Adeno-associated viruses (AAVs) are increasingly used as gene therapy vectors. AAVs package their genome in a non-enveloped T = 1 icosahedral capsid of ~3.8 megaDalton, consisting of 60 subunits of 3 ...distinct viral proteins (VPs), which vary only in their N-terminus. While all three VPs play a role in cell-entry and transduction, their precise stoichiometry and structural organization in the capsid has remained elusive. Here we investigate the composition of several AAV serotypes by high-resolution native mass spectrometry. Our data reveal that the capsids assemble stochastically, leading to a highly heterogeneous population of capsids of variable composition, whereby even the single-most abundant VP stoichiometry represents only a small percentage of the total AAV population. We estimate that virtually every AAV capsid in a particular preparation has a unique composition. The systematic scoring of the simulations against experimental native MS data offers a sensitive new method to characterize these therapeutically important heterogeneous capsids.
The World Health Organization has declared the ongoing outbreak of COVID-19, which is caused by a novel coronavirus SARS-CoV-2, a pandemic. There is currently a lack of knowledge about the antibody ...response elicited from SARS-CoV-2 infection. One major immunological question concerns antigenic differences between SARS-CoV-2 and SARS-CoV. We address this question by analyzing plasma from patients infected by SARS-CoV-2 or SARS-CoV and from infected or immunized mice. Our results show that, although cross-reactivity in antibody binding to the spike protein is common, cross-neutralization of the live viruses may be rare, indicating the presence of a non-neutralizing antibody response to conserved epitopes in the spike. Whether such low or non-neutralizing antibody response leads to antibody-dependent disease enhancement needs to be addressed in the future. Overall, this study not only addresses a fundamental question regarding antigenicity differences between SARS-CoV-2 and SARS-CoV but also has implications for immunogen design and vaccine development.
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•Cross-reactive antigen binding is common between SARS-CoV and SARS-CoV-2•Cross-reactive antibody responses target both RBD and non-RBD regions•Cross-neutralization of live viruses may be rare between SARS-CoV and SARS-CoV-2
Lv et al. examine the antibody responses from patients infected by SARS-CoV-2 or SARS-CoV and from infected or immunized mice. The results show that cross-reactive binding to the spike protein is common, whereas cross-neutralization of the live viruses may be rare.
The Hsp90 molecular chaperone and its Cdc37 cochaperone help stabilize and activate more than half of the human kinome. However, both the mechanism by which these chaperones assist their "client" ...kinases and the reason why some kinases are addicted to Hsp90 while closely related family members are independent are unknown. Our structural understanding of these interactions is lacking, as no full-length structures of human Hsp90, Cdc37, or either of these proteins with a kinase have been elucidated. Here we report a 3.9 angstrom cryo–electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex. Surprisingly, the two lobes of Cdk4 are completely separated with the β4-β5 sheet unfolded. Cdc37 mimics part of the kinase N lobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase β5 strand and interacts with exposed N- and C-lobe interfaces, protecting the kinase in a trapped unfolded state. On the basis of this structure and an extensive amount of previously collected data, we propose unifying conceptual and mechanistic models of chaperone-kinase interactions.
The immune system can mount T cell responses against tumors; however, the antigen specificities of tumor-infiltrating lymphocytes (TILs) are not well understood. We used yeast-display libraries of ...peptide-human leukocyte antigen (pHLA) to screen for antigens of “orphan” T cell receptors (TCRs) expressed on TILs from human colorectal adenocarcinoma. Four TIL-derived TCRs exhibited strong selection for peptides presented in a highly diverse pHLA-A∗02:01 library. Three of the TIL TCRs were specific for non-mutated self-antigens, two of which were present in separate patient tumors, and shared specificity for a non-mutated self-antigen derived from U2AF2. These results show that the exposed recognition surface of MHC-bound peptides accessible to the TCR contains sufficient structural information to enable the reconstruction of sequences of peptide targets for pathogenic TCRs of unknown specificity. This finding underscores the surprising specificity of TCRs for their cognate antigens and enables the facile indentification of tumor antigens through unbiased screening.
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•Development of a human leukocyte antigen library for TCR ligand identification•Single-cell sequencing and phenotyping of T cells infiltrating human colon cancer•Ligand discovery for four tumor-derived T cell receptors•Identification of a shared non-mutated tumor antigen between two patients
A new approach for identifying T cell receptor ligands reveals insights into the specificity of tumor-infiltrating lymphocytes.
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•Vip3Aa is internalized into the Sf9 cells.•Vip3Aa is not associated with endosomes and mitochondria after internalization.•Vip3Aa interferes cell division and induces apoptosis in ...Sf9 cells.
Vip3Aa protein is produced by Bacillus thuringiensis during vegetative growth and displays high toxicity against a wide range of lepidopteran insect larvae such as Spodoptera exigua and Spodoptera frugiperda, both important insect pests worldwide. Vip3Aa protein is synthesized as a protoxin (proVip3Aa) and becomes activated by digestion with either trypsin or insect gut proteases. The activated Vip3Aa protein (actVip3Aa) binds to a specific receptor in the brush border epithelial midgut cells, causing cell death via apoptosis, possibly induced by its pore-forming activity. Here we investigated the actVip3Aa intracellular localization to explain the molecular mechanism leading to the cytotoxicity of Vip3Aa toxin. The Spodoptera frugiperda (Sf9) cell line was incubated with fluorescently labeled Vip3Aa, namely Alexa488-actVip3Aa, and the intracellular localization was analyzed through a laser scanning confocal microscope. The Alexa488-actVip3Aa was internalized into the Sf9 cells. Immunofluorescence detection demonstrated that Alexa488-actVip3Aa did not colocalize with early endosomes which is usually implicated in clathrin-mediated endocytosis, suggesting that the actVip3Aa does not use clathrin-dependent endocytosis to transport into the cytosol. Intracellular visualization also shows that actVip3Aa does not directly target to mitochondria upon entry into the cytosol. Following internalization, actVip3Aa causes cell division disruption that subsequently could trigger cell death via apoptosis.
Most vertebrate oocytes contain a Balbiani body, a large, non-membrane-bound compartment packed with RNA, mitochondria, and other organelles. Little is known about this compartment, though it ...specifies germline identity in many non-mammalian vertebrates. We show Xvelo, a disordered protein with an N-terminal prion-like domain, is an abundant constituent of Xenopus Balbiani bodies. Disruption of the prion-like domain of Xvelo, or substitution with a prion-like domain from an unrelated protein, interferes with its incorporation into Balbiani bodies in vivo. Recombinant Xvelo forms amyloid-like networks in vitro. Amyloid-like assemblies of Xvelo recruit both RNA and mitochondria in binding assays. We propose that Xenopus Balbiani bodies form by amyloid-like assembly of Xvelo, accompanied by co-recruitment of mitochondria and RNA. Prion-like domains are found in germ plasm organizing proteins in other species, suggesting that Balbiani body formation by amyloid-like assembly could be a conserved mechanism that helps oocytes function as long-lived germ cells.
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•The organelle content of the Balbiani body is held together by an Xvelo matrix•Xvelo forms amyloid-like networks in vitro, which can recruit RNA and mitochondria•Prion-like domain of Xvelo dictates specificity in amyloid assembly•Amyloid-like polymerization is conserved among vertebrate Balbiani body organizers
Amyloid-like self-assembly of a specific protein drives formation of a cellular compartment in oocytes.
•The successful infection and replication of MrNV in Sf9 cells should facilitate long-term and in-depth investigation of MrNV infection pathway.•MrNV internalization favors caveolin (CAV)-mediated ...pathway which can be halted and reactivated by genistein and okadaic acid.•Replication of MrNV (at 72h p.i.) resulted in cytopathic effects (CPE) and multiplication of virion number in the infected cells.
In this study we demonstrated that Macrobrachium rosenbergii nodavirus (MrNV) was able to internalize and replicate in Sf9 insect cells, with levels of infection altered by substances affecting the caveolin-(CAV) mediated endocytosis pathway. The use of Sf9 cells for efficient MrNV replication and propagation was demonstrated by confocal microscopy and PCR amplification, through which early viral binding and internalization were initially detectable at 30min post-infection; whereas at 72h, the distinguishable sign of late-MrNV infection was observable as the gradual accumulation of a cytopathic effect (CPE) in the cells, ultimately resulting in cellular disruption. Moreover, during the early period of infection, the MrNV signals were highly co-localized with CAV1 signals of the CAV-mediated endocytosis pathway. The use of genistein as an inhibitor of the CAV-mediated endocytosis pathway significantly reduced MrNV and CAV1 co-localization, and also reduced the levels of MrNV infection in Sf9 cells as shown by PCR and ELISA. Moreover, the addition of the pathway agonist okadaic acid not only recovered but also augmented both the levels of MrNV co-localization with CAV1 and of Sf9 infection in the presence of genistein inhibition; therefore demonstrating that MrNV infection in Sf9 cells was associated with the CAV-mediated endocytosis pathway machinery.
Efficient immune responses against viral infection are determined by sufficient activation of nucleic acid sensor-mediated innate immunity
. Coronavirus disease 2019, caused by severe acute ...respiratory syndrome coronavirus 2 (SARS-CoV-2), remains an ongoing global pandemic. It is an urgent challenge to clarify the innate recognition mechanism to control this virus. Here we show that retinoic acid-inducible gene-I (RIG-I) sufficiently restrains SARS-CoV-2 replication in human lung cells in a type I/III interferon (IFN)-independent manner. RIG-I recognizes the 3' untranslated region of the SARS-CoV-2 RNA genome via the helicase domains, but not the C-terminal domain. This new mode of RIG-I recognition does not stimulate its ATPase, thereby aborting the activation of the conventional mitochondrial antiviral-signaling protein-dependent pathways, which is in accordance with lack of cytokine induction. Nevertheless, the interaction of RIG-I with the viral genome directly abrogates viral RNA-dependent RNA polymerase mediation of the first step of replication. Consistently, genetic ablation of RIG-I allows lung cells to produce viral particles that expressed the viral spike protein. By contrast, the anti-SARS-CoV-2 activity was restored by all-trans retinoic acid treatment through upregulation of RIG-I protein expression in primary lung cells derived from patients with chronic obstructive pulmonary disease. Thus, our findings demonstrate the distinctive role of RIG-I as a restraining factor in the early phase of SARS-CoV-2 infection in human lung cells.
Cytoplasmic dynein-1 binds dynactin and cargo adaptor proteins to form a transport machine capable of long-distance processive movement along microtubules. However, it is unclear why dynein-1 moves ...poorly on its own or how it is activated by dynactin. Here, we present a cryoelectron microscopy structure of the complete 1.4-megadalton human dynein-1 complex in an inhibited state known as the phi-particle. We reveal the 3D structure of the cargo binding dynein tail and show how self-dimerization of the motor domains locks them in a conformation with low microtubule affinity. Disrupting motor dimerization with structure-based mutagenesis drives dynein-1 into an open form with higher affinity for both microtubules and dynactin. We find the open form is also inhibited for movement and that dynactin relieves this by reorienting the motor domains to interact correctly with microtubules. Our model explains how dynactin binding to the dynein-1 tail directly stimulates its motor activity.
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•Cryo-EM shows human cytoplasmic dynein-1 in its auto-inhibited, phi-particle form•Phi-particle disruption in vitro and in cells reveals its role in dynein regulation•There is a transition from phi-particle to open-dynein: both forms are inhibited•Dynactin binds open-dynein and aligns its motors to activate processive movement
Cryo-EM of human cytoplasmic dynein-1 reveals the mechanism underlying its auto-inhibition and activation.
Pathological degeneration of axons disrupts neural circuits and represents one of the hallmarks of neurodegeneration
. Sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) ...is a central regulator of this neurodegenerative process
, and its Toll/interleukin-1 receptor (TIR) domain exerts its pro-neurodegenerative action through NADase activity
. However, the mechanisms by which the activation of SARM1 is stringently controlled are unclear. Here we report the cryo-electron microscopy structures of full-length SARM1 proteins. We show that NAD
is an unexpected ligand of the armadillo/heat repeat motifs (ARM) domain of SARM1. This binding of NAD
to the ARM domain facilitated the inhibition of the TIR-domain NADase through the domain interface. Disruption of the NAD
-binding site or the ARM-TIR interaction caused constitutive activation of SARM1 and thereby led to axonal degeneration. These findings suggest that NAD
mediates self-inhibition of this central pro-neurodegenerative protein.
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