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.

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.

Innate lymphoid cells and COVID-19 severity in SARS-CoV-2 infection.

Background: Risk of severe COVID-19 increases with age, is greater in males, and is associated with lymphopenia, but not with higher burden of SARS-CoV-2. It is unknown whether effects of age and sex on abundance of specific lymphoid subsets explain these correlations. Methods: Multiple regression was used to determine the relationship between abundance of specific blood lymphoid cell types, age, sex, requirement for hospitalization, duration of hospitalization, and elevation of blood markers of systemic inflammation, in adults hospitalized for severe COVID-19 (n = 40), treated for COVID-19 as outpatients (n = 51), and in uninfected controls (n = 86), as well as in children with COVID-19 (n = 19), recovering from COVID-19 (n = 14), MIS-C (n = 11), recovering from MIS-C (n = 7), and pediatric controls (n = 17). Results: This observational study found that the abundance of innate lymphoid cells (ILCs) decreases more than 7-fold over the human lifespan - T cell subsets decrease less than 2-fold - and is lower in males than in females. After accounting for effects of age and sex, ILCs, but not T cells, were lower in adults hospitalized with COVID-19, independent of lymphopenia. Among SARS-CoV-2-infected adults, the abundance of ILCs, but not of T cells, correlated inversely with odds and duration of hospitalization, and with severity of inflammation. ILCs were also uniquely decreased in pediatric COVID-19 and the numbers of these cells did not recover during follow-up. In contrast, children with MIS-C had depletion of both ILCs and T cells, and both cell types increased during follow-up. In both pediatric COVID-19 and MIS-C, ILC abundance correlated inversely with inflammation. Blood ILC mRNA and phenotype tracked closely with ILCs from lung. Importantly, blood ILCs produced amphiregulin, a protein implicated in disease tolerance and tissue homeostasis. Among controls, the percentage of ILCs that produced amphiregulin was higher in females than in males, and people hospitalized with COVID-19 had a lower percentage of ILCs that produced amphiregulin than did controls. Conclusions: These results suggest that, by promoting disease tolerance, homeostatic ILCs decrease morbidity and mortality associated with SARS-CoV-2 infection, and that lower ILC abundance contributes to increased COVID-19 severity with age and in males. Funding: This work was supported in part by the Massachusetts Consortium for Pathogen Readiness and NIH grants R37AI147868, R01AI148784, F30HD100110, 5K08HL143183.

Innate lymphoid cells and disease tolerance in SARS-CoV-2 infection.

Risk of severe COVID-19 increases with age, is greater in males, and is associated with lymphopenia, but not with higher burden of SARS-CoV-2. It is unknown whether effects of age and sex on abundance of specific lymphoid subsets explain these correlations. This study found that the abundance of innate lymphoid cells (ILCs) decreases more than 7-fold over the human lifespan - T cell subsets decrease less than 2-fold - and is lower in males than in females. After accounting for effects of age and sex, ILCs, but not T cells, were lower in adults hospitalized with COVID-19, independent of lymphopenia. Among SARS-CoV-2-infected adults, the abundance of ILCs, but not of T cells, correlated inversely with odds and duration of hospitalization, and with severity of inflammation. ILCs were also uniquely decreased in pediatric COVID-19 and the numbers of these cells did not recover during follow-up. In contrast, children with MIS-C had depletion of both ILCs and T cells, and both cell types increased during follow-up. In both pediatric COVID-19 and MIS-C, ILC abundance correlated inversely with inflammation. Blood ILC mRNA and phenotype tracked closely with ILCs from lung. Importantly, blood ILCs produced amphiregulin, a protein implicated in disease tolerance and tissue homeostasis, and the percentage of amphiregulin-producing ILCs was higher in females than in males. These results suggest that, by promoting disease tolerance, homeostatic ILCs decrease morbidity and mortality associated with SARS-CoV-2 infection, and that lower ILC abundance accounts for increased COVID-19 severity with age and in males.

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.

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.

Constrained Mutational Sampling of Amino Acids in HIV-1 Protease Evolution.

The evolution of HIV-1 protein sequences should be governed by a combination of factors including nucleotide mutational probabilities, the genetic code, and fitness. The impact of these factors on protein sequence evolution is interdependent, making it challenging to infer the individual contribution of each factor from phylogenetic analyses alone. We investigated the protein sequence evolution of HIV-1 by determining an experimental fitness landscape of all individual amino acid changes in protease. We compared our experimental results to the frequency of protease variants in a publicly available data set of 32,163 sequenced isolates from drug-naïve individuals. The most common amino acids in sequenced isolates supported robust experimental fitness, indicating that the experimental fitness landscape captured key features of selection acting on protease during viral infections of hosts. Amino acid changes requiring multiple mutations from the likely ancestor were slightly less likely to support robust experimental fitness than single mutations, consistent with the genetic code favoring chemically conservative amino acid changes. Amino acids that were common in sequenced isolates were predominantly accessible by single mutations from the likely protease ancestor. Multiple mutations commonly observed in isolates were accessible by mutational walks with highly fit single mutation intermediates. Our results indicate that the prevalence of multiple-base mutations in HIV-1 protease is strongly influenced by mutational sampling.

Human Anti-HIV-1 gp120 Monoclonal Antibodies with Neutralizing Activity Cloned from Humanized Mice Infected with HIV-1.

Broadly neutralizing, anti-HIV-1 gp120 mAbs have been isolated from infected individuals, and there is considerable interest in developing these reagents for Ab-based immunoprophylaxis and treatment. As a means to identify potentially new anti-HIV Abs, we exploited humanized NOD-scid IL2rγnull mice systemically infected with HIV-1 to generate a wide variety of Ag-specific human mAbs. The Abs were encoded by a diverse range of variable gene families and Ig classes, including IgA, and several showed significant levels of somatic mutation. Moreover, the isolated Abs not only bound target Ags with similar affinity as broadly neutralizing Abs, they also demonstrated neutralizing ability against multiple HIV-1 clades. The use of humanized mice will allow us to use our knowledge of HIV-1 gp120 structure and function, and the immune response targeting this protein, to generate native human prophylactic Abs to reduce the infection and spread of HIV-1.

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.

Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration.

HIV-2 and SIVMAC are AIDS-causing, zoonotic lentiviruses that jumped to humans and rhesus macaques, respectively, from SIVSM-bearing sooty mangabey monkeys. Cross-species transmission events such as these sometimes necessitate virus adaptation to species-specific, host restriction factors such as TRIM5. Here, a new human restriction activity is described that blocks viruses of the SIVSM/SIVMAC/HIV-2 lineage. Human T, B, and myeloid cell lines, peripheral blood mononuclear cells and dendritic cells were 4 to >100-fold less transducible by VSV G-pseudotyped SIVMAC, HIV-2, or SIVSM than by HIV-1. In contrast, transduction of six epithelial cell lines was equivalent to that by HIV-1. Substitution of HIV-1 CA with the SIVMAC or HIV-2 CA was sufficient to reduce HIV-1 transduction to the level of the respective vectors. Among such CA chimeras there was a general trend such that CAs from epidemic HIV-2 Group A and B isolates were the most infectious on human T cells, CA from a 1° sooty mangabey isolate was the least infectious, and non-epidemic HIV-2 Group D, E, F, and G CAs were in the middle. The CA-specific decrease in infectivity was observed with either HIV-1, HIV-2, ecotropic MLV, or ALV Env pseudotypes, indicating that it was independent of the virus entry pathway. As2O3, a drug that suppresses TRIM5-mediated restriction, increased human blood cell transduction by SIVMAC but not by HIV-1. Nonetheless, elimination of TRIM5 restriction activity did not rescue SIVMAC transduction. Also, in contrast to TRIM5-mediated restriction, the SIVMAC CA-specific block occurred after completion of reverse transcription and the formation of 2-LTR circles, but before establishment of the provirus. Transduction efficiency in heterokaryons generated by fusing epithelial cells with T cells resembled that in the T cells, indicative of a dominant-acting SIVMAC restriction activity in the latter. These results suggest that the nucleus of human blood cells possesses a restriction factor specific for the CA of HIV-2/SIVMAC/SIVSM and that cross-species transmission of SIVSM to human T cells necessitated adaptation of HIV-2 to this putative restriction factor.

Evidence for a pathogenic determinant in HIV-1 Nef involved in B cell dysfunction in HIV/AIDS.

B lymphocyte hyperactivation and elevated immunoglobulin levels (hypergammaglobulinemia) are pathogenic manifestations of HIV-1 infection. Here we provide evidence that these hallmarks are caused by a soluble factor whose production by infected macrophages is induced by the HIV-1 Nef protein. In vitro, HIV-1-infected macrophages or macrophages expressing Nef promoted B cell activation and differentiation to immunoglobulin-secreting cells. Nef-mediated activation of NF-kappaB in macrophages induced secretion of the acute-phase protein ferritin, and ferritin was necessary and sufficient for the observed Nef-dependent B cell changes. The extent of hypergammaglobulinemia in HIV-1-infected individuals correlated directly with plasma ferritin levels and with viral load. Furthermore, the induction of ferritin production and hypergammaglobulinemia was recapitulated when Nef was specifically expressed in macrophages and T cells of transgenic mice. Collectively, these results indicate that the HIV-1 Nef protein carries a pathogenic determinant that governs B cell defects in HIV-1 infection.