Protein determinants of RNA binding by DNA polymerase of the T4-related bacteriophage RB69

DNA polymerase (gp43) of phage T4 plays two biological roles, one as an essential DNA binding replication enzyme and the other as an mRNA-specific autogenous translational repressor. Binding of T4 gp43 to its mRNA target (translational operator RNA) interferes with gp43-DNA interactions, but it is unclear how the protein determinants for binding DNA are affected by the dynamics of gp43-mRNA interactions. We have used RB69 gp43, a natural variant of the T4 enzyme whose crystal structure has been determined to identify protein sites that respond to the interaction with specific RNA. We used protein phosphorylation markers, photocross-linking studies, protease sensitivity assays, and mutational analyses to examine the effects of operator RNA on the enzyme's five structural domains (N, exo, palm, fingers, and thumb). Our studies suggest that this RNA affects gp43-DNA interactions through global effects on protein structure that occlude DNA-binding sites but leave the enzyme accessible to interactions with the sliding clamp (RB69 gp45) and possibly other polymerase accessory proteins. We discuss the possible biological significance of putative RNA-binding motifs in the N and palm domains of RB69 gp43.     

Development of a high-throughput fluorescence polarization assay for Bcl-x(L)

Antiapoptotic protein Bcl-x(L) has been demonstrated to play a very important role in a variety of diseases such as cancer. Its biological function can be inhibited by proapoptotic proteins such Bak, Bad, and Bax by forming complexes mediated primarily by the Bcl-2 homology 3 (BH3) domain. To facilitate drug discovery for Bcl-x(L) inhibitors, we have developed and optimized a fluorescence polarization assay based on the interaction between Bcl-x(L) and BH3 domain peptides. We observed that the fluorescein-labeled Bad BH3 peptide [NLWAAQRYGRELRRMSDK(fluorescein)FVD or fluorescent Bad peptide] generates best overall results. Fluorescent Bad peptide interacts strongly with Bcl-x(L) with a K(d) of 21.48nM. The assay is stable over a 24-h period and can tolerate the presence of dimethyl sulfoxide up to 8%. By using a competition assay, several peptides derived from the BH3 region of Bak, Bad, Bax, and Bcl-2 were investigated. Bad and Bak BH3 peptides compete efficiently with IC(50) values of 0.048 and 1.14 microM, respectively, while the peptides from the BH3 region of Bcl-2 and Bax compete weakly. A mutated Bak peptide, which has been shown to be inactive for binding to Bcl-x(L), did not compete. The relative binding order of the peptides (Bad>Bak>Bcl-2>Bax>mutated Bak) correlates well with previously published results. When tested in high-throughput formats, the assay has a signal-to-noise ratio of 15.37 and a Z(') factor of at least 0.73. The plate-to-plate variability for free peptide control and bound peptide control is minimal. This validates the assay not only for investigating the nature of Bcl-x(L)-peptide interaction, but also for high-throughput screening of Bcl-x(L) inhibitors.     

In vitro selection and characterization of influenza A (A/N9) virus variants resistant to a novel neuraminidase inhibitor, A-315675

With the recent introduction of neuraminidase (NA) inhibitors into clinical practice for the treatment of influenza virus infections, considerable attention has been focused on the potential for resistance development and cross-resistance between different agents from this class. A-315675 is a novel influenza virus NA inhibitor that has potent enzyme activity and is highly active in cell culture against a variety of strains of influenza A and B viruses. To further assess the therapeutic potential of this compound, in vitro resistance studies have been conducted and a comparative assessment has been made relative to oseltamivir carboxylate. The development of viral resistance to A-315675 was studied by in vitro serial passage of influenza A/N9 virus strains grown in MDCK cells in the presence of increasing concentrations of A-315675. Parallel passaging experiments were conducted with oseltamivir carboxylate, the active form of a currently marketed oral agent for the treatment of influenza virus infections. Passage experiments with A-315675 identified a variant at passage 8 that was 60-fold less susceptible to the compound. Sequencing of the viral population identified an E119D mutation in the NA gene, but no mutations were observed in the hemagglutinin (HA) gene. However, by passage 10 (2.56 microM A-315675), two mutations (R233K, S339P) in the HA gene appeared in addition to the E119D mutation in the NA gene, resulting in a 310-fold-lower susceptibility to A-315675. Further passaging at higher drug concentrations had no effect on the generation of further NA or HA mutations (20.5 microM A-315675). This P15 virus displayed 355-fold-lower susceptibility to A-315675 and >175-fold-lower susceptibility to zanamivir than did wild-type virus, but it retained a high degree of susceptibility to oseltamivir carboxylate. By comparison, virus variants recovered from passaging against oseltamivir carboxylate (passage 14) harbored an E119V mutation and displayed a 6,000-fold-lower susceptibility to oseltamivir carboxylate and a 175-fold-lower susceptibility to zanamivir than did wild-type virus. Interestingly, this mutant still retained susceptibility to A-315675 (42-fold loss). This suggests that cross-resistance between A-315675- and oseltamivir carboxylate-selected variants in vitro is minimal.     

Skeletal defects in VEGF(120/120) mice reveal multiple roles for VEGF in skeletogenesis

Angiogenesis is an essential component of skeletal development and VEGF signaling plays an important if not pivotal role in this process. Previous attempts to examine the roles of VEGF in vivo have been largely unsuccessful because deletion of even one VEGF allele leads to embryonic lethality before skeletal development is initiated. The availability of mice expressing only the VEGF120 isoform (which do survive to term) has offered an opportunity to explore the function of VEGF during embryonic skeletal development. Our study of these mice provides new in vivo evidence for multiple important roles of VEGF in both endochondral and intramembranous bone formation, as well as some insights into isoform-specific functions. There are two key differences in vascularization of developing bones between wild-type and VEGF(120/120) mice. VEGF(120/120) mice have not only a delayed recruitment of blood vessels into the perichondrium but also show delayed invasion of vessels into the primary ossification center, demonstrating a significant role of VEGF at both an early and late stage of cartilage vascularization. These findings are the basis for a two-step model of VEGF-controlled vascularization of the developing skeleton, a hypothesis that is supported by the new finding that VEGF is expressed robustly in the perichondrium and surrounding tissue of cartilage templates of future bones well before blood vessels appear in these regions. We also describe new in vivo evidence for a possible role of VEGF in chondrocyte maturation, and document that VEGF has a direct role in regulating osteoblastic activity based on in vivo evidence and organ culture experiments.