The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope.

The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, at least in theory, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate the levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV-1) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody- and drug-escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s induction. Our data indicate that this phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly, and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is induced, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation.

HSF1 Activation Can Restrict HIV Replication.

Host protein folding stress responses can play important roles in RNA virus replication and evolution. Prior work suggested a complicated interplay between the cytosolic proteostasis stress response, controlled by the transcriptional master regulator heat shock factor 1 (HSF1), and human immunodeficiency virus-1 (HIV-1). We sought to uncouple HSF1 transcription factor activity from cytotoxic proteostasis stress and thereby better elucidate the proposed role(s) of HSF1 in the HIV-1 lifecycle. To achieve this objective, we used chemical genetic, stress-independent control of HSF1 activity to establish whether and how HSF1 influences HIV-1 replication. Stress-independent HSF1 induction decreased both the total quantity and infectivity of HIV-1 virions. Moreover, HIV-1 was unable to escape HSF1-mediated restriction over the course of several serial passages. These results clarify the interplay between the host's heat shock response and HIV-1 infection and motivate continued investigation of chaperones as potential antiviral therapeutic targets.

Host proteostasis modulates influenza evolution.

Predicting and constraining RNA virus evolution require understanding the molecular factors that define the mutational landscape accessible to these pathogens. RNA viruses typically have high mutation rates, resulting in frequent production of protein variants with compromised biophysical properties. Their evolution is necessarily constrained by the consequent challenge to protein folding and function. We hypothesized that host proteostasis mechanisms may be significant determinants of the fitness of viral protein variants, serving as a critical force shaping viral evolution. Here, we test that hypothesis by propagating influenza in host cells displaying chemically-controlled, divergent proteostasis environments. We find that both the nature of selection on the influenza genome and the accessibility of specific mutational trajectories are significantly impacted by host proteostasis. These findings provide new insights into features of host-pathogen interactions that shape viral evolution, and into the potential design of host proteostasis-targeted antiviral therapeutics that are refractory to resistance.

Transportable, Chemical Genetic Methodology for the Small Molecule-Mediated Inhibition of Heat Shock Factor 1.

Proteostasis in the cytosol is governed by the heat shock response. The master regulator of the heat shock response, heat shock factor 1 (HSF1), and key chaperones whose levels are HSF1-regulated have emerged as high-profile targets for therapeutic applications ranging from protein misfolding-related disorders to cancer. Nonetheless, a generally applicable methodology to selectively and potently inhibit endogenous HSF1 in a small molecule-dependent manner in disease model systems remains elusive. Also problematic, the administration of even highly selective chaperone inhibitors often has the side effect of activating HSF1 and thereby inducing a compensatory heat shock response. Herein, we report a ligand-regulatable, dominant negative version of HSF1 that addresses these issues. Our approach, which required engineering a new dominant negative HSF1 variant, permits dosable inhibition of endogenous HSF1 with a selective small molecule in cell-based model systems of interest. The methodology allows us to uncouple the pleiotropic effects of chaperone inhibitors and environmental toxins from the concomitantly induced compensatory heat shock response. Integration of our method with techniques to activate HSF1 enables the creation of cell lines in which the cytosolic proteostasis network can be up- or down-regulated by orthogonal small molecules. Selective, small molecule-mediated inhibition of HSF1 has distinctive implications for the proteostasis of both chaperone-dependent globular proteins and aggregation-prone intrinsically disordered proteins. Altogether, this work provides critical methods for continued exploration of the biological roles of HSF1 and the therapeutic potential of heat shock response modulation.

Photoactivatable fluorescein derivatives caged with a (3-hydroxy-2-naphthalenyl)methyl group.

The (3-hydroxy-2-naphthalenyl)methyl (NQMP) group represents an efficient photocage for fluorescein-based dyes. Thus, irradiation of the 6-NQMP ether of 2'-hydroxymethylfluorescein with low-intensity UVA light results in a 4-fold increase in emission intensity. Photoactivation of nonfluorescent NQMP-caged 3-allyloxyfluorescein produces a highly emissive fluorescein monoether. To facilitate conjugation of the caged dye to the substrate of interest via click chemistry, the allyloxy appendage was functionalized with an azide moiety.

Selective and Reversible Photochemical Derivatization of Cysteine Residues in Peptides and Proteins.

Selective derivatization of solvent-exposed cysteine residues in peptides and proteins is achieved by brief irradiation of an aqueous solution containing 3-(hydroxymethyl)-2-naphthol derivatives (NQMPs) with 350 nm fluorescent lamp. NQMP can be conjugated with various moieties, such as PEG, dyes, carbohydrates, or possess a fragment for further selective derivatization, e.g., biotin, azide, alkyne, etc. Attractive features of this labeling approach include an exceptionally fast rate of the reaction and a requirement for low equivalence of the reagent. The NQMP-thioether linkage is stable under ambient conditions, survives protein digestion and MS analysis. Irradiation of NQMP-labeled protein in a dilute solution (<40 µM) or in the presence of a vinyl ether results in a traceless release of the substrate. The reversible biotinylation of bovine serum albumin, as well as capture and release of this protein using NeutrAvidin Agarose resin beads has been demonstrated.

9-Aryl-9-xanthenols: a convenient platform for the design of fluorimetric and colorimetric pH indicators.

In aqueous and alcohol solutions, colorless and non-fluorescent derivatives of 9-aryl-9H-xanthen-9-ol equilibrate with brightly colored and fluorescent 9-arylxanthylium cations, thus offering a convenient platform for the design of dual-mode indicators for emission and absorption-based pH measurements. The position of the prototropic equilibrium depends only on the hydronium ion concentration and is not affected by general acids or other ions. Furthermore, the equilibrium equivalence point can be readily adjusted by introducing substituents in the xanthenol core. As dehydroxylation of 3,6-dialkoxy-9-(o-tolyl)-9-xanthenol occurs at pH = 6.5, indicators of this type are well suited for biological applications as illustrated by in vitro cell culture studies with NIH 3T3 cells.