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Primate lentiviruses (PLVs), including human immunodeficiency virus type 1 (HIV-1), HIV type 2 (HIV-2), and the simian immunodeficiency viruses (SIVs), cause persistent lifelong infections despite the presence of virus-specific adaptive immune responses. The target of antibodies is the viral envelope glycoprotein (Env), which is expressed on the surface of virions and infected cells as a trimer of gp120:gp41 heterodimers. SIV env sequence variation arising from evasion of antibodies is well established and closely mimics the pattern observed in HIV-infected human patients, yet despite the experimental advantages of the macaque model, the viral dynamics during acute and early infection leading to escape within env have not been well defined. To examine the evolutionary dynamics of SIV env during early infection (< 6 months post-infection), we obtained longitudinal plasma samples from a small cohort of SIVmac251-infected rhesus macaques. We deep-sequenced the env gene from longitudinal samples spanning acute and early infection (2-29 weeks post-infection) from four SIV-infected rhesus macaques and the inoculum (a swarm stock of uncloned SIVmac251) that was used to infect the cohort (by repeated low-dose challenge). Using high-resolution next generation sequencing (NGS), we captured a population bottleneck at the point of transmission from the stock into each animal, tracked the subsequent emergence of Env diversity from the initially homogeneous population, and correlated changes with the onset of Env-specific antibodies. We identified a pattern of common substitutions, insertions, and deletions in env of animals with antibody responses, which repeatedly emerge in SIV-infected primate hosts, and found that adaptive changes clustered within short regions of the V1 and V4 loops of gp120. Surprisingly, we found multiple large in-frame deletions in V4 emerge to become dominant in the viral population in two animals with detectable antibody responses to Env. Furthermore, we developed a novel deep sequencing based viral fitness assay (Fit-Seq) and measured the relative fitness of several key in vivo antibody escape adaptations. Using Fit-Seq, we found that Env adaptations in V1 and V4 that confer antibody escape had no associated fitness costs in the absence of antibody, but rather, replicated to the same level as SIVmac239 WT. Surprisingly, we also found that Fit-Seq was able to detect antibody-mediated neutralization of SIVmac239 even in cases where activity was undetectable by standard neutralization assays. Our observation of reproducible patterns of Env variation clustered in V1 and V4, together with measurements of relative fitness, suggest that antibody responses can select for mutations that confer viral escape yet have little or no associated replicative fitness cost in the absence of antibody, even while providing a clear fitness advantage in the environment of constant antibody selection encountered in vivo. Thus, sites that can change with little or no impact on relative fitness may have evolved as an immune evasion mechanism to facilitate rapid and early escape from Env-specific antibody., Medical Sciences

Mitochondrial outer membrane permeabilization is a key step in the apoptotic cell death program, regulating life-death decisions in response to cytotoxic drugs and other forms of cell stress. In this thesis I use mathematical modeling of a reconstituted biochemical system to identify and integrate mechanisms of apoptotic pore formation. A key bottleneck in using mathematical models to characterize mechanisms has been the difficulty of efficiently creating and revising alternative models and evaluating them against data. This problem is addressed through the use of a software framework, PySB, that allows ensembles of models to be transparently described using tools and approaches from computer programming. These alternative hypotheses can then be evaluated against data using methods from Bayesian statistics for discrimination of models with varying numbers of (possibly non-identifiable) parameters. Using this framework, calibration of a set of models to in vitro kinetic measurements of the membrane insertion of Bax identifies a conformational intermediate associated with BH3-only:Bax complex formation and membrane association but not pore formation. Functional measurements of Bax point mutants from tumors show that the pore formation process can be blocked at the transition into or out of this intermediate, preventing mitochondrial permeabilization. In another study, model-based analysis of Bax insertion and permeabilization kinetics across a range of BH3-only, Bax, and liposome concentrations reveals the context-dependence of the mechanisms regulating pore formation. Bax recruitment is shown to depend on liposome concentration kinetically but not stoichiometrically, whereas cBid recruitment is shown to be limited at high cBid:liposome concentrations. I show that Bax distribution among liposomes is dependent on the presence of pre-existing pores, and that pores grow to include large numbers of Bax monomers but have a minimum size of four subunits. More generally, these studies serve as examples of how ensemble modeling can be used to integrate information about complex mechanisms from disparate sets of experimental observations., Systems Biology

Recent improvements in mass spectrometry instrumentation have stimulated the fusion of this technology with protein crosslinking to advance the structural proteomics field. However, analysis of complex datasets from crosslinking experiments remains a bottleneck. The majority of crosslinking studies for structural characterization of protein- protein interactions have been conducted with reagents specific for discrete amino acids. While this approach simplifies data analysis, the requirement for specific functionalities to be present at the interaction interface limits resolution. Herein, we report the application of stapled peptides for the development of photoaffinity reagents for mass spectrometric characterization of BCL-2 family protein interactions. To validate this approach, we synthesized photoreactive stabilized alpha-helices of BH3 domains (pSAHBs) incorporating a benzophenone containing amino acid, and demonstrated that the photo crosslinking specificity of these reagents paralleled the interaction specificity of the native proteins. We show that the standard SEQUEST algorithm is effective at identifying specific amino acids crosslinked by pSAHBs, and that this information can be used to create distance restraints for characterizing interaction interfaces by in silico docking. The pSAHB approach is applied to characterize previously elusive activating interactions between BH3 domains and the proapoptotic proteins BAX and BAK. We demonstrate that full-length BAK requires a direct activation stimulus, and that this involves interaction at a canonical surface groove at the C-terminal face of BAK. We confirmed that initiation of direct BAX activation occurs at a geographically distinct site at the N-terminal face of BAX, but further find that induced release of its C-terminus from the canonical groove exposes these residues for secondary BH3 interaction. These data suggest that BAX may be subject to a two-step activation mechanism within distinct cytosolic and mitochondrial compartments. Finally, we report the structural characterization of an interaction between BAD and glucokinase, the first description of a BH3 domain interaction with a non-BCL-2 family member. We identify the active site region of glucokinase as the BAD interaction site, establishing this region as a novel target for development of glucokinase activators. We conclude that the pSAHB approach represents a rapid and powerful approach to protein interaction site identification that complements conventional structural biology techniques.