SRP313155 PRJNA719172 GSE171356 The Host Cell's ER Proteostasis Network Shapes HIV-1 Envelope Protein Mutational Tolerance Buffering of deleterious mutations by molecular chaperones and degradation of aberrant proteins by quality control systems are both major factors that can impact the mutational landscape available to a client protein. The impacts of the proteostasis network on protein evolution are not limited to just endogenous clients, but can also shape the mutational landscapes accessible to rapidly evolving viral proteins. Here, we test the hypothesis that the composition of the host cell's endoplasmic reticulum (ER) proteostasis network shapes the evolution of RNA viruses by focusing on human immunodeficiency virus-1 envelope (Env), a membrane glycoprotein that folds and matures in the host cell's secretory pathway. We apply chemical genetic methods to activate the IRE1-XBP1s and/or the ATF6 transcriptional arms of the unfolded protein response in a stress-independent manner. We then quantitatively assess the impact of the resulting altered host cell ER proteostasis environments on the relative enrichment of all Env single amino acid substitutions using deep mutational scanning. We find that upregulation of host ER proteostasis factors globally reduces the mutational tolerance of HIV-1 Env, particularly upon induction of the IRE1-XBP1s transcriptional arm of the UPR. The effects of ATF6 activation are less global, but still significant at particular Env sites. The impact of the XBP1s-induced ER proteostasis environment is disparate for diverse structural elements of Env. Conserved, functionally important regions generally exhibit the largest decreases in mutational tolerance upon XBP1s activation. In contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display greatly enhanced mutational tolerance when XBP1s is activated. Altogether, these data reveal a new set of host factors that specifically shape the mutational space accessible to HIV Env and, more generally, provide compelling evidence that UPR-regulated proteostasis mechanisms play critical roles in membrane protein evolution. Overall design: RNA-seq characterizing a clonal SupT1 cell line stably expressing both the gene encoding XBP1s under control of the tetracycline repressor (tetR) and the gene encoding E. coli dihydrofolate reductase (DHFR) destabilizing domain fused to the active form of ATF6 (amino acid residues 1-373). These cells were treated with small molecules for 24 hours in quadruplicate to modulate the proteostasis environment in a stress-independent manner. The cells were treated with: 1) 0.1% DMSO for vehicle treatment condition, 2) 2 ug/mL doxycycline (dox) to activate XBP1s, 3) 10 uM trimethoprim (TMP) to activate ATF6, and 4) 2 ug/mL dox and 10 uM TMP to induce both XBP1s and ATF6 concomitantly. GSE171356 pubmed 35180219