S:D614G and S:H655Y are gateway mutations that act epistatically to promote SARS-CoV-2 variant fitness.

SARS-CoV-2 variants bearing complex combinations of mutations that confer increased transmissibility, COVID-19 severity, and immune escape, were first detected after S:D614G had gone to fixation, and likely originated during persistent infection of immunocompromised hosts. To test the hypothesis that S:D614G facilitated emergence of such variants, S:D614G was reverted to the ancestral sequence in the context of sequential Spike sequences from an immunocompromised individual, and within each of the major SARS-CoV-2 variants of concern. In all cases, infectivity of the S:D614G revertants was severely compromised. The infectivity of atypical SARS-CoV-2 lineages that propagated in the absence of S:D614G was found to be dependent upon either S:Q613H or S:H655Y. Notably, Gamma and Omicron variants possess both S:D614G and S:H655Y, each of which contributed to infectivity of these variants. Among sarbecoviruses, S:Q613H, S:D614G, and S:H655Y are only detected in SARS-CoV-2, which is also distinguished by a polybasic S1/S2 cleavage site. Genetic and biochemical experiments here showed that S:Q613H, S:D614G, and S:H655Y each stabilize Spike on virions, and that they are dispensable in the absence of S1/S2 cleavage, consistent with selection of these mutations by the S1/S2 cleavage site. CryoEM revealed that either S:D614G or S:H655Y shift the Spike receptor binding domain (RBD) towards the open conformation required for ACE2-binding and therefore on pathway for infection. Consistent with this, an smFRET reporter for RBD conformation showed that both S:D614G and S:H655Y spontaneously adopt the conformation that ACE2 induces in the parental Spike. Data from these orthogonal experiments demonstrate that S:D614G and S:H655Y are convergent adaptations to the polybasic S1/S2 cleavage site which stabilize S1 on the virion in the open RBD conformation and act epistatically to promote the fitness of variants bearing complex combinations of clinically significant mutations.

Microbiota-dependent proteolysis of gluten subverts diet-mediated protection against type 1 diabetes.

Diet and commensals can affect the development of autoimmune diseases like type 1 diabetes (T1D). However, whether dietary interventions are microbe-mediated was unclear. We found that a diet based on hydrolyzed casein (HC) as a protein source protects non-obese diabetic (NOD) mice in conventional and germ-free (GF) conditions via improvement in the physiology of insulin-producing cells to reduce autoimmune activation. The addition of gluten (a cereal protein complex associated with celiac disease) facilitates autoimmunity dependent on microbial proteolysis of gluten: T1D develops in GF animals monocolonized with Enterococcus faecalis harboring secreted gluten-digesting proteases but not in mice colonized with protease deficient bacteria. Gluten digestion by E. faecalis generates T cell-activating peptides and promotes innate immunity by enhancing macrophage reactivity to lipopolysaccharide (LPS). Gnotobiotic NOD Toll4-negative mice monocolonized with E. faecalis on an HC + gluten diet are resistant to T1D. These findings provide insights into strategies to develop dietary interventions to help protect humans against autoimmunity.

Multimodal surveillance of SARS-CoV-2 at a university enables development of a robust outbreak response framework.

BACKGROUND: Universities are vulnerable to infectious disease outbreaks, making them ideal environments to study transmission dynamics and evaluate mitigation and surveillance measures. Here, we analyze multimodal COVID-19-associated data collected during the 2020-2021 academic year at Colorado Mesa University and introduce a SARS-CoV-2 surveillance and response framework. METHODS: We analyzed epidemiological and sociobehavioral data (demographics, contact tracing, and WiFi-based co-location data) alongside pathogen surveillance data (wastewater and diagnostic testing, and viral genomic sequencing of wastewater and clinical specimens) to characterize outbreak dynamics and inform policy. We applied relative risk, multiple linear regression, and social network assortativity to identify attributes or behaviors associated with contracting SARS-CoV-2. To characterize SARS-CoV-2 transmission, we used viral sequencing, phylogenomic tools, and functional assays. FINDINGS: Athletes, particularly those on high-contact teams, had the highest risk of testing positive. On average, individuals who tested positive had more contacts and longer interaction durations than individuals who never tested positive. The distribution of contacts per individual was overdispersed, although not as overdispersed as the distribution of phylogenomic descendants. Corroboration via technical replicates was essential for identification of wastewater mutations. CONCLUSIONS: Based on our findings, we formulate a framework that combines tools into an integrated disease surveillance program that can be implemented in other congregate settings with limited resources. FUNDING: This work was supported by the National Science Foundation, the Hertz Foundation, the National Institutes of Health, the Centers for Disease Control and Prevention, the Massachusetts Consortium on Pathogen Readiness, the Howard Hughes Medical Institute, the Flu Lab, and the Audacious Project.

Analysis of 6.4 million SARS-CoV-2 genomes identifies mutations associated with fitness.

Repeated emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased fitness underscores the value of rapid detection and characterization of new lineages. We have developed PyR0, a hierarchical Bayesian multinomial logistic regression model that infers relative prevalence of all viral lineages across geographic regions, detects lineages increasing in prevalence, and identifies mutations relevant to fitness. Applying PyR0 to all publicly available SARS-CoV-2 genomes, we identify numerous substitutions that increase fitness, including previously identified spike mutations and many nonspike mutations within the nucleocapsid and nonstructural proteins. PyR0 forecasts growth of new lineages from their mutational profile, ranks the fitness of lineages as new sequences become available, and prioritizes mutations of biological and public health concern for functional characterization.

Conformational dynamics and allosteric modulation of the SARS-CoV-2 spike.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells through binding to angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural and dynamic data have shown that S can adopt multiple conformations, which controls the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging, we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and in the presence of the D614G mutation. We find that D614G modulates the energetics of the RBD position in a manner similar to ACE2 binding. We also find that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicate antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

Conformational dynamics and allosteric modulation of the SARS-CoV-2 spike.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells through binding to angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural and dynamic data have shown that S can adopt multiple conformations, which controls the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We find that D614G modulates the energetics of the RBD position in a manner similar to ACE2 binding. We also find that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicate antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant.

The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide, reaching near fixation in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and on cells rendered permissive by ectopic expression of human ACE2 or of ACE2 orthologs from various mammals, including Chinese rufous horseshoe bat and Malayan pangolin. D614G did not alter S protein synthesis, processing, or incorporation into SARS-CoV-2 particles, but D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts an interprotomer contact and that the conformation is shifted toward an ACE2 binding-competent state, which is modeled to be on pathway for virion membrane fusion with target cells. Consistent with this more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated.

Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant.

The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and cells rendered permissive by ectopic expression of various mammalian ACE2 orthologs. Nonetheless, D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts a critical interprotomer contact and that this dramatically shifts the S protein trimer conformation toward an ACE2-binding and fusion-competent state. Consistent with the more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated. These results indicate that D614G adopts conformations that make virion membrane fusion with the target cell membrane more probable but that D614G retains susceptibility to therapies that disrupt interaction of the SARS-CoV-2 S protein with the ACE2 receptor.

Polymorphic Immune Mechanisms Regulate Commensal Repertoire.

Environmental influences (infections and diet) strongly affect a host's microbiota. However, host genetics may influence commensal communities, as suggested by the greater similarity between the microbiomes of identical twins compared to non-identical twins. Variability of human genomes and microbiomes complicates the understanding of polymorphic mechanisms regulating the commensal communities. Whereas animal studies allow genetic modifications, they are sensitive to influences known as "cage" or "legacy" effects. Here, we analyze ex-germ-free mice of various genetic backgrounds, including immunodeficient and major histocompatibility complex (MHC) congenic strains, receiving identical input microbiota. The host's polymorphic mechanisms affect the gut microbiome, and both innate (anti-microbial peptides, complement, pentraxins, and enzymes affecting microbial survival) and adaptive (MHC-dependent and MHC-independent) pathways influence the microbiota. In our experiments, polymorphic mechanisms regulate only a limited number of microbial lineages (independently of their abundance). Our comparative analyses suggest that some microbes may benefit from the specific immune responses that they elicit.

Cyclophilin A protects HIV-1 from restriction by human TRIM5α.

The HIV-1 capsid (CA) protein lattice encases viral genomic RNA and regulates steps essential to target-cell invasion1. Cyclophilin A (CypA) has interacted with the CA of lentiviruses related to HIV-1 for millions of years2-7. Disruption of the CA-CypA interaction decreases HIV-1 infectivity in human cells8-12 but stimulates infectivity in non-human primate cells13-15. Genetic and biochemical data suggest that CypA protects HIV-1 from a CA-specific restriction factor in human cells16-20. Discovery of the CA-specific restriction factor tripartite-containing motif 5α (TRIM5α)21 and multiple, independently derived, TRIM5-CypA fusion genes4,5,15,22-26 pointed to human TRIM5α being the CypA-sensitive restriction factor. However, HIV-1 restriction by human TRIM5α in tumour cell lines is minimal21 and inhibition of such activity by CypA has not been detected27. Here, by exploiting reverse genetic tools optimized for primary human blood cells, we demonstrate that disruption of the CA-CypA interaction renders HIV-1 susceptible to potent restriction by human TRIM5α, with the block occurring before reverse transcription. Endogenous TRIM5α associated with virion cores as they entered the cytoplasm, but only when the CA-CypA interaction was disrupted. These experiments resolve the long-standing mystery of the role of CypA in HIV-1 replication by demonstrating that this ubiquitous cellular protein shields HIV-1 from previously inapparent restriction by human TRIM5α.

Intron-containing RNA from the HIV-1 provirus activates type I interferon and inflammatory cytokines.

HIV-1-infected people who take drugs that suppress viremia to undetectable levels are protected from developing AIDS. Nonetheless, HIV-1 establishes proviruses in long-lived CD4+ memory T cells, and perhaps other cell types, that preclude elimination of the virus even after years of continuous antiviral therapy. Here we show that the HIV-1 provirus activates innate immune signaling in isolated dendritic cells, macrophages, and CD4+ T cells. Immune activation requires transcription from the HIV-1 provirus and expression of CRM1-dependent, Rev-dependent, RRE-containing, unspliced HIV-1 RNA. If rev is provided in trans, all HIV-1 coding sequences are dispensable for activation except those cis-acting sequences required for replication or splicing. Our results indicate that the complex, post-transcriptional regulation intrinsic to HIV-1 RNA is detected by the innate immune system as a danger signal, and that drugs which disrupt HIV-1 transcription or HIV-1 RNA metabolism would add qualitative benefit to current antiviral drug regimens.

Primate immunodeficiency virus proteins Vpx and Vpr counteract transcriptional repression of proviruses by the HUSH complex.

Host factors that silence provirus transcription in CD4+ memory T cells help HIV-1 escape eradication by the host immune system and by antiviral drugs1. These same factors, however, must be overcome for HIV-1 to propagate. Here we show that Vpx and Vpr encoded by diverse primate immunodeficiency viruses activate provirus transcription. Vpx and Vpr are adaptor proteins for the DCAF1-CUL4A/B E3 ubiquitin ligase that degrade SAMHD1 and increase reverse transcription2-4. Nonetheless, Vpx and Vpr have effects on reporter gene expression that are not explained by SAMHD1 degradation5-8. A screen for factors that mimic these effects identified the human silencing hub (HUSH) complex, FAM208A (TASOR/RAP140), MPHOSPH8 (MPP8), PPHLN1 (PERIPHILIN) and MORC29-13. Vpx associated with the HUSH complex and decreased steady-state level of these proteins in a DCAF1/CUL4A/B/proteasome-dependent manner14,15. Replication kinetics of HIV-1 and SIVMAC was accelerated to a similar extent by vpx or FAM208A knockdown. Finally, vpx increased steady-state levels of LINE-1 ORF1p, as previously described for FAM208A disruption11. These results demonstrate that the HUSH complex represses primate immunodeficiency virus transcription, and that, to counteract this restriction, viral Vpx or Vpr proteins degrade the HUSH complex.

Cutting Edge: Commensal Microbiota Has Disparate Effects on Manifestations of Polyglandular Autoimmune Inflammation.

Polyglandular autoimmune inflammation accompanies type 1 diabetes (T1D) in NOD mice, affecting organs like thyroid and salivary glands. Although commensals are not required for T1D progression, germ-free (GF) mice had a very low degree of sialitis, which was restored by colonization with select microbial lineages. Moreover, unlike T1D, which is blocked in mice lacking MyD88 signaling adaptor under conventional, but not GF, housing conditions, sialitis did not develop in MyD88(-/-) GF mice. Thus, microbes and MyD88-dependent signaling are critical for sialitis development. The severity of sialitis did not correlate with the degree of insulitis in the same animal and was less sensitive to a T1D-reducing diet, but it was similar to T1D with regard to microbiota-dependent sexual dimorphism. The unexpected distinction in requirements for the microbiota for different autoimmune pathologies within the same organism is crucial for understanding the nature of microbial involvement in complex autoimmune disorders, including human autoimmune polyglandular syndromes.

Microbiota and autoimmunity: exploring new avenues.

Given the recognized role of the commensal microbiota in regulating host immunity to pathogens, it is not surprising that microbiota are also capable of regulating autoimmune responses. The underlying mechanisms of autoimmune regulation by the microbiota are just beginning to emerge. Here, we discuss possible pressure points toward the development of autoimmune diseases that can be influenced by the microbiota. Besides acting on the adaptive and innate arms of the immune response, the microbiota can affect the targets of autoimmunity directly, even during development in utero, and be involved in regulation of autoimmunity via interactions with hormones.

Gender bias in autoimmunity is influenced by microbiota.

Gender bias and the role of sex hormones in autoimmune diseases are well established. In specific pathogen-free nonobese diabetic (NOD) mice, females have 1.3-4.4 times higher incidence of type 1 diabetes (T1D). Germ-free (GF) mice lost the gender bias (female-to-male ratio 1.1-1.2). Gut microbiota differed in males and females, a trend reversed by male castration, confirming that androgens influence gut microbiota. Colonization of GF NOD mice with defined microbiota revealed that some, but not all, lineages overrepresented in male mice supported a gender bias in T1D. Although protection of males did not correlate with blood androgen concentration, hormone-supported expansion of selected microbial lineages may work as a positive-feedback mechanism contributing to the sexual dimorphism of autoimmune diseases. Gene-expression analysis suggested pathways involved in protection of males from T1D by microbiota. Our results favor a two-signal model of gender bias, in which hormones and microbes together trigger protective pathways.

Innate immune sensing of retroviral infection via Toll-like receptor 7 occurs upon viral entry.

Innate immune sensors are required for induction of pathogen-specific immune responses. Retroviruses are notorious for their ability to evade immune defenses and establish long-term persistence in susceptible hosts. However, some infected animals are able to develop efficient virus-specific immune responses, and thus can be employed for identification of critical innate virus-sensing mechanisms. With mice from two inbred strains that control retroviruses via adaptive immune mechanisms, we found that of all steps in viral replication, the ability to enter the host cell was sufficient to induce antivirus humoral immune responses. Virus sensing occurred in endosomes via a MyD88-Toll-like receptor 7-dependent mechanism and stimulated virus-neutralizing immunity independently of type I interferons. Thus, efficient adaptive immunity to retroviruses is induced in vivo by innate sensing of the early stages of retroviral infection.

Replication of beta- and gammaretroviruses is restricted in I/LnJ mice via the same genetic mechanism.

Mice of the I/LnJ inbred strain are unique in their ability to mount a robust and sustained humoral immune response capable of neutralizing infection with a betaretrovirus, mouse mammary tumor virus (MMTV). Virus-neutralizing antibodies (Abs) coat MMTV virions secreted by infected cells, preventing virus spread and hence the formation of mammary tumors. To investigate whether I/LnJ mice resist infection with other retroviruses besides MMTV, the animals were infected with murine leukemia virus (MuLV), a gammaretrovirus. MuLV-infected I/LnJ mice produced virus-neutralizing Abs that block virus transmission and virally induced disease. Generation of virus-neutralizing Abs required gamma interferon but was independent of interleukin-12. This unique mechanism of retrovirus resistance is governed by a single recessive gene, virus infectivity controller 1 (vic1), mapped to chromosome 17. In addition to controlling the antivirus humoral immune response, vic1 is also required for an antiviral cytotoxic response. Both types of responses were maintained in mice of the susceptible genetic background but congenic for the I/LnJ vic1 locus. Although the vic1-mediated resistance to MuLV resembles the mechanism of retroviral recovery controlled by the resistance to Friend virus 3 (rfv3) gene, the rfv3 gene has been mapped to chromosome 15 and confers resistance to MuLV but not to MMTV. Thus, we have identified a unique virus resistance mechanism that controls immunity against two distinct retroviruses.