B cell genomics behind cross-neutralization of SARS-CoV-2 variants and SARS-CoV.

Monoclonal antibodies (mAbs) are a focus in vaccine and therapeutic design to counteract severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. Here, we combined B cell sorting with single-cell VDJ and RNA sequencing (RNA-seq) and mAb structures to characterize B cell responses against SARS-CoV-2. We show that the SARS-CoV-2-specific B cell repertoire consists of transcriptionally distinct B cell populations with cells producing potently neutralizing antibodies (nAbs) localized in two clusters that resemble memory and activated B cells. Cryo-electron microscopy structures of selected nAbs from these two clusters complexed with SARS-CoV-2 spike trimers show recognition of various receptor-binding domain (RBD) epitopes. One of these mAbs, BG10-19, locks the spike trimer in a closed conformation to potently neutralize SARS-CoV-2, the recently arising mutants B.1.1.7 and B.1.351, and SARS-CoV and cross-reacts with heterologous RBDs. Together, our results characterize transcriptional differences among SARS-CoV-2-specific B cells and uncover cross-neutralizing Ab targets that will inform immunogen and therapeutic design against coronaviruses.

Small-molecule inhibitors directly target CARD9 and mimic its protective variant in inflammatory bowel disease.

Advances in human genetics have dramatically expanded our understanding of complex heritable diseases. Genome-wide association studies have identified an allelic series of CARD9 variants associated with increased risk of or protection from inflammatory bowel disease (IBD). The predisposing variant of CARD9 is associated with increased NF-κB-mediated cytokine production. Conversely, the protective variant lacks a functional C-terminal domain and is unable to recruit the E3 ubiquitin ligase TRIM62. Here, we used biochemical insights into CARD9 variant proteins to create a blueprint for IBD therapeutics and recapitulated the mechanism of the CARD9 protective variant using small molecules. We developed a multiplexed bead-based technology to screen compounds for disruption of the CARD9-TRIM62 interaction. We identified compounds that directly and selectively bind CARD9, disrupt TRIM62 recruitment, inhibit TRIM62-mediated ubiquitinylation of CARD9, and demonstrate cellular activity and selectivity in CARD9-dependent pathways. Taken together, small molecules targeting CARD9 illustrate a path toward improved IBD therapeutics.

Exceptionally high-affinity Ras binders that remodel its effector domain.

The Ras proteins are aberrantly activated in a wide range of human cancers, often endowing tumors with aggressive properties and resistance to therapy. Decades of effort to develop direct Ras inhibitors for clinical use have thus far failed, largely because of a lack of adequate small-molecule-binding pockets on the Ras surface. Here, we report the discovery of Ras-binding miniproteins from a naïve library and their evolution to afford versions with midpicomolar affinity to Ras. A series of biochemical experiments indicated that these miniproteins bind to the Ras effector domain as dimers, and high-resolution crystal structures revealed that these miniprotein dimers bind Ras in an unprecedented mode in which the Ras effector domain is remodeled to expose an extended pocket that connects two isolated pockets previously found to engage small-molecule ligands. We also report a Ras point mutant that stabilizes the protein in the open conformation trapped by these miniproteins. These findings provide new tools for studying Ras structure and function and present opportunities for the development of both miniprotein and small-molecule inhibitors that directly target the Ras proteins.

The Autophagy-Related Beclin-1 Protein Requires the Coiled-Coil and BARA Domains To Form a Homodimer with Submicromolar Affinity.

Beclin-1 (BECN1) is an essential component of macroautophagy. This process is a highly conserved survival mechanism that recycles damaged cellular components or pathogens by encasing them in a bilayer vesicle that fuses with a lysosome to allow degradation of the vesicular contents. Mutations or altered expression profiles of BECN1 have been linked to various cancers and neurodegenerative diseases. Viruses, including HIV and herpes simplex virus 1 (HSV-1), are also known to specifically target BECN1 as a means of evading host defense mechanisms. Autophagy is regulated by the interaction between BECN1 and Bcl-2, a pro-survival protein in the apoptotic pathway that stabilizes the BECN1 homodimer. Disruption of the homodimer by phosphorylation or competitive binding promotes autophagy through an unknown mechanism. We report here the first recombinant synthesis (3-5 mg/L in an Escherichia coli culture) and characterization of full-length, human BECN1. Our analysis reveals that full-length BECN1 exists as a soluble homodimer (KD ∼ 0.45 µM) that interacts with Bcl-2 (KD = 4.3 ± 1.2 µM) and binds to lipid membranes. Dimerization is proposed to be mediated by a coiled-coil region of BECN1. A construct lacking the C-terminal BARA domain but including the coiled-coil region exhibits a homodimer KD 3.5-fold weaker than that of full-length BECN1, indicating that both the BARA domain and the coiled-coil region of BECN1 contribute to dimer formation. Using site-directed mutagenesis, we show that residues at the C-terminus of the coiled-coil region previously shown to interact with the BARA domain play a key role in dimerization and mutations weaken the interface by ∼5-fold.

An equilibrium-dependent retroviral mRNA switch regulates translational recoding.

Most retroviruses require translational recoding of a viral messenger RNA stop codon to maintain a precise ratio of structural (Gag) and enzymatic (Pol) proteins during virus assembly. Pol is expressed exclusively as a Gag-Pol fusion either by ribosomal frameshifting or by read-through of the gag stop codon. Both of these mechanisms occur infrequently and only affect 5-10% of translating ribosomes, allowing the virus to maintain the critical Gag to Gag-Pol ratio. Although it is understood that the frequency of the recoding event is regulated by cis RNA motifs, no mechanistic explanation is currently available for how the critical protein ratio is maintained. Here we present the NMR structure of the murine leukaemia virus recoding signal and show that a protonation-dependent switch occurs to induce the active conformation. The equilibrium is such that at physiological pH the active, read-through permissive conformation is populated at approximately 6%: a level that correlates with in vivo protein quantities. The RNA functions by a highly sensitive, chemo-mechanical coupling tuned to ensure an optimal read-through frequency. Similar observations for a frameshifting signal indicate that this novel equilibrium-based mechanism may have a general role in translational recoding.

An mpox virus mRNA-lipid nanoparticle vaccine confers protection against lethal orthopoxviral challenge.

Mpox virus (MPXV) caused a global outbreak in 2022. Although smallpox vaccines were rapidly deployed to curb spread and disease among those at highest risk, breakthrough disease was noted after complete immunization. Given the threat of additional zoonotic events and the virus's evolving ability to drive human-to-human transmission, there is an urgent need for an MPXV-specific vaccine that confers protection against evolving MPXV strains and related orthopoxviruses. Here, we demonstrate that an mRNA-lipid nanoparticle vaccine encoding a set of four highly conserved MPXV surface proteins involved in virus attachment, entry, and transmission can induce MPXV-specific immunity and heterologous protection against a lethal vaccinia virus (VACV) challenge. Compared with modified vaccinia virus Ankara (MVA), which forms the basis for the current MPXV vaccine, immunization with an mRNA-based MPXV vaccine generated superior neutralizing activity against MPXV and VACV and more efficiently inhibited spread between cells. We also observed greater Fc effector TH1-biased humoral immunity to the four MPXV antigens encoded by the vaccine, as well as to the four VACV homologs. Single MPXV antigen-encoding mRNA vaccines provided partial protection against VACV challenge, whereas multivalent vaccines combining mRNAs encoding two, three, or four MPXV antigens protected against disease-related weight loss and death equal or superior to MVA vaccination. These data demonstrate that an mRNA-based MPXV vaccine confers robust protection against VACV.

Gut microbiome ADP-ribosyltransferases are widespread phage-encoded fitness factors.

Bacterial ADP-ribosyltransferases (ADPRTs) have been described as toxins involved in pathogenesis through the modification of host proteins. Here, we report that ADPRTs are not pathogen restricted but widely prevalent in the human gut microbiome and often associated with phage elements. We validated their biochemical activity in a large clinical isolate collection and further examined Bxa, a highly abundant ADPRT in Bacteroides. Bxa is expressed, secreted, and enzymatically active in Bacteroides and can ADP-ribosylate non-muscle myosin II proteins. Addition of Bxa to epithelial cells remodeled the actin cytoskeleton and induced secretion of inosine. Bxa-encoding B. stercoris can use inosine as a carbon source and colonizes the gut to significantly greater numbers than a bxa-deleted strain in germ-free and altered Schaedler flora (ASF) mice. Colonization correlated with increased inosine concentrations in the feces and tissues. Altogether, our results show that ADPRTs are abundant in the microbiome and act as bacterial fitness factors.

An alternate conformation of the hyperthermostable HU protein from Thermotoga maritima has unexpectedly high flexibility.

The homodimeric HU protein from the hyperthermophile Thermotoga maritima (HUTmar) is a model system which can yield insights into the molecular determinants of thermostability in proteins. Unusually for a thermostable protein, HUTmar exists in a structurally heterogeneous state as evidenced by the assignment of two distinct and approximately equally populated forms in solution. Relaxation measurements combined with chemical shift, hydrogen exchange, and nuclear Overhauser enhancement data confirm the main structural features of both forms. In addition, these data support a two-state model for HUTmar in which the major form closely resembles the X-ray structure while the very flexible minor form is less structured. HUTmar may therefore be a new example of the small class of hyperthermostable proteins with unexpected flexibility.

HIV-1: packaging a shifty genome?

In this issue of Cell Host & Microbe, Chamanian et al. (2013) show that the frameshifting region in the HIV-1 genome influences the efficiency of genome packaging. This study may provide insights into mechanisms that constrain retroviruses into packaging only two copies of the genome during retroviral assembly.

Preformed protein-binding motifs in 7SK snRNA: structural and thermodynamic comparisons with retroviral TAR.

The 7SK small nuclear RNA is a highly conserved non-coding RNA that regulates transcriptional elongation. 7SK utilizes the HEXIM proteins to sequester the transcription factor P-TEFb by a mechanism similar to that used by retroviral TAR RNA to engage Tat and P-TEFb. Tat has also recently been shown to bind 7SK directly and recruit P-TEFb to TAR. We report here the solution structures of the free and arginine-bound forms of stem loop 4 of 7SK (7SK-SL4). Comparison of the 7SK-SL4 and TAR structures demonstrates the presence of a common arginine sandwich motif. However, arginine binding to 7SK-SL4 is mechanistically distinct and occurs via docking into a pre-organized pocket resulting in a 1000-fold increased affinity. Furthermore, whereas formation of the binding pocket in TAR requires a critical base-triple, hydrogen-bond formation between the equivalent bases in 7SK-SL4 is not essential and the pocket is stabilized solely by a pseudo base-triple platform. In addition, this theme of preformed protein binding motifs also extends into the pentaloop. The configuration of the loop suggests that 7SK-SL4 is poised to make ternary contacts with P-TEFb and HEXIM or Tat. These key differences between 7SK-SL4 and TAR present an opportunity to understand RNA structural adaptation and have implications for understanding differential interactions with Tat.