixed infection, polymerase may not trans-complement efficiently. To continue the extensive analysis to identify where the block in replication of rtM204I occurs, the next step tested was RNA encapsidation. However, encapsidation has not been characterized in the HBV recombinant baculovirus system. Detectable levels of encapsidation by Southern blot first becomes visible approximately 10h post-transduction, with HBV SS DNA arising by 14h, RC/DS DNA by 24h, and EC DNA by 48h. Once encapsidation was characterized, WT and rtM204I levels of RNA encapsidation were measured. It is clear that the block in rtM204I replication does not occur at the level of RNA encapsidation, suggesting that the defect is either at the step of reverse transcription or DNA-to-DNA synthesis. Additionally, partial restoration of rtM204I replication by the WT polymerase construct, PCFE, does not cause an increase in RNA encapsidation compared to rtM204I alone, suggesting that more than one polymerase molecule may be packaged within a nucleocapsid, at least when excess WT polymerase is over-expressed from a packaging-defective RNA during trans-complementation of a drug-resistant mutant. To begin to directly compare the function of the WT and rtM204I polymerases, the endogenous polymerase reaction was used. The rtM204I polymerase does not show increased activity when dNTP concentrations exceed 20μM, in contrast to a previous report. The rtM204I polymerase appears to be functionally hindered and unaffected by dNTP concentrations, although the effect of increasing dNTP concentrations in vivo still needs to be tested. Collectively, the research described within this Dissertation narrows down the step where the defect in rtM204I replication occurs. That is, at the level of reverse transcription or DNA-to-DNA synthesis. Results generated from trans-complementation of rtM204I by a WT polymerase demonstrate that replication of rtM204I can be partially restored. In addition, results suggest that more than one polymerase molecule may be packaged within a nucleocapsid under certain conditions. Finally, the rtM204I polymerase appears to also be defective in the in vitro endogenous polymerase reaction.