Entecavir (ETV) exhibits potent antiviral activity in patients chronically infected with wild-type or lamivudine (3TC)-resistant (3TC r ) hepatitis B virus (HBV). Among the patients treated in phase II ETV clinical trials, two patients for whom previous therapies had failed exhibited virologic breakthrough while on ETV.Isolates from these patients (arbitrarily designated patients A and B) were analyzed genotypically for emergent substitutions in HBV reverse transcriptase (RT) and phenotypically for reduced susceptibility in cultures and in HBV polymerase assays. After 54 weeks of 3TC therapy, patient A (AI463901-A) received 0.5 mg of ETV for 52 weeks followed by a combination of ETV and 100 mg of 3TC for 89 weeks. Viral rebound occurred at 133 weeks after ETV was started. The 3TC r RT substitutions rtV173L, rtL180M, and rtM204V were present at study entry, and the additional substitutions rtI169T and rtM250V emerged during ETV-3TC combination treatment. Reduced ETV susceptibility in vitro required the rtM250V substitution in addition to the 3TC r substitutions. For liver transplant patient B (AI463015-B), previous famciclovir, ganciclovir, foscarnet, and 3TC therapies had failed, and RT changes rtS78S/T, rtV173L, rtL180M, rtT184S, and rtM204V were present at study entry. Viral rebound occurred after 76 weeks of therapy with ETV at 1.0 mg, with the emergence of rtT184G, rtI169T, and rtS202I substitutions within the preexisting 3TC r background. Reduced susceptibility in vitro was highest when both the rtT184G and the rtS202I changes were combined with the 3TC r substitutions. In summary, infrequent ETV resistance can emerge during prolonged therapy, with selection of additional RT substitutions within a 3TC r HBV background, leading to reduced ETV susceptibility and treatment failure.Nearly 400 million people are chronically infected with hepatitis B virus (HBV) worldwide (16,19). After prolonged infections, often lasting decades, patients frequently develop severe liver disease that can lead to cirrhosis and hepatocellular carcinoma. Chronically infected patients also serve as sources of HBV transmission. There are currently three approved therapies for chronic HBV infections: interferon, lamivudine (3TC; ␤-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine), and adefovir-dipivoxil, the prodrug of adefovir [ADV; 9-(2-phosphonylmethoxyethyl)adenine] (18). Interferon is administered subcutaneously and is associated with numerous adverse events, some of which can be severe, and a sustained antiviral response in only 30 to 40% of treated patients (30). 3TC treatment, administered orally, is effective in reducing viral loads but results in the frequent emergence of drug-resistant HBV due to substitutions at the Tyr-Met-Asp-Asp (YMDD) nucleotide binding site motif of viral DNA polymerase. Data from four large clinical trials revealed 3TC resistance (3TC r ) mutations in 24, 42, 53, and 70% of patients after 1, 2, 3, and 4 years of therapy, respectively (15). Treatment with ADV, recently approved by the U.S. Food and Drug Administration,...

Apoptotic stimuli activate and oligomerize the proapoptotic proteins Bak and Bax, resulting in mitochondrial outer-membrane permeabilization and subsequent cell death. This activation can occur when certain BH3-only proteins interact directly with Bak and Bax. Recently published crystal structures reveal that Bax separates into core and latch domains in response to BH3 peptides. The distinguishing characteristics of BH3 peptides capable of directly activating Bax were also elucidated. Here we identify specific BH3 peptides capable of "unlatching" Bak and describe structural insights into Bak activation and oligomerization. Crystal structures and crosslinking experiments demonstrate that Bak undergoes a conformational change similar to that of Bax upon activation. A structure of the Bak core domain dimer provides a high-resolution image of this key intermediate in the pore-forming oligomer. Our results confirm an analogous mechanism for activation and dimerization of Bak and Bax in response to certain BH3 peptides.

The Bcl-2 family of proteins plays a central role in the control of apoptosis via the intrinsic pathway (1). The family is composed of two opposing groups, one promoting cell survival (comprising Bcl-2, Bcl-x L , Bcl-w, Mcl-1, and A1, each containing up to four Bcl-2 homology (BH) 3 domains) and another promoting cell death. This latter group includes Bax and Bak, proteins similar in sequence and structure to the pro-survival family members, and the so-called BH3-only proteins. In response to stress or damage signals, BH3-only proteins are up-regulated either transcriptionally or post-translationally, or both. Apoptosis results when the pro-survival proteins are overwhelmed and Bax/Bak undergo a conformational change at the mitochondrial membrane. Apoptogenic factors such as cytochrome c are then released from the mitochondrial intramembrane space into the cytosol, resulting in the activation of caspases.Many biochemical, structural, and cellular studies support an interaction between pro-survival proteins and both subgroups of pro-apoptotic proteins. The helical BH3 domain of pro-apoptotic proteins engages a cognate groove on the prosurvival proteins (2-5). Four hydrophobic amino acids from the BH3 domain insert into pockets in the groove, and a conserved aspartyl residue in the BH3 domain is hydrogen-bonded with a conserved arginyl residue on the pro-survival protein.Although the BH3-only proteins are mostly unstructured in solution (6), Bak and Bax have well characterized globular structures (7,8), remarkably similar to the pro-survival proteins, in which the BH3 domain is located within an amphipathic helix (␣2) with its hydrophobic face buried. Thus Bak and Bax cannot bind to pro-survival proteins without first everting their BH3 domains. The natural trigger for the exposure of the BH3 domain of Bak/Bax is not known, but it could be a chemical stimulus such as H 2 O 2 (9), a physical stimulus such as heat (10), or an interaction between Bak/Bax and a BH3-only protein that is up-regulated in response to cellular stresses. Some evidence supports such interactions, at least between a subset of the BH3-only proteins and Bax/Bak (11-15). The isolation of complexes of this type has proven elusive, but a recent NMR study suggests the BH3 domain of the BH3-only protein Bim binds Bax on the opposite face as compared with where a BH3 peptide binds a pro-survival protein (16). It has been proposed that this interaction triggers a structural rearrangement in Bax or Bak into large homo-oligomeric complexes at the mitochondrial membrane. Other data support a model in

The BH3-only protein Bim is a potent direct activator of the proapoptotic effector protein Bax, but the structural basis for its activity has remained poorly defined. Here we describe the crystal structure of the BimBH3 peptide bound to BaxΔC26 and structure-based mutagenesis studies. Similar to BidBH3, the BimBH3 peptide binds into the cognate surface groove of Bax using the conserved hydrophobic BH3 residues h1–h4. However, the structure and mutagenesis data show that Bim is less reliant compared with Bid on its ‘h0' residues for activating Bax and that a single amino-acid difference between Bim and Bid encodes a fivefold difference in Bax-binding potency. Similar to the structures of BidBH3 and BaxBH3 bound to BaxΔC21, the structure of the BimBH3 complex with BaxΔC displays a cavity surrounded by Bax α1, α2, α5 and α8. Our results are consistent with a model in which binding of an activator BH3 domain to the Bax groove initiates separation of its core (α2–α5) and latch (α6–α8) domains, enabling its subsequent dimerisation and the permeabilisation of the mitochondrial outer membrane.

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