RESUMEN
The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.
Asunto(s)
COVID-19/patología , COVID-19/virología , Fusión de Membrana , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Serina Endopeptidasas/metabolismo , Internalización del Virus , Adulto , Anciano , Anciano de 80 o más Años , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Línea Celular , Membrana Celular/metabolismo , Membrana Celular/virología , Chlorocebus aethiops , Convalecencia , Femenino , Humanos , Sueros Inmunes/inmunología , Intestinos/patología , Intestinos/virología , Pulmón/patología , Pulmón/virología , Masculino , Persona de Mediana Edad , Mutación , Mucosa Nasal/patología , Mucosa Nasal/virología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Técnicas de Cultivo de Tejidos , Virulencia , Replicación ViralRESUMEN
During pathogen infection, antibodies can be carried into the infected cell, where they are detected by the ubiquitously expressed cytosolic antibody receptor TRIM21. Here we found that recognition of intracellular antibodies by TRIM21 activated immune signaling. TRIM21 catalyzed the formation of Lys63 (K63)-linked ubiquitin chains and stimulated the transcription factor pathways of NF-κB, AP-1, IRF3, IRF5 and IRF7. Activation resulted in the production of proinflammatory cytokines, modulation of natural killer stress ligands and induction of an antiviral state. Intracellular antibody signaling was abrogated by genetic deletion of TRIM21 and was restored by ectopic expression of TRIM21. The sensing of antibodies by TRIM21 was stimulated after infection by DNA or RNA nonenveloped viruses or intracellular bacteria. Thus, the antibody-TRIM21 detection system provides potent, comprehensive activation of the innate immune system independently of known pattern-recognition receptors.
Asunto(s)
Anticuerpos Antivirales/inmunología , Espacio Intracelular/inmunología , Espacio Intracelular/metabolismo , Receptores Fc/metabolismo , Ribonucleoproteínas/inmunología , Transducción de Señal , Virus/inmunología , Adenoviridae/inmunología , Animales , Anticuerpos Antivirales/química , Anticuerpos Antivirales/metabolismo , Complejo Antígeno-Anticuerpo/inmunología , Complejo Antígeno-Anticuerpo/metabolismo , Bacterias/inmunología , Línea Celular , Reacciones Cruzadas , Citocinas/biosíntesis , Humanos , Mediadores de Inflamación/metabolismo , Factores Reguladores del Interferón/metabolismo , Ratones , Simulación del Acoplamiento Molecular , FN-kappa B/metabolismo , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores de Reconocimiento de Patrones/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
HIV-1 uses inositol hexakisphosphate (IP6) to build a metastable capsid capable of delivering its genome into the host nucleus. Here, we show that viruses that are unable to package IP6 lack capsid protection and are detected by innate immunity, resulting in the activation of an antiviral state that inhibits infection. Disrupting IP6 enrichment results in defective capsids that trigger cytokine and chemokine responses during infection of both primary macrophages and T-cell lines. Restoring IP6 enrichment with a single mutation rescues the ability of HIV-1 to infect cells without being detected. Using a combination of capsid mutants and CRISPR-derived knockout cell lines for RNA and DNA sensors, we show that immune sensing is dependent upon the cGAS-STING axis and independent of capsid detection. Sensing requires the synthesis of viral DNA and is prevented by reverse transcriptase inhibitors or reverse transcriptase active-site mutation. These results demonstrate that IP6 is required to build capsids that can successfully transit the cell and avoid host innate immune sensing.
Asunto(s)
Cápside , Infecciones por VIH , Humanos , Cápside/metabolismo , Interacciones Huésped-Patógeno , Inmunidad Innata , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Proteínas de la Membrana/metabolismoRESUMEN
The HIV capsid self-assembles a protective conical shell that simultaneously prevents host sensing whilst permitting the import of nucleotides to drive DNA synthesis. This is accomplished through the construction of dynamic, highly charged pores at the centre of each capsid multimer. The clustering of charges required for dNTP import is strongly destabilising and it is proposed that HIV uses the metabolite IP6 to coordinate the pore during assembly. Here we have investigated the role of inositol phosphates in coordinating a ring of positively charged lysine residues (K25) that forms at the base of the capsid pore. We show that whilst IP5, which can functionally replace IP6, engages an arginine ring (R18) at the top of the pore, the lysine ring simultaneously binds a second IP5 molecule. Dose dependent removal of K25 from the pore severely inhibits HIV infection and concomitantly prevents DNA synthesis. Cryo-tomography reveals that K25A virions have a severe assembly defect that inhibits the formation of mature capsid cones. Monitoring both the kinetics and morphology of capsids assembled in vitro reveals that while mutation K25A can still form tubes, the ability of IP6 to drive assembly of capsid cones has been lost. Finally, in single molecule TIRF microscopy experiments, capsid lattices in permeabilised K25 mutant virions are rapidly lost and cannot be stabilised by IP6. These results suggest that the coordination of IP6 by a second charged ring in mature hexamers drives the assembly of conical capsids capable of reverse transcription and infection.
Asunto(s)
Cápside/metabolismo , VIH-1/fisiología , Lisina/metabolismo , Ácido Fítico/metabolismo , Ensamble de Virus/fisiología , Línea Celular , ADN Viral/biosíntesis , VIH-1/genética , VIH-1/metabolismo , Humanos , Microscopía Fluorescente , Nucleótidos/metabolismoRESUMEN
Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor ACE2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors may reduce but not abolish viral spread.
Asunto(s)
Fusión Celular , Furina/genética , Glicoproteína de la Espiga del Coronavirus/química , Internalización del Virus , Animales , COVID-19 , Sistemas CRISPR-Cas , Chlorocebus aethiops , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Estructura Terciaria de Proteína , SARS-CoV-2 , Serina Endopeptidasas , Células VeroRESUMEN
The majority of SARS-CoV-2 vaccines in use or advanced development are based on the viral spike protein (S) as their immunogen. S is present on virions as prefusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described against both open and closed conformations. The long-term success of vaccination strategies depends upon inducing antibodies that provide long-lasting broad immunity against evolving SARS-CoV-2 strains. Here, we have assessed the results of immunization in a mouse model using an S protein trimer stabilized in the closed state to prevent full exposure of the receptor binding site and therefore interaction with the receptor. We compared this with other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induced a T cell response and long-lived, strongly neutralizing antibody responses against 2019 SARS-CoV-2 and variants of concern P.1 and B.1.351. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralizing responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, immune responses than open spikes and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. We suggest that closed spikes, together with their improved stability and storage properties, may be a valuable component of refined, next-generation vaccines. IMPORTANCE Vaccines in use against SARS-CoV-2 induce immune responses against the spike protein. There is intense interest in whether the antibody response induced by vaccines will be robust against new variants, as well as in next-generation vaccines for use in previously infected or immunized individuals. We assessed the use as an immunogen of a spike protein engineered to be conformationally stabilized in the closed state where the receptor binding site is occluded. Despite occlusion of the receptor binding site, the spike induces potently neutralizing sera against multiple SARS-CoV-2 variants. Antibodies are raised against a different pattern of epitopes to those induced by other spike constructs, preferring conformational epitopes present in the closed conformation. Closed spikes, or mRNA vaccines based on their sequence, can be a valuable component of next-generation vaccines.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Epítopos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/inmunología , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Vacunas contra la COVID-19/química , Vacunas contra la COVID-19/inmunología , Epítopos/química , Epítopos/inmunología , Células HEK293 , Humanos , Ratones , Estabilidad Proteica , SARS-CoV-2/química , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Tripartite motif (TRIM) 21 is a cytosolic antibody receptor that neutralizes antibody-coated viruses that penetrate the cell and simultaneously activates innate immunity. Here we show that the conjugation of TRIM21 with K63-linked ubiquitin (Ub-(63)Ub) catalyzed by the sequential activity of nonredundant E2 Ub enzymes is required for its dual antiviral functions. TRIM21 is first labeled with monoubiquitin (monoUb) by the E2 Ube2W. The monoUb is a substrate for the heterodimeric E2 Ube2N/Ube2V2, resulting in TRIM21-anchored Ub-(63)Ub. Depletion of either E2 abolishes Ub-(63)Ub and Ub-(48)Ub conjugation of TRIM21, NF-κB signaling, and virus neutralization. The formation of TRIM21-Ub-(63)Ub precedes proteasome recruitment, and we identify an essential role for the 19S-resident and degradation-coupled deubiquitinase Poh1 in TRIM21 neutralization, signaling, and cytokine induction. This study elucidates a complex mechanism of step-wise ubiquitination and deubiquitination activities that allows contemporaneous innate immune signaling and neutralization by TRIM21.
Asunto(s)
Ribonucleoproteínas/metabolismo , Ubiquitinación , Animales , Línea Celular , Citocinas/genética , Humanos , Lisina/metabolismo , Ratones , Modelos Biológicos , FN-kappa B/metabolismo , Pruebas de Neutralización , Poliubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal , Especificidad por Sustrato , Transactivadores/metabolismo , Transcripción Genética , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
IgA is the most prevalent antibody type on mucosal surfaces and the second most prevalent antibody in circulation, yet its role in immune defense is not fully understood. Here we show that IgA is carried inside cells during virus infection, where it activates intracellular virus neutralization and innate immune signaling. Cytosolic IgA-virion complexes colocalize with the high-affinity antibody receptor tripartite motif-containing protein 21 (TRIM21) and are positive for lysine-48 ubiquitin chains. IgA neutralizes adenovirus infection in a TRIM21- and proteasome-dependent manner in both human and mouse cells. Translocated IgA also potently activates NF-κB signaling pathways in cells expressing TRIM21, whereas viral infection in the absence of antibody or TRIM21 is undetected. TRIM21 recognizes an epitope in IgG Fc that is not conserved in IgA; however, fluorescence anisotropy experiments demonstrate that direct binding to IgA is maintained. We use molecular modeling to show that TRIM21 forms a nonspecific hydrophobic seal around a ß-loop structure that is present in IgG, IgM, and IgA, explaining how TRIM21 achieves such remarkable broad antibody specificity. The findings demonstrate that the antiviral protection afforded by IgA extends to the intracellular cytosolic environment.
Asunto(s)
Infecciones por Adenoviridae/inmunología , Anticuerpos Neutralizantes/metabolismo , Inmunidad Innata , Inmunoglobulina A/metabolismo , Pruebas de Neutralización , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Citocinas/metabolismo , Células HeLa , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina M/metabolismo , Espacio Intracelular/metabolismo , Ratones , Modelos Moleculares , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Proteína que Contiene Valosina , Replicación ViralRESUMEN
Tripartite motif-containing 21 (TRIM21) is a cytosolic IgG receptor that mediates intracellular virus neutralization by antibody. TRIM21 targets virions for destruction in the proteasome, but it is unclear how a substrate as large as a viral capsid is degraded. Here, we identify the ATPase p97/valosin-containing protein (VCP), an enzyme with segregase and unfoldase activity, as a key player in this process. Depletion or catalytic inhibition of VCP prevents capsid degradation and reduces neutralization. VCP is required concurrently with the proteasome, as addition of inhibitor after proteasomal degradation has no effect. Moreover, our results suggest that it is the challenging nature of virus as a substrate that necessitates involvement of VCP, since intracellularly expressed IgG Fc is degraded in a VCP-independent manner. These results implicate VCP as an important host factor in antiviral immunity.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Pruebas de Neutralización , Ribonucleoproteínas/fisiología , Catálisis , Humanos , Proteína que Contiene ValosinaRESUMEN
Protection against bacterial and viral pathogens by antibodies has always been thought to end at the cell surface. Once inside the cell, a pathogen was understood to be safe from humoral immunity. However, it has now been found that antibodies can routinely enter cells attached to viral particles and mediate an intracellular immune response. Antibody-coated virions are detected inside the cell by means of an intracellular antibody receptor, TRIM21, which directs their degradation by recruitment of the ubiquitin-proteasome system. In this article we assess how this discovery alters our view of the way in which antibodies neutralise viral infection. We also consider the antiviral function of TRIM21 in the context of its other reported roles in immune signalling and autoimmunity. Finally, we discuss the conceptual implications of intracellular antibody immunity and how it alters our view of the discrete separation of extracellular and intracellular environments.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Inmunidad Humoral , Ribonucleoproteínas/inmunología , Adenoviridae/inmunología , Animales , Complejo Antígeno-Anticuerpo/inmunología , Autoantígenos/inmunología , Citosol/inmunología , Citosol/virología , Humanos , Ratones , Complejo de la Endopetidasa Proteasomal/inmunología , Transducción de Señal , Ubiquitinación , Acoplamiento Viral , Internalización del VirusRESUMEN
Antibodies provide effective antiviral immunity despite the fact that viruses escape into cells when they infect. Here we show that antibodies remain attached to viruses after cell infection and mediate an intracellular immune response that disables virions in the cytosol. We have discovered that cells possess a cytosolic IgG receptor, tripartite motif-containing 21 (TRIM21), which binds to antibodies with a higher affinity than any other IgG receptor in the human body. TRIM21 rapidly recruits to incoming antibody-bound virus and targets it to the proteasome via its E3 ubiquitin ligase activity. Proteasomal targeting leads to rapid degradation of virions in the cytosol before translation of virally encoded genes. Infection experiments demonstrate that at physiological antibody concentrations TRIM21 neutralizes viral infection. These results reveal an intracellular arm of adaptive immunity in which the protection mediated by antibodies does not end at the cell membrane but continues inside the cell to provide a last line of defense against infection.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Inmunidad/inmunología , Espacio Intracelular/inmunología , Ribonucleoproteínas/inmunología , Línea Celular , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Microesferas , Pruebas de Neutralización , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Inactivación de VirusRESUMEN
HIV-1 Gag metamorphoses inside each virion, from an immature lattice that forms during viral production to a mature capsid that drives infection. Here we show that the immature lattice is required to concentrate the cellular metabolite inositol hexakisphosphate (IP6) into virions to catalyze mature capsid assembly. Disabling the ability of HIV-1 to enrich IP6 does not prevent immature lattice formation or production of the virus. However, without sufficient IP6 molecules inside each virion, HIV-1 can no longer build a stable capsid and fails to become infectious. IP6 cannot be replaced by other inositol phosphate (IP) molecules, as substitution with other IPs profoundly slows mature assembly kinetics and results in virions with gross morphological defects. Our results demonstrate that while HIV-1 can become independent of IP6 for immature assembly, it remains dependent upon the metabolite for mature capsid formation.
Asunto(s)
VIH-1 , VIH-1/metabolismo , Cápside/metabolismo , Ensamble de Virus , Proteínas de la Cápside/metabolismo , Ácido Fítico/metabolismo , ViriónRESUMEN
The assembly of an HIV-1 particle begins with the construction of a spherical lattice composed of hexamer subunits of the Gag polyprotein. The cellular metabolite inositol hexakisphosphate (IP6) binds and stabilizes the immature Gag lattice via an interaction with the six-helix bundle (6HB), a crucial structural feature of Gag hexamers that modulates both virus assembly and infectivity. The 6HB must be stable enough to promote immature Gag lattice formation, but also flexible enough to be accessible to the viral protease, which cleaves the 6HB during particle maturation. 6HB cleavage liberates the capsid (CA) domain of Gag from the adjacent spacer peptide 1 (SP1) and IP6 from its binding site. This pool of IP6 molecules then promotes the assembly of CA into the mature conical capsid that is required for infection. Depletion of IP6 in virus-producer cells results in severe defects in assembly and infectivity of wild-type (WT) virions. Here we show that in an SP1 double mutant (M4L/T8I) with a hyperstable 6HB, IP6 can block virion infectivity by preventing CA-SP1 processing. Thus, depletion of IP6 in virus-producer cells markedly increases M4L/T8I CA-SP1 processing and infectivity. We also show that the introduction of the M4L/T8I mutations partially rescues the assembly and infectivity defects induced by IP6 depletion on WT virions, likely by increasing the affinity of the immature lattice for limiting IP6. These findings reinforce the importance of the 6HB in virus assembly, maturation, and infection and highlight the ability of IP6 to modulate 6HB stability.
Asunto(s)
VIH-1 , Ácido Fítico , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana , Proteínas de la Cápside/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , VIH-1/metabolismo , Mutación , Péptidos/metabolismo , Ácido Fítico/metabolismo , Virión/genética , Virión/metabolismoRESUMEN
Immunocompromised patients often fail to raise protective vaccine-induced immunity against the global emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Although monoclonal antibodies have been authorized for clinical use, most have lost their ability to potently neutralize the evolving Omicron subvariants. Thus, there is an urgent need for treatment strategies that can provide protection against these and emerging SARS-CoV-2 variants to prevent the development of severe coronavirus disease 2019. Here, we report on the design and characterization of a long-acting viral entry-blocking angiotensin-converting enzyme 2 (ACE2) dimeric fusion molecule. Specifically, a soluble truncated human dimeric ACE2 variant, engineered for improved binding to the receptor-binding domain of SARS-CoV-2, was fused with human albumin tailored for favorable engagement of the neonatal fragment crystallizable receptor (FcRn), which resulted in enhanced plasma half-life and allowed for needle-free transmucosal delivery upon nasal administration in human FcRn-expressing transgenic mice. Importantly, the dimeric ACE2-fused albumin demonstrated potent neutralization of SARS-CoV-2 immune escape variants.
RESUMEN
COVID-19 has stimulated the rapid development of new antibody and small molecule therapeutics to inhibit SARS-CoV-2 infection. Here we describe a third antiviral modality that combines the drug-like advantages of both. Bicycles are entropically constrained peptides stabilized by a central chemical scaffold into a bi-cyclic structure. Rapid screening of diverse bacteriophage libraries against SARS-CoV-2 Spike yielded unique Bicycle binders across the entire protein. Exploiting Bicycles' inherent chemical combinability, we converted early micromolar hits into nanomolar viral inhibitors through simple multimerization. We also show how combining Bicycles against different epitopes into a single biparatopic agent allows Spike from diverse variants of concern (VoC) to be targeted (Alpha, Beta, Delta and Omicron). Finally, we demonstrate in both male hACE2-transgenic mice and Syrian golden hamsters that both multimerized and biparatopic Bicycles reduce viraemia and prevent host inflammation. These results introduce Bicycles as a potential antiviral modality to tackle new and rapidly evolving viruses.
Asunto(s)
COVID-19 , SARS-CoV-2 , Masculino , Animales , Cricetinae , Ratones , Antivirales/farmacología , Péptidos/farmacología , Anticuerpos , Mesocricetus , Ratones Transgénicos , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
TRIM7 catalyzes the ubiquitination of multiple substrates with unrelated biological functions. This cross-reactivity is at odds with the specificity usually displayed by enzymes, including ubiquitin ligases. Here we show that TRIM7's extreme substrate promiscuity is due to a highly unusual binding mechanism, in which the PRYSPRY domain captures any ligand with a C-terminal helix that terminates in a hydrophobic residue followed by a glutamine. Many of the non-structural proteins found in RNA viruses contain C-terminal glutamines as a result of polyprotein cleavage by 3C protease. This viral processing strategy generates novel substrates for TRIM7 and explains its ability to inhibit Coxsackie virus and norovirus replication. In addition to viral proteins, cellular proteins such as glycogenin have evolved C-termini that make them a TRIM7 substrate. The 'helix-ΦQ' degron motif recognized by TRIM7 is reminiscent of the N-end degron system and is found in ~1% of cellular proteins. These features, together with TRIM7's restricted tissue expression and lack of immune regulation, suggest that viral restriction may not be its physiological function.
Asunto(s)
Infecciones por Caliciviridae , Glutamina , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas , Proteasas Virales 3C , Enterovirus , Humanos , Norovirus , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Virales/genéticaRESUMEN
HIV virion assembly begins with the construction of an immature lattice consisting of Gag hexamers. Upon virion release, protease-mediated Gag cleavage leads to a maturation event in which the immature lattice disassembles and the mature capsid assembles. The cellular metabolite inositiol hexakisphosphate (IP6) and maturation inhibitors (MIs) both bind and stabilize immature Gag hexamers, but whereas IP6 promotes virus maturation, MIs inhibit it. Here we show that HIV is evolutionarily constrained to maintain an immature lattice stability that ensures IP6 packaging without preventing maturation. Replication-deficient mutant viruses with reduced IP6 recruitment display increased infectivity upon treatment with the MI PF46396 (PF96) or the acquisition of second-site compensatory mutations. Both PF96 and second-site mutations stabilise the immature lattice and restore IP6 incorporation, suggesting that immature lattice stability and IP6 binding are interdependent. This IP6 dependence suggests that modifying MIs to compete with IP6 for Gag hexamer binding could substantially improve MI antiviral potency.
RESUMEN
Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, leads to respiratory symptoms that can be fatal. However, neurological symptoms have also been observed in some patients. The cause of these complications is currently unknown. Here, we use human-pluripotent-stem-cell-derived brain organoids to examine SARS-CoV-2 neurotropism. We find expression of viral receptor ACE2 in mature choroid plexus cells expressing abundant lipoproteins, but not in neurons or other cell types. We challenge organoids with SARS-CoV-2 spike pseudovirus and live virus to demonstrate viral tropism for choroid plexus epithelial cells but little to no infection of neurons or glia. We find that infected cells are apolipoprotein- and ACE2-expressing cells of the choroid plexus epithelial barrier. Finally, we show that infection with SARS-CoV-2 damages the choroid plexus epithelium, leading to leakage across this important barrier that normally prevents entry of pathogens, immune cells, and cytokines into cerebrospinal fluid and the brain.
Asunto(s)
Barrera Hematoencefálica/virología , Plexo Coroideo/virología , SARS-CoV-2/fisiología , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Chlorocebus aethiops , Células HEK293 , Humanos , Modelos Biológicos , Organoides/virología , Células Vero , Tropismo Viral , Internalización del VirusRESUMEN
The cytosolic antibody receptor TRIM21 possesses unique ubiquitination activity that drives broad-spectrum anti-pathogen targeting and underpins the protein depletion technology Trim-Away. This activity is dependent on formation of self-anchored, K63-linked ubiquitin chains by the heterodimeric E2 enzyme Ube2N/Ube2V2. Here we reveal how TRIM21 facilitates ubiquitin transfer and differentiates this E2 from other closely related enzymes. A tri-ionic motif provides optimally distributed anchor points that allow TRIM21 to wrap an Ube2N~Ub complex around its RING domain, locking the closed conformation and promoting ubiquitin discharge. Mutation of these anchor points inhibits ubiquitination with Ube2N/Ube2V2, viral neutralization and immune signalling. We show that the same mechanism is employed by the anti-HIV restriction factor TRIM5 and identify spatially conserved ionic anchor points in other Ube2N-recruiting RING E3s. The tri-ionic motif is exclusively required for Ube2N but not Ube2D1 activity and provides a generic E2-specific catalysis mechanism for RING E3s.
Asunto(s)
Lisina/metabolismo , Ribonucleoproteínas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/fisiología , Secuencias de Aminoácidos/genética , Factores de Restricción Antivirales , Biocatálisis , Cristalografía por Rayos X , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Mutación , Resonancia Magnética Nuclear Biomolecular , Unión Proteica/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
HIV-1 hijacks host proteins to promote infection. Here we show that HIV is also dependent upon the host metabolite inositol hexakisphosphate (IP6) for viral production and primary cell replication. HIV-1 recruits IP6 into virions using two lysine rings in its immature hexamers. Mutation of either ring inhibits IP6 packaging and reduces viral production. Loss of IP6 also results in virions with highly unstable capsids, leading to a profound loss of reverse transcription and cell infection. Replacement of one ring with a hydrophobic isoleucine core restores viral production, but IP6 incorporation and infection remain impaired, consistent with an independent role for IP6 in stable capsid assembly. Genetic knockout of biosynthetic kinases IPMK and IPPK reveals that cellular IP6 availability limits the production of diverse lentiviruses, but in the absence of IP6, HIV-1 packages IP5 without loss of infectivity. Together, these data suggest that IP6 is a critical cofactor for HIV-1 replication.