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Highlights d NTD-targeting antibodies are a key part of immunity to SARS-CoV-2 d NTD neutralizing antibodies target a single antigenic site of vulnerability d Neutralizing NTD antibodies protect hamsters from SARS-CoV-2 challenge d Variants of concern have mutations in the NTD that escape neutralization

Highlights d The receptor-binding motif (RBM) is a highly variable region of SARS-CoV-2 spike d RBM mutation N439K has emerged independently in multiple lineages d N439K increases spike affinity for hACE2; viral fitness and disease are unchanged d N439K confers resistance to several mAbs and escapes some polyclonal responses

An ideal anti-SARS-CoV-2 antibody would resist viral escape [1][2][3] , have activity against diverse SARS-related coronaviruses (sarbecoviruses) [4][5][6][7] , and be highly protective through viral neutralization [8][9][10][11] and effector functions 12,13 . Understanding how these properties relate to each other and vary across epitopes would aid development of antibody therapeutics and guide vaccine design. Here, we comprehensively characterize escape, breadth, and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a tradeoff between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a previously undescribed cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies targeting the ACE2 receptor binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we characterize one potent RBM antibody (S2E12 8 ) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth, and potency among antibodies targeting the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.The most potently neutralizing antibodies to SARS-CoV-2-including those in clinical use 14 and dominant in polyclonal sera 15,16 -target the spike receptor-binding domain (RBD). Mutations in the RBD that reduce binding by antibodies have emerged among SARS-CoV-2 variants [17][18][19][20][21] , highlighting the need for antibodies and vaccines that are robust to viral escape. We have previously described an antibody, S309 4 , that exhibits potent effector functions and neutralizes all current SARS-CoV-2 variants 22,23 and the divergent sarbecovirus SARS-CoV-1. S309 forms the basis for an antibody therapy (VIR-7831, recently renamed sotrovimab) that has received Emergency Use Authorization from the FDA for treatment of COVID-19 24 . Longer term, antibodies with broad activity across SARS-related coronaviruses (sarbecoviruses) would be useful to combat potential future spillovers 6 . These efforts would be aided by a systematic understanding of the relationships among antibody epitope,

SUMMARY Major features of transcription by human RNA Polymerase II (Pol II) remain poorly defined due to a lack of quantitative approaches for visualizing Pol II progress at nucleotide resolution. We developed a simple and powerful approach for performing native elongating transcript sequencing (NET-seq) in human cells that globally maps strand-specific Pol II density at nucleotide resolution. NET-seq exposes a mode of antisense transcription that originates downstream and converges on transcription from the canonical promoter. Convergent transcription is associated with a distinctive chromatin configuration and is characteristic of lower-expressed genes. Integration of NET-seq with genomic footprinting data reveals stereotypic Pol II pausing coincident with transcription factor occupancy. Finally, exons retained in mature transcripts display Pol II pausing signatures that differ markedly from skipped exons, indicating an intrinsic capacity for Pol II to recognize exons with different processing fates. Together, human NET-seq exposes the topography and regulatory complexity of human gene expression.

SARS-CoV-2 entry into host cells is orchestrated by the spike (S) glycoprotein that contains an immunodominant receptor-binding domain (RBD) targeted by the largest fraction of neutralizing antibodies (Abs) in COVID-19 patient plasma. Little is known about neutralizing Abs binding to epitopes outside the RBD and their contribution to protection. Here, we describe 41 human monoclonal Abs (mAbs) derived from memory B cells, which recognize the SARS-CoV-2 S N-terminal domain (NTD) and show that a subset of them neutralize SARS-CoV-2 ultrapotently. We define an antigenic map of the SARS-CoV-2 NTD and identify a supersite recognized by all known NTD-specific neutralizing mAbs. These mAbs inhibit cell-to-cell fusion, activate effector functions, and protect Syrian hamsters from SARS-CoV-2 challenge. SARS-CoV-2 variants, including the 501Y.V2 and B.1.1.7 lineages, harbor frequent mutations localized in the NTD supersite suggesting ongoing selective pressure and the importance of NTD-specific neutralizing mAbs to protective immunity.

SUMMARY Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of T cell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.