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 towards 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.

Repeated emergence of SARS-CoV-2 variants with increased fitness underscores the value of rapid detection and characterization of new lineages. We have developed PyR 0 , 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 PyR 0 to all publicly available SARS-CoV-2 genomes, we identify numerous substitutions that increase fitness, including previously identified spike mutations and many non-spike mutations within the nucleocapsid and nonstructural proteins. PyR 0 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.

AbstractVirus genome sequence variants that appear over the course of an outbreak can be exploited to map the trajectory of the virus from one susceptible host to another. While such variants are usually of no functional significance, in some cases they may allow the virus to transmit faster, change disease severity, or confer resistance to antiviral therapies. Since the discovery of SARS-CoV-2 as the cause of COVID-19, the virus has spread around the globe, and thousands of SARS-CoV-2 genomes have been sequenced. The rate of sequence variation among SARS-CoV-2 isolates is modest for an RNA virus but the enormous number of human-to-human transmission events has provided abundant opportunity for selection of sequence variants. Among these, the SARS-CoV-2 Spike protein variant, D614G, was not present in the presumptive common ancestor of this zoonotic virus but was first detected in late January in Germany and China. The D614G variant steadily increased in frequency and now constitutes >97% of isolates world-wide, raising the question whether D614G confers a replication advantage to SARS-CoV-2. Structural models predict that D614G would disrupt contacts between the S1 and S2 domains of the Spike protein and cause significant shifts in conformation. Using single-cycle vectors we showed that D614G is three to nine-fold more infectious than the ancestral form on human lung and colon cell lines, as well as on other human cell lines rendered permissive by ectopic expression of human ACE2 and TMPRSS2, or by ACE2 orthologues from pangolin, pig, dog, or cat. Nonetheless, monoclonal antibodies targeting the receptor binding domain of the SARS-CoV-2 Spike protein retain full neutralization potency. These results suggest that D614G was selected for increased human-to-human transmission, that it contributed to the rapidity of SARS-CoV-2 spread around the world, and that it does not confer resistance to antiviral therapies targeting the receptor binding domain.

HIV-1 infection is associated with heightened inflammation and excess risk of cardiovascular disease, cancer, and other complications. These pathologies persist despite antiretroviral therapy (ART). In two independent cohorts, we found that innate lymphoid cells (ILCs) were depleted in the blood and gut of people with HIV-1, even with effective ART. ILC depletion was associated with neutrophil infiltration of the gut lamina propria, type 1 interferon activation, increased microbial translocation, and natural killer (NK) cell skewing towards an inflammatory state with chromatin structure and phenotype typical of WNT transcription factor TCF7-dependent memory T cells. Cytokines that are elevated during acute HIV-1 infection reproduced the ILC and NK cell abnormalities ex vivo . These results demonstrate that inflammatory cytokines associated with HIV-1 infection irreversibly disrupt ILCs. This results in loss of gut epithelial integrity, microbial translocation, and memory NK cells with heightened inflammatory potential, and explains the chronic inflammation in people with HIV-1.

Recently, a large subfamily of nucleotide-binding and oligomerization domain-containing proteins that have an N-terminal pyrin-like domain and C-terminal leucine-rich repeats has been described. In this study, we identified PYNOD, a novel member of this family that lacks the leucine-rich repeats. We found that human PYNOD mRNA is expressed in various tissues and at high levels in heart, skeletal muscle and brain. It is also expressed in various cell lines, including haematopoietic cell lines. PYNOD oligomerizes and binds to ASC, an adaptor protein that plays a role in apoptotic and inflammatory signal transduction, and to caspase-1 and IL-1beta. PYNOD inhibits apoptosis-associated speck-like protein containing a CARD (ASC)-mediated NF-kappaB activation and apoptosis, and caspase-1-mediated IL-1beta maturation, and it does so in the presence and absence of constitutively active mutants of CARD12 and PYPAF1, which are enhancers of these processes. Thus, PYNOD is a novel regulator of apoptosis and inflammation.

PYPAF3 is a member of the PYRIN-containing apoptotic protease-activating factor-1-like proteins (PYPAFs, also called NALPs). Among the members of this family, PYPAF1, PYPAF5, PYPAF7, and NALP1 have been shown to induce caspase-1-dependent interleukin-1␤ secretion and NF-B activation in the presence of the adaptor molecule ASC. On the other hand, we recently discovered that PYNOD, another member of this family, is a suppressor of these responses. Here, we show that PYPAF3 is the second member that inhibits caspase-1-dependent interleukin-1␤ secretion. In contrast, PYPAF2/NALP2 does not inhibit this response but rather inhibits the NF-B activation that is induced by the combined expression of PYPAF1 and ASC. Both PYPAF2 and PYPAF3 mRNAs are broadly expressed in a variety of tissues; however, neither is expressed in skeletal muscle, and only PYPAF2 mRNA is expressed in heart and brain. They are also expressed in many cell lines of both hematopoietic and non-hematopoietic lineages. Stimulation of monocytic THP-1 cells with lipopolysaccharide or interleukin-1␤ induced PYPAF3 mRNA expression. Furthermore, the stable expression of PYPAF3 in THP-1 cells abrogated the ability of the cells to produce interleukin-1␤ in response to lipopolysaccharide. These results suggest that PYPAF3 is a feedback regulator of interleukin-1␤ secretion. Thus, PYPAF2 and PYPAF3, together with PYNOD, constitute an anti-inflammatory subgroup of PYPAFs.

Many members of the nucleotide-binding and oligomerization domain (NOD)- and leucine-rich-repeat–containing protein (NLR) family play important roles in pathogen recognition and inflammation. However, we previously reported that human PYNOD/NLRP10, an NLR-like protein consisting of a pyrin domain and a NOD, inhibits inflammatory signal mediated by caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in reconstitution experiments using HEK293 cells. In this study, we investigated the molecular mechanism of PYNOD’s anti-inflammatory activity in vitro and its expression and function in mice. Human PYNOD inhibited the autoprocessing of caspase-1 and caspase-1–mediated IL-1β processing and suppressed the aggregation of ASC, a hallmark of ASC activation. Interestingly, the NOD of human PYNOD was sufficient to inhibit caspase-1–mediated IL-1β secretion, whereas its pyrin domain was sufficient to inhibit ASC-mediated NF-κB activation and apoptosis and to reduce ASC’s ability to promote caspase-1–mediated IL-1β production. Mouse PYNOD protein was detected in the skin, tongue, heart, colon, peritoneal macrophages, and several cell lines of hematopoietic and myocytic lineages. Mouse PYNOD colocalized with ASC aggregates in LPS + R837-stimulated macrophages; however, unlike human PYNOD, mouse PYNOD failed to inhibit ASC aggregation. Macrophages and neutrophils from PYNOD-transgenic mice exhibited reduced IL-1β processing and secretion upon microbial infection, although mouse PYNOD failed to inhibit caspase-1 processing, which was inhibited by caspase-4 inhibitor z-LEED-fluoromethylketone. These results suggest that mouse PYNOD colocalizes with ASC and inhibits caspase-1–mediated IL-1β processing without inhibiting caspase-4 (mouse caspase-11)–mediated caspase-1 processing. Furthermore, PYNOD-transgenic mice were resistant to lethal endotoxic shock. Thus, PYNOD is the first example of an NLR that possesses an anti-inflammatory function in vivo.

Ndfip1 functions as both a recruiter and an activator of multiple HECT domain E3 ubiquitin ligases of the Nedd4 family. In this study, we demonstrate that Ndfip1 is involved in the ubiquitin-mediated degradation of mitochondrial antiviral signaling (MAVS), which is a key adaptor protein in RIG-I–like receptor–mediated immune signaling. We found that overexpression of Ndfip1 severely impaired MAVS and Sendai virus–mediated activation of IFN-stimulated response element, NF-κB, IFN-β promoter, and polyinosinic-polycytidylic acid or influenza virus RNA–stimulated IRF-3 phosphorylation, as well as the transcription of IFN-β. This functional interaction was confirmed by knockdown of Ndfip1, which facilitated MAVS-mediated downstream signaling and elevated MAVS protein levels. Further analysis indicated that Ndfip1 enhances both self-ubiquitination of HECT domain-containing E3 ubiquitin ligase Smurf1 and its interaction with MAVS, and eventually promotes MAVS degradation. In addition, the activation of IFN-β by MAVS, influenza virus RNA, polyinosinic-polycytidylic acid, and Sendai virus was enhanced in Ndfip1 knockdown cells. These results reveal that Ndfip1 is a potent inhibitor of MAVS-mediated antiviral response.