Coping with cellular stressors, manifesting as either intrinsic cues or environmental insults, is key to preserving cellular and organismal health. One strategy is to activate the Integrated Stress Response (ISR), a conserved eukaryotic signaling pathway that reprograms translation towards damage mitigation and recovery, or apoptosis when stress is irremediable. Although an inherently cytoprotective process, dysregulation of the ISR underlies a number of neurological disorders, and detailing its mechanism of action and precisely understanding modulators of the pathway is of critical importance. The ISR integrates diverse stresses through a set of stress-sensing kinases. Upon activation, they converge on a common task: the phosphorylation of a single serine in the translation initiation factor eIF2, the protein responsible for bringing the first amino acid to a translating ribosome. Phosphorylated eIF2 (eIF2-p), instead of acting as a substrate, inhibits its dedicated nucleotide exchange factor eIF2B, which is a two-fold symmetric heterodecameric complex. Inhibition of eIF2B by eIF2-p impairs the cycling of eIF2 from its inactive GDP-bound state to its active GTP-bound state, leading to diminished translation of most (and selective translation of a few) select mRNAs, the latter containing regulatory upstream open reading frames. Thus eIF2B serves as the central node regulating the critical first step of protein synthesis.In Chapter 1 I will be describing how through a suite of in vitro and in vivo tools, we uncovered the mechanism by which eIF2B activity is modulated. We discovered that, contrary to prior hypotheses, eIF2B activity is regulated through its conformation. eIF2-p binding to eIF2B induces a conformational change in eIF2B that disfavors substrate (eIF2) engagement by pulling the two halves of the complex apart and disrupting the eIF2 binding site. eIF2-p therefore activates the ISR by shifting eIF2B from its enzymatically active ‘A-State’ towards a conformationally inhibited ‘I-State’ with diminished substrate binding and enzymatic activity. This conformational inhibition of eIF2B can be overcome by the small-molecule Integrated Stress Response Inhibitor (ISRIB) that our lab discovered. ISRIB binds across the symmetry interface of eIF2B and staples the complex into its active conformation, allosterically competing off the inhibitor eIF2-p. We find orthogonal support for the A/I-State model of ISR activation through viral effector proteins and point mutations in eIF2B. In Chapter 2 I will describe how the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade activation of the ISR. Mechanistically, NSs tightly binds to eIF2B and directly competes with inhibitory eIF2-p binding, restoring eIF2B’s conformation to its active A-State. Having the opposite effect (and detailed in Chapter 3), a single histidine to aspartate point mutation at eIF2B’s dimerization interface mimics the effects of eIF2-p binding by promoting an I-State like conformation. Through charge repulsion, this point mutation results in eIF2-p independent modulation of eIF2B’s conformation and concomitant activation of the ISR in vitro and in cells. Collectively, these data establish that eIF2B is a highly dynamic complex through which protein translation and cellular functioning can be modulated by small molecules (ISRIB), endogenous binding partners (eIF2 and eIF2-P), evolved viral proteins, and point mutations. My work on ISR regulation came to an abrupt halt when SARS-CoV-2 took over the world. During this involuntary pause, all non-Covid work stopped at UCSF. In an effort to address the global threat, I assembled and led an interdisciplinary team to develop agents that powerfully neutralize the virus (detailed in Chapter 4). In particular, we obtained single-domain antibodies (nanobodies) by screening a yeast surface-displayed library of synthetic nanobodies. We identified nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction. One exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a humanized trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains powerful binding and neutralization properties against the Delta-SARS-CoV-2. In hamster models of infection, mNb6-tri delivered via nasal-spray reduced the severity of infection in animals exposed to SARS-CoV-2, and served as a potent post-exposure prophylaxis in animals co-housed with infected individuals. Further, in initial safety trials in >200 human subjects after administration via a nasal spray, no safety concerns were observed. Most importantly mNb6-tri is fully resistant to aerosolization, lyophilization, and heat, and it can be readily and economically produced in yeast at industrial scale. These properties may allow these or similar agents to serve as a widely deployable, patient-friendly prophylactic and/or early infection therapeutic that can be self-administered directly to the initial sites of infection. Such a therapeutic is particularly needed in the developing world where vaccination levels remain low and access to expensive therapeutics is significantly limited.