Risky Party and Nightclub Attendance during the COVID-19 Nightlife Shutdown in New York City

From March 2020 through May 2021, nightlife venues were shut down and large gatherings were deemed illegal in New York City (NYC) due to COVID-19. This study sought to determine the extent of risky party attendance during the COVID-19 shutdown among people who attend electronic dance music parties in NYC. During the first four months that venues were permitted to reopen (June through September 2021), time–space sampling was used to survey adults (n = 278) about their party attendance during the first year of the shutdown (March 2020–March 2021). We examined prevalence and correlates of attendance and mask-wearing at such parties. A total of 43.9% attended private parties with more than 10 people, 27.3% attended nightclubs, and 20.5% attended other parties such as raves. Among those who attended any, 32.3% never wore a mask and 19.3% reported attending parties in which no one wore a mask. Past-year ecstasy use was associated with increased risk for attending private (aPR = 1.51, 95% CI: 1.00–2.28) or other parties (aPR = 2.75, 95% CI: 1.48–5.13), and use of 2C series drugs was associated with increased risk for attending nightclubs (aPR = 2.67, 95% CI: 1.24–5.77) or other parties (aPR = 2.50, 95% CI: 1.06–5.87). Attending >10 parties was associated with increased risk for never wearing a mask (aPR = 2.74, 95% CI: 1.11–6.75) and for no other attendees wearing masks (aPR = 4.22, 95% CI: 1.26–14.07). Illegal dance parties continued in NYC during the COVID-19 shutdown. Prevention and harm reduction efforts to mitigate risk of COVID-19 transmission during such shutdowns are sorely needed.     

Impact of natural selection on global patterns of genetic variation and association with clinical phenotypes at genes involved in SARS-CoV-2 infection

Human genomic diversity has been shaped by both ancient and ongoing challenges from viruses. The current coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a devastating impact on population health. However, genetic diversity and evolutionary forces impacting host genes related to SARS-CoV-2 infection are not well understood. We investigated global patterns of genetic variation and signatures of natural selection at host genes relevant to SARS-CoV-2 infection (angiotensin converting enzyme 2 [ACE2], transmembrane protease serine 2 [TMPRSS2], dipeptidyl peptidase 4 [DPP4], and lymphocyte antigen 6 complex locus E [LY6E]). We analyzed data from 2,012 ethnically diverse Africans and 15,977 individuals of European and African ancestry with electronic health records and integrated with global data from the 1000 Genomes Project. At ACE2, we identified 41 nonsynonymous variants that were rare in most populations, several of which impact protein function. However, three nonsynonymous variants (rs138390800, rs147311723, and rs145437639) were common among central African hunter-gatherers from Cameroon (minor allele frequency 0.083 to 0.164) and are on haplotypes that exhibit signatures of positive selection. We identify signatures of selection impacting variation at regulatory regions influencing ACE2 expression in multiple African populations. At TMPRSS2, we identified 13 amino acid changes that are adaptive and specific to the human lineage compared with the chimpanzee genome. Genetic variants that are targets of natural selection are associated with clinical phenotypes common in patients with COVID-19. Our study provides insights into global variation at host genes related to SARS-CoV-2 infection, which have been shaped by natural selection in some populations, possibly due to prior viral infections.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronaviruses are enveloped, positive-sense, and single-stranded RNA viruses, many of which are zoonotic pathogens that crossed over into humans. Seven coronavirus species, including SARS-CoV-2, have been discovered that, depending on the virus and host physiological condition, may cause mild or lethal respiratory disease. There is considerable variation in disease prevalence and severity across populations and communities. Importantly, minority populations in the United States appear to have been disproportionally affected by COVID-19 (1, 2). For example, in Chicago, more than 50% of COVID-19 cases and nearly 70% of COVID-19 deaths are in African Americans (who make up 30% of the population of Chicago) (1). While social and economic factors are largely responsible for driving COVID-19 health disparities, investigating genetic diversity at host genes related to SARS-CoV-2 infection could help identify functionally important variation, which may play a role in individual risk for severe COVID-19 infection.In this study, we focused on four key genes playing a role in SARS-CoV-2 infection (3). The ACE2 gene, encoding the angiotensin-converting enzyme-2 protein, was reported to be a main binding site for severe acute respiratory syndrome coronavirus (SARS-CoV) during an outbreak in 2003, and evidence showed stronger binding affinity to SARS-CoV-2, which enters the target cells via ACE2 receptors (3, 4). The ACE2 gene is located on the X chromosome (chrX); its expression level varies among populations (5); and it is ubiquitously expressed in the lung, blood vessels, gut, kidney, testis, and brain, all organs that appear to be affected as part of the COVID-19 clinical spectrum (6). SARS-CoV-2 infects cells through a membrane fusion mechanism, which in the case of SARS-CoV, is known to induce down-regulation of ACE2 (7). Such down-regulation has been shown to cause inefficient counteraction of angiotensin II effects, leading to enhanced pulmonary inflammation and intravascular coagulation (7). Additionally, altered expression of ACE2 has been associated with cardiovascular and cerebrovascular disease, which is highly relevant to COVID-19 as several cardiovascular conditions are associated with severe disease. TMPRSS2, located on the outer membrane of host target cells, binds to and cleaves ACE2, resulting in activation of spike proteins on the viral envelope and facilitating membrane fusion and endocytosis (8). Two additional genes, DPP4 and LY6E, have been shown to play an important role in the entry of SARS-CoV-2 virus into host cells. DPP4 is a known functional receptor for the Middle East respiratory syndrome coronavirus (MERS-CoV), causing a severe respiratory illness with high mortality (9, 10). LY6E encodes a glycosylphosphatidylinositol-anchored cell surface protein, which is a critical antiviral immune effector that controls coronavirus infection and pathogenesis (11). Mice lacking LY6E in hematopoietic cells were susceptible to murine coronavirus infection (11).Previous studies of genetic diversity at ACE2 and TMPRSS2 in global human populations did not include an extensive set of African populations (5, 12–14). No common coding variants (defined here as minor allele frequency [MAF] > 0.05) at ACE2 were identified in any prior population studies. However, few studies included diverse indigenous African populations whose genomes harbor the greatest diversity among humans. This leads to a substantial disparity in the representation of African ancestries in human genetic studies of COVID-19, impeding health equity as the transferability of findings based on non-African ancestries to African populations can be low (15). Including more African populations in studying the genetic diversity of genes involved in SARS-CoV-2 infection is extremely necessary. Additionally, the evolutionary forces underlying global patterns of genetic diversity at host genes related to SARS-CoV-2 infection are not well understood. Using methods to detect natural selection signatures at host genes related to viral infections helps identify putatively functional variants that could play a role in disease risk.We characterized genetic variation and studied natural selection signatures at ACE2, TMPRSS2, DPP4, and LY6E in ethnically diverse human populations by analyzing 2,012 genomes from ethnically diverse Africans (referred to as the “African diversity” dataset), 2,504 genomes from the 1000 Genomes Project (1KG), and whole-exome sequencing of 15,977 individuals of European ancestry (EA) and African ancestry from the Penn Medicine BioBank (PMBB) dataset (SI Appendix, Fig. S1). The African diversity dataset includes populations with diverse subsistence patterns (hunter-gatherers, pastoralists, agriculturalists) and speaking languages belonging to the four major language families in Africa (Khoesan; Niger–Congo, of which Bantu is the largest subfamily; Afroasiatic; and Nilo-Saharan). We identify functionally relevant variation, compare the patterns of variation across global populations, and provide insight into the evolutionary forces underlying these patterns of genetic variation. In addition, we perform an association study using the variants identified from whole-exome sequencing at the four genes and clinical traits derived from electronic health record (EHR) data linked to the subjects enrolled in the PMBB. The EHR data include diseases related to organ dysfunctions associated with severe COVID-19, such as respiratory, cardiovascular, liver, and renal complications. Our study of genetic variation in genes involved in SARS-CoV-2 infection provides data to investigate infection susceptibility within and between populations and indicates that variants in these genes may play a role in comorbidities relevant to COVID-19 severity.     

A two-component protein condensate of the EGFR cytoplasmic tail and Grb2 regulates Ras activation by SOS at the membrane

We reconstitute a phosphotyrosine-mediated protein condensation phase transition of the ∼200 residue cytoplasmic tail of the epidermal growth factor receptor (EGFR) and the adaptor protein, Grb2, on a membrane surface. The phase transition depends on phosphorylation of the EGFR tail, which recruits Grb2, and crosslinking through a Grb2-Grb2 binding interface. The Grb2 Y160 residue plays a structurally critical role in the Grb2-Grb2 interaction, and phosphorylation or mutation of Y160 prevents EGFR:Grb2 condensation. By extending the reconstitution experiment to include the guanine nucleotide exchange factor, SOS, and its substrate Ras, we further find that the condensation state of the EGFR tail controls the ability of SOS, recruited via Grb2, to activate Ras. These results identify an EGFR:Grb2 protein condensation phase transition as a regulator of signal propagation from EGFR to the MAPK pathway.

Recently, a class of phenomena known as protein condensation phase transitions has begun to emerge in biology. Originally identified in the context of nuclear organization (1) and gene expression (2), a distinct two-dimensional protein condensation on the cell membrane has now been discovered in the T cell receptor (TCR) signaling system involving the scaffold protein LAT (3–5). TCR activation results in phosphorylation of LAT on at least four distinct tyrosine sites, which subsequently recruit the adaptor protein Grb2 and the signaling molecule PLCγ via selective binding interactions with their SH2 domains. Additional scaffold and signaling molecules, including SOS, GADS, and SLP76, are recruited to Grb2 and PLCγ through further specific protein–protein interactions (6, 7). Multivalency among some of these binding interactions can crosslink LAT molecules in a two-dimensional bond percolation network on the membrane surface. The resulting LAT protein condensate resembles the nephrin:NCK:N-WASP condensate (8) in that both form on the membrane surface under control of tyrosine phosphorylation and exert at least one aspect of functional control over signaling output via a distinct type of kinetic regulatory mechanism (9–11). The basic molecular features controlling the LAT and nephrin protein condensates are common among biological signaling machinery, and other similar condensates continue to be discovered (12, 13). The LAT condensation shares downstream signaling molecules with the EGF-receptor (EGFR) signaling system, raising the question if EGFR may participate in a signaling-mediated protein condensation itself.EGFR signals to the mitogen-activated protein kinase (MAPK) pathway and controls key cellular functions, including growth and proliferation (14–16). EGFR is a paradigmatic model system in studies of signal transduction, and immense, collective scientific effort has revealed the inner workings of its signaling mechanism down to the atomic level (17). EGFR is autoinhibited in its monomeric form. Ligand-driven activation is achieved through formation of an asymmetric receptor dimer in which one kinase activates the other to phosphorylate the nine tyrosine sites in the C-terminal tails (17, 18). There is an obvious conceptual connection between EGFR and the LAT signaling system in T cells. The ∼200-residue–long cytoplasmic tail of EGFR resembles LAT in that both are intrinsically disordered and contain multiple sites of tyrosine phosphorylation that recruit adaptor proteins, including Grb2, upon receptor activation (19). Phosphorylation at tyrosine residues Y1068, Y1086, Y1148, and Y1173 in the EGFR tail creates sites to which Grb2 can bind via its SH2 domain. EGFR-associated Grb2 subsequently recruits SOS, through binding of its SH3 domains to the proline-rich domain of SOS. Once at the membrane, SOS undergoes a multistep autoinhibition-release process and begins to catalyze nucleotide exchange of RasGDP to RasGTP, activating Ras and the MAPK pathway (20).While these most basic elements of the EGFR activation mechanism are widely accepted, larger-scale features of the signaling complex remain enigmatic. A number of studies have reported higher-ordered multimers of EGFR during activation, including early observations by Förster Resonance Energy Transfer and fluorescence lifetime studies (21–23), as have more recent studies using single molecule (24, 25) and computational methods (26). Structural analyses and point mutation studies on EGFR have identified a binding interface enabling EGFR asymmetric dimers to associate (27), but the role of these higher-order assemblies remains unclear. At the same time, many functional properties of the signaling system remain unexplained as well. For example, EGFR is a frequently altered oncogene in human cancers, and drugs (including tyrosine kinase inhibitors) targeting EGFR signaling have produced impressive initial patient responses (28). All too often, however, these drugs fail to offer sustained patient benefits, in large part because of poorly understood resistance mechanisms (29). Physical aspects of the cellular microenvironment have been implicated as possible contributors to resistance development (30), and there is a growing realization that EGFR possesses kinase-independent (e.g., signaling independent) prosurvival functions in cancer cells (31). These points fuel speculation that additional layers of regulation over the EGFR signaling mechanism exist, including at the level of the receptor signaling complex itself.Here we report that EGFR undergoes a protein condensation-phase transition upon activation. We reconstituted the cytoplasmic tails of EGFR on supported bilayers and characterized the system behavior upon interaction with Grb2 and SOS, using total internal reflection fluorescence (TIRF) imaging. This experimental platform has been highly effective for revealing both phase-transition characteristics and functional signaling aspects of LAT protein condensates (4, 5, 10, 32–34). Published reports on the LAT system to date have emphasized SOS (or the SOS proline-rich [PR] domain) as a critical crosslinking element. Titrating the SOS PR domain into an initially homogeneous mixture of phosphorylated LAT and Grb2 revealed a sharp transition to the condensed phase, which we have also observed with the EGFR:Grb2:SOS system. Under slightly different conditions, however, we report observations of an EGFR:Grb2 condensation-phase transition without any SOS or other crosslinking molecule. We show that crosslinking is achieved through a Grb2–Grb2 binding interface. Phosphorylation on Grb2 at Y160 as well as a Y160E mutation [both reported to disrupt Grb2–Grb2 binding (35, 36)] were observed to prevent formation of EGFR condensates. We note that the evidence of Grb2–Grb2 binding we observed occurred in the context of EGFR-associated Grb2, which is localized to the membrane surface; free Grb2 dimers are not necessary.The consequence of EGFR condensation on downstream signaling is characterized by mapping the catalytic efficiency of SOS to activate Ras as a function of the EGFR condensation state. SOS is the primary Ras guanine nucleotide exchange factor (GEF) responsible for activating Ras in the EGFR-to-MAPK signaling pathway (37–40). At the membrane, SOS undergoes a multistep process of autoinhibition release before beginning to activate Ras. Once fully activated, SOS is highly processive, and a single SOS molecule can activate hundreds of Ras molecules before disengaging from the membrane (41–43). Autoinhibition release in SOS is a slow process, which necessitates that SOS be retained at the membrane for an extended time in order for Ras activation to begin (5, 10). This delay between initial recruitment of SOS and subsequent initiation of its Ras GEF activity provides a kinetic proofreading mechanism that essentially requires SOS to achieve multivalent engagement with the membrane (e.g., through multiple Grb2 or other interactions) in order for it to activate any Ras molecules.Experimental results described here reveal that Ras activation by SOS is strongly enhanced by EGFR condensation. Calibrated measurements of both SOS recruitment and Ras activation confirmed enhanced SOS catalytic activity on a per-molecule basis, in addition to enhanced recruitment to the condensates. These results suggest that a Grb2-mediated EGFR protein condensation-phase transition is a functional element controlling signal propagation from EGFR downstream to the MAPK signaling pathway.