GAILLARD RK, BARNARD J, LOPEZ V, HODGES P, BOURNE E, JOHNSON L, ALLEN MI, CONDREAY P, MILLER WH, CONDREAY LD; Interscience Conference on Antimicrobial Agents and Chemotherapy.
Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 1999 Sep 26-29; 39: 446 (abstract no. 2056).
GlaxoWellcome Inc., RTP, NC.
Lamivudine [(-)2'-deoxy-3'-thiacytidine, LAM, Epiver[TM]-HBV[TM]] is an approved antiviral agent for the treatment of hepatitis B infection. Variant HBV species containing mutations in the YMDD motif of the viral polymerase emerge in some patients during therapy and decrease the sensitivity of the virus to LAM in vitro. Clinical evidence suggests that this YMDD variant HBV has a reduced 'fitness'. While emergence of YMDD variant HBV is associated with reduced therapeutic response in some patients, serum HBV DNA and ALT levels remain below baseline levels in many of these patients. Also, when therapy is stopped in patients with YMDD variant HBV, wt HBV reappears in the serum and levels of YMDD variant HBV decline. To understand this phenomenon, we evaluated selected kinetic parameters of the polymerase within viral core particles isolated from HepG2 cells transfected with plasmids containing wt and each of the following variant constructs, L528M/M552V, L528M/ M552I, M552I, L528M, and M552V. Although the turnover rate for wt and variant polymerase was the same, the Km's of the variants for the natural dNTP's were markedly different. The variant Km's ranged from 1.3 to 6.4x wt Km depending on the dNTP and AA change(s). Variants with the greatest increase in Km from wt (M552I and M552V) were the most replication deficient and produced the least amount of extra cellular virions and intracellular core particles. Radiolabeled products of in vitro polymerase reactions revealed that variants with the highest Km's produced a higher percentage of first strand synthesis, indicating more immature particles. These results indicate that the decreased replication of the variants may be, in part, due to inefficient use of the natural dNTP substrates by the variant polymerases. Preliminary evidence indicates that in HepG2 cells dNTP pool sizes are in the 1 to 2 uM range, a range in which the wt enzyme will approach the maximum rate of polymerization while the variants will b e slower. This shift in rate of elongation could explain the apparent replication defect in the emerging YMDD variants and reduction in their fitness.
Publication Types:
Keywords:
- Alanine Transaminase
- Antiviral Agents
- Gene Products, pol
- Hepatitis B
- Hepatitis B Antibodies
- Hepatitis B Surface Antigens
- Hepatitis B e Antigens
- Hepatitis B virus
- Humans
- In Vitro
- Kinetics
- Lamivudine
- Mutation
- P protein, Hepatitis B virus
- Plasmids
- analysis
- genetics
- immunology
Other ID:
UI: 102245667
From Meeting Abstracts