ABSTRACT
SARS-CoV-2 Delta and Omicron are globally relevant variants of concern. Although individuals infected with Delta are at risk of developing severe lung disease, infection with Omicron often causes milder symptoms, especially in vaccinated individuals1,2. The question arises of whether widespread Omicron infections could lead to future cross-variant protection, accelerating the end of the pandemic. Here we show that without vaccination, infection with Omicron induces a limited humoral immune response in mice and humans. Sera from mice overexpressing the human ACE2 receptor and infected with Omicron neutralize only Omicron, but not other variants of concern, whereas broader cross-variant neutralization was observed after WA1 and Delta infections. Unlike WA1 and Delta, Omicron replicates to low levels in the lungs and brains of infected animals, leading to mild disease with reduced expression of pro-inflammatory cytokines and diminished activation of lung-resident T cells. Sera from individuals who were unvaccinated and infected with Omicron show the same limited neutralization of only Omicron itself. By contrast, Omicron breakthrough infections induce overall higher neutralization titres against all variants of concern. Our results demonstrate that Omicron infection enhances pre-existing immunity elicited by vaccines but, on its own, may not confer broad protection against non-Omicron variants in unvaccinated individuals.
Subject(s)
COVID-19 , Cross Protection , SARS-CoV-2 , Vaccination , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/administration & dosage , Cross Protection/immunology , Cytokines , Humans , Mice , SARS-CoV-2/classification , SARS-CoV-2/immunology , Vaccination/statistics & numerical dataABSTRACT
Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the role of Mac1 catalytic activity in viral replication, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wild-type. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, the N40D mutant replicated at >1000-fold lower levels compared to the wild-type virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection. Our data validate the critical role of SARS-CoV-2 NSP3 Mac1 catalytic activity in viral replication and as a promising therapeutic target to develop antivirals.
Subject(s)
Coronavirus Papain-Like Proteases , SARS-CoV-2 , Virus Replication , Animals , Humans , Mice , Alanine , Antiviral Agents , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/genetics , Coronavirus Papain-Like Proteases/metabolismABSTRACT
An ability to activate latent HIV-1 expression could benefit many HIV cure strategies, but the first generation of latency reversing agents (LRAs) has proven disappointing. We evaluated AKT/mTOR activators as a potential new class of LRAs. Two glycogen synthase kinase-3 inhibitors (GSK-3i's), SB-216763 and tideglusib (the latter already in phase II clinical trials) that activate AKT/mTOR signaling were tested. These GSK-3i's reactivated latent HIV-1 present in blood samples from aviremic individuals on antiretroviral therapy (ART) in the absence of T cell activation, release of inflammatory cytokines, cell toxicity, or impaired effector function of cytotoxic T lymphocytes or NK cells. However, when administered in vivo to SIV-infected rhesus macaques on suppressive ART, tideglusib exhibited poor pharmacodynamic properties and resulted in no clear evidence of significant SIV latency reversal. Whether alternative pharmacological formulations or combinations of this drug with other classes of LRAs will lead to an effective in vivo latency-reversing strategy remains to be determined.IMPORTANCE If combined with immune therapeutics, latency reversing agents (LRAs) have the potential to reduce the size of the reservoir sufficiently that an engineered immune response can control the virus in the absence of antiretroviral therapy. We have identified a new class of LRAs that do not induce T-cell activation and that are able to potentiate, rather than inhibit, CD8+ T and NK cell cytotoxic effector functions. This new class of LRAs corresponds to inhibitors of glycogen synthase kinase-3. In this work, we have also studied the effects of one member of this drug class, tideglusib, in SIV-infected rhesus monkeys. When tested in vivo, however, tideglusib showed unfavorable pharmacokinetic properties, which resulted in lack of SIV latency reversal. The disconnect between our ex vivo and in vivo results highlights the importance of developing next generation LRAs with pharmacological properties that allow systemic drug delivery in relevant anatomical compartments harboring latent reservoirs.
ABSTRACT
It is unclear what mechanisms govern latent HIV infection in vivo or in primary cell models. To investigate these questions, we compared the HIV and cellular transcription profile in three primary cell models and peripheral CD4+ T cells from HIV-infected ART-suppressed individuals using RT-ddPCR and RNA-seq. All primary cell models recapitulated the block to HIV multiple splicing seen in cells from ART-suppressed individuals, suggesting that this may be a key feature of HIV latency in primary CD4+ T cells. Blocks to HIV transcriptional initiation and elongation were observed more variably among models. A common set of 234 cellular genes, including members of the minor spliceosome pathway, was differentially expressed between unstimulated and activated cells from primary cell models and ART-suppressed individuals, suggesting these genes may play a role in the blocks to HIV transcription and splicing underlying latent infection. These genes may represent new targets for therapies designed to reactivate or silence latently-infected cells.
Subject(s)
CD4-Positive T-Lymphocytes/virology , HIV Infections/virology , HIV-1/genetics , Transcriptome , Virus Latency/genetics , Anti-Retroviral Agents/therapeutic use , HIV-1/physiology , Humans , RNA, Viral/geneticsABSTRACT
BACKGROUND: HIV-infected cell lines are widely used to study latent HIV infection, which is considered the main barrier to HIV cure. We hypothesized that these cell lines differ from each other and from cells from HIV-infected individuals in the mechanisms underlying latency. RESULTS: To quantify the degree to which HIV expression is inhibited by blocks at different stages of HIV transcription, we employed a recently-described panel of RT-ddPCR assays to measure levels of 7 HIV transcripts ("read-through," initiated, 5' elongated, mid-transcribed/unspliced [Pol], distal-transcribed [Nef], polyadenylated, and multiply-sliced [Tat-Rev]) in bulk populations of latently-infected (U1, ACH-2, J-Lat) and productively-infected (8E5, activated J-Lat) cell lines. To assess single-cell variation and investigate cellular genes associated with HIV transcriptional blocks, we developed a novel multiplex qPCR panel and quantified single cell levels of 7 HIV targets and 89 cellular transcripts in latently- and productively-infected cell lines. The bulk cell HIV transcription profile differed dramatically between cell lines and cells from ART-suppressed individuals. Compared to cells from ART-suppressed individuals, latent cell lines showed lower levels of HIV transcriptional initiation and higher levels of polyadenylation and splicing. ACH-2 and J-Lat cells showed different forms of transcriptional interference, while U1 cells showed a block to elongation. Single-cell studies revealed marked variation between/within cell lines in expression of HIV transcripts, T cell phenotypic markers, antiviral factors, and genes implicated in latency. Expression of multiply-spliced HIV Tat-Rev was associated with expression of cellular genes involved in activation, tissue retention, T cell transcription, and apoptosis/survival. CONCLUSIONS: HIV-infected cell lines differ from each other and from cells from ART-treated individuals in the mechanisms governing latent HIV infection. These differences in viral and cellular gene expression must be considered when gauging the suitability of a given cell line for future research on HIV. At the same time, some features were shared across cell lines, such as low expression of antiviral defense genes and a relationship between productive infection and genes involved in survival. These features may contribute to HIV latency or persistence in vivo, and deserve further study using novel single cell assays such as those described in this manuscript.
Subject(s)
HIV-1/genetics , HIV-1/physiology , Transcriptome , Virus Activation/genetics , Virus Latency/genetics , Cell Line , DNA, Viral/analysis , Gene Expression Regulation, Viral , Host Microbial Interactions/genetics , Humans , Jurkat Cells , Multiplex Polymerase Chain Reaction , RNA, Viral/genetics , Transcription, Genetic , U937 CellsABSTRACT
Long interspersed element-1 (LINE-1 or L1) retrotransposons account for nearly 17% of human genomic DNA and represent a major evolutionary force that has reshaped the structure and function of the human genome. However, questions remain concerning both the frequency and the developmental timing of L1 retrotransposition in vivo and whether the mobility of these retroelements commonly results in insertional and post-insertional mechanisms of genomic injury. Cells exhibiting high rates of L1 retrotransposition might be especially at risk for such injury. We assessed L1 mRNA expression and L1 retrotransposition in two biologically relevant cell types, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), as well as in control parental human dermal fibroblasts (HDFs). Full-length L1 mRNA and the L1 open reading frame 1-encoded protein (ORF1p) were readily detected in hESCs and iPSCs, but not in HDFs. Sequencing analysis proved the expression of human-specific L1 element mRNAs in iPSCs. Bisulfite sequencing revealed that the increased L1 expression observed in iPSCs correlates with an overall decrease in CpG methylation in the L1 promoter region. Finally, retrotransposition of an engineered human L1 element was ~10-fold more efficient in iPSCs than in parental HDFs. These findings indicate that somatic cell reprogramming is associated with marked increases in L1 expression and perhaps increases in endogenous L1 retrotransposition, which could potentially impact the genomic integrity of the resultant iPSCs.
Subject(s)
Embryonic Stem Cells/cytology , Induced Pluripotent Stem Cells/cytology , Long Interspersed Nucleotide Elements , Mutagenesis, Insertional , Cell Differentiation , DNA Methylation , Humans , Promoter Regions, GeneticABSTRACT
Despite the success of combination antiretroviral therapy (ART) for individuals living with HIV, mild forms of HIV-associated neurocognitive disorder (HAND) continue to occur. Brain microglia form the principal target for HIV infection in the brain. It remains unknown how infection of these cells leads to neuroinflammation, neuronal dysfunction, and/or death observed in HAND. Utilizing two different inducible pluripotent stem cell-derived brain organoid models (cerebral and choroid plexus [ChP] organoids) containing microglia, we investigated the pathogenic changes associated with HIV infection. Infection of microglia was associated with a sharp increase in CCL2 and CXCL10 chemokine gene expression and the activation of many type I interferon stimulated genes (MX1, ISG15, ISG20, IFI27, IFITM3 and others). Production of the proinflammatory chemokines persisted at low levels after treatment of the cell cultures with ART, consistent with the persistence of mild HAND following clinical introduction of ART. Expression of multiple members of the S100 family of inflammatory genes sharply increased following HIV infection of microglia measured by single-cell RNA-seq. However, S100 gene expression was not limited to microglia but was also detected more broadly in uninfected stromal cells, mature and immature ChP cells, neural progenitor cells and importantly in bystander neurons suggesting propagation of the inflammatory response to bystander cells. Neurotransmitter transporter expression declined in uninfected neurons, accompanied by increased expression of genes promoting cellular senescence and cell death. Together, these studies underscore how an inflammatory response generated in HIV-infected microglia is propagated to multiple uninfected bystander cells ultimately resulting in the dysfunction and death of bystander neurons.
ABSTRACT
Hybrid immunity against SARS-CoV-2 has not been well studied in pregnancy. We conducted a comprehensive analysis of neutralizing antibodies (nAb) and binding antibodies in pregnant individuals who received mRNA vaccination, natural infection, or both. A third vaccine dose augmented nAb levels compared to the two-dose regimen or natural infection alone; this effect was more pronounced in hybrid immunity. There was reduced anti-Omicron nAb, but the maternal-fetal transfer efficiency remained comparable to that of other variants. Vaccine-induced nAbs were transferred more efficiently than infection-induced nAbs. Anti-spike receptor binding domain (RBD) IgG was associated with nAb against wild-type (Wuhan-Hu-1) following breakthrough infection. Both vaccination and infection-induced anti-RBD IgA, which was more durable than anti-nucleocapsid IgA. IgA response was attenuated in pregnancy compared to non-pregnant controls. These data provide additional evidence of augmentation of humoral immune responses in hybrid immunity in pregnancy.
ABSTRACT
SARS-CoV-2 continues to pose a threat to public health. Current therapeutics remain limited to direct acting antivirals that lack distinct mechanisms of action and are already showing signs of viral resistance. The virus encodes an ADP-ribosylhydrolase macrodomain (Mac1) that plays an important role in the coronaviral lifecycle by suppressing host innate immune responses. Genetic inactivation of Mac1 abrogates viral replication in vivo by potentiating host innate immune responses. However, it is unknown whether this can be achieved by pharmacologic inhibition and can therefore be exploited therapeutically. Here we report a potent and selective lead small molecule, AVI-4206, that is effective in an in vivo model of SARS-CoV-2 infection. Cellular models indicate that AVI-4206 has high target engagement and can weakly inhibit viral replication in a gamma interferon- and Mac1 catalytic activity-dependent manner; a stronger antiviral effect for AVI-4206 is observed in human airway organoids. In an animal model of severe SARS-CoV-2 infection, AVI-4206 reduces viral replication, potentiates innate immune responses, and leads to a survival benefit. Our results pharmacologically validate Mac1 as a therapeutic target via a novel immune-restoring mechanism that could potentially synergize with existing therapies targeting distinct, essential aspects of the coronaviral life cycle. This approach could be more widely used to target other viral macrodomains to develop antiviral therapeutics beyond COVID-19.
ABSTRACT
Halogenated phenolic compounds (HPCs) including hydroxylated polychlorobiphenyls (OH-PCBs) and hydroxylated polybromodiphenyl-ethers (OH-PBDEs) can be persistent organic pollutant (POP) metabolites or natural marine compounds. Structurally similar to thyroid hormones (THs), they are retained in blood, transported through selective barriers, and the cause of endocrine and neuronal POP effects. This study presents a meta-analysis of HPC burdens in human and wildlife tissues, including OH-PCBs, OH-PBDEs, Pentachlorophenol, and polybromophenols. HPC blood plasma levels were also compared to known in vitro and in vivo toxicological effect concentrations. Blood, highly perfused, and fetal tissues contained the highest levels of HPCs. Plasma concentrations of analyzed OH-PCBs/PBDEs ranged from 0.1 to 100 nM in humans and up to 240, 454, 800, and 7650 nM for birds, fish, cetaceans, and other mammals, respectively. These concentrations fully fall within the in vitro effect concentrations reported in literature for HPCs of 0.05-10000 nM. We strongly advise further study of HPC blood levels in the general population, children, and fetal tissue to establish background levels and the risk at sensitive development stages. As not all HPCs are, or can be, chemically analyzed, the application of additional bioanalysis might reveal an even greater toxicological relevance of HPCs. In addition, metabolic activation should always be included within in vitro hazard assessment of POPs.
Subject(s)
Environmental Pollutants/toxicity , Phenols/toxicity , Animals , Environmental Monitoring , Environmental Pollutants/metabolism , Humans , Phenols/metabolism , Polychlorinated Biphenyls/metabolism , Polychlorinated Biphenyls/toxicityABSTRACT
Traditional sediment risk assessment predominantly considers the hazard derived from legacy contaminants that are present in nonpolar sediment extracts, such as polychlorinated biphenyls (PCBs), dioxins, furans (PCDD/Fs), and polyaromatic hydrocarbons (PAHs). Although in vivo experiments with these compounds have shown to be thyroid hormone disrupting (THD), in vitro their THD potency is not observed in nonpolar sediment extracts. This is hypothesized to be due to the absence of in vitro biotransformation which will result in bioactivation of the lipophilic compounds into THD hydroxyl metabolites. This study reveals that indeed metabolically activated nonpolar contaminants in sediments can competitively bind to thyroid hormone transport proteins. Sediment fractions were incubated with S9 rat microsomes, and the metabolites were extracted with a newly developed method that excludes most of the lipids to avoid interference in the applied nonradioactive 96-well plate TTR competitive binding assay. Metabolic activation increased the TTR binding potency of nonpolar fractions of POP-polluted sediments up to 100 times, resulting in potencies up to 240 nmol T4 equivalents/g sediment equivalent (nmol T4-Eq/g SEQ). This demonstrates that a more realistic in vitro sediment THD risk characterization should also include testing of both polar and medium polar sediment extracts for THD, as well as bioactivated nonpolar sediment fractions to prevent underestimation of its toxic potency.
Subject(s)
Biotransformation , Geologic Sediments/chemistry , Hydrocarbons, Chlorinated/chemistry , Polycyclic Aromatic Hydrocarbons/chemistry , Thyroid Hormones/physiology , Animals , Chromatography, Gel , Hydrocarbons, Chlorinated/pharmacokinetics , Microsomes, Liver/metabolism , Polycyclic Aromatic Hydrocarbons/pharmacokinetics , Rats , Spectrometry, FluorescenceABSTRACT
Progress in understanding long COVID and developing effective therapeutics is hampered in part by the lack of suitable animal models. Here we used ACE2-transgenic mice recovered from Omicron (BA.1) infection to test for pulmonary and behavioral post-acute sequelae. Through in-depth phenotyping by CyTOF, we demonstrate that naïve mice experiencing a first Omicron infection exhibit profound immune perturbations in the lung after resolving acute infection. This is not observed if mice were first vaccinated with spike-encoding mRNA. The protective effects of vaccination against post-acute sequelae were associated with a highly polyfunctional SARS-CoV-2-specific T cell response that was recalled upon BA.1 breakthrough infection but not seen with BA.1 infection alone. Without vaccination, the chemokine receptor CXCR4 was uniquely upregulated on multiple pulmonary immune subsets in the BA.1 convalescent mice, a process previously connected to severe COVID-19. Taking advantage of recent developments in machine learning and computer vision, we demonstrate that BA.1 convalescent mice exhibited spontaneous behavioral changes, emotional alterations, and cognitive-related deficits in context habituation. Collectively, our data identify immunological and behavioral post-acute sequelae after Omicron infection and uncover a protective effect of vaccination against post-acute pulmonary immune perturbations.
ABSTRACT
Although the SARS-CoV-2 Omicron variant (BA.1) spread rapidly across the world and effectively evaded immune responses, its viral fitness in cell and animal models was reduced. The precise nature of this attenuation remains unknown as generating replication-competent viral genomes is challenging because of the length of the viral genome (~30 kb). Here, we present a plasmid-based viral genome assembly and rescue strategy (pGLUE) that constructs complete infectious viruses or noninfectious subgenomic replicons in a single ligation reaction with >80% efficiency. Fully sequenced replicons and infectious viral stocks can be generated in 1 and 3 weeks, respectively. By testing a series of naturally occurring viruses as well as Delta-Omicron chimeric replicons, we show that Omicron nonstructural protein 6 harbors critical attenuating mutations, which dampen viral RNA replication and reduce lipid droplet consumption. Thus, pGLUE overcomes remaining barriers to broadly study SARS-CoV-2 replication and reveals deficits in nonstructural protein function underlying Omicron attenuation.
Subject(s)
COVID-19 , Coronavirus Nucleocapsid Proteins , SARS-CoV-2 , Animals , Coronavirus Nucleocapsid Proteins/genetics , Genome, Viral/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Subgenomic RNA/geneticsABSTRACT
Although the SARS-CoV-2 Omicron variant (BA.1) spread rapidly across the world and effectively evaded immune responses, its viral fitness in cell and animal models was reduced. The precise nature of this attenuation remains unknown as generating replication-competent viral genomes is challenging because of the length of the viral genome (30kb). Here, we designed a plasmid-based viral genome assembly and resc ue strategy (pGLUE) that constructs complete infectious viruses or noninfectious subgenomic replicons in a single ligation reaction with >80% efficiency. Fully sequenced replicons and infectious viral stocks can be generated in 1 and 3 weeks, respectively. By testing a series of naturally occurring viruses as well as Delta-Omicron chimeric replicons, we show that Omicron nonstructural protein 6 harbors critical attenuating mutations, which dampen viral RNA replication and reduce lipid droplet consumption. Thus, pGLUE overcomes remaining barriers to broadly study SARS-CoV-2 replication and reveals deficits in nonstructural protein function underlying Omicron attenuation.
ABSTRACT
The emergence of SARS-CoV-2 recombinants is of particular concern as they can result in a sudden increase in immune evasion due to antigenic shift. Recent recombinants XBB and XBB.1.5 have higher transmissibility than previous recombinants such as "Deltacron." We hypothesized that immunity to a SARS-CoV-2 recombinant depends on prior exposure to its parental strains. To test this hypothesis, we examined whether Delta or Omicron (BA.1 or BA.2) immunity conferred through infection, vaccination, or breakthrough infection could neutralize Deltacron and XBB/XBB.1.5 recombinants. We found that Delta, BA.1, or BA.2 breakthrough infections provided better immune protection against Deltacron and its parental strains than did the vaccine booster. None of the sera were effective at neutralizing the XBB lineage or its parent BA.2.75.2, except for the sera from the BA.2 breakthrough group. These results support our hypothesis. In turn, our findings underscore the importance of multivalent vaccines that correspond to the antigenic profile of circulating variants of concern and of variant-specific diagnostics that may guide public health and individual decisions in response to emerging SARS-CoV-2 recombinants.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/prevention & control , Vaccination , Antigenic Drift and Shift , Breakthrough Infections , Antibodies, Neutralizing , Antibodies, ViralABSTRACT
Members of the APOBEC3 (A3) family of cytidine deaminase enzymes act as host defense mechanisms limiting both infections by exogenous retroviruses and mobilization of endogenous retrotransposons. Previous studies revealed that the overexpression of some A3 proteins could restrict engineered human Long INterspersed Element-1 (LINE-1 or L1) retrotransposition in HeLa cells. However, whether endogenous A3 proteins play a role in restricting L1 retrotransposition remains largely unexplored. Here, we show that HeLa cells express endogenous A3B and A3C, whereas human embryonic stem cells (hESCs) express A3B, A3C, A3DE, A3F, and A3G. To study the relative contribution of endogenous A3 proteins in restricting L1 retrotransposition, we first generated small hairpin RNAs (shRNAs) to suppress endogenous A3 mRNA expression, and then assessed L1 mobility using a cell-based L1 retrotransposition assay. We demonstrate that in both HeLa and hESCs, shRNA-based knockdown of A3B promotes a â¼2-3.7-fold increase in the retrotransposition efficiency of an engineered human L1. Knockdown of the other A3s produced no significant increase in L1 activity. Thus, A3B appears to restrict engineered L1 retrotransposition in a broad range of cell types, including pluripotent cells.
Subject(s)
Cytidine Deaminase/metabolism , Embryonic Stem Cells/metabolism , Long Interspersed Nucleotide Elements/physiology , Pluripotent Stem Cells/metabolism , Cell Line, Transformed , Cytidine Deaminase/genetics , Embryonic Stem Cells/cytology , Gene Expression Regulation, Enzymologic/physiology , HeLa Cells , Humans , Minor Histocompatibility Antigens , Pluripotent Stem Cells/cytology , RNA, Messenger/biosynthesis , RNA, Messenger/geneticsABSTRACT
Rfv3 is an autosomal dominant gene that influences the recovery of resistant mice from Friend retrovirus (FV) infection by limiting viremia and promoting a more potent neutralizing antibody response. We previously reported that Rfv3 is encoded by Apobec3, an innate retrovirus restriction factor. However, it was recently suggested that the Rfv3 susceptible phenotype of high viremia at 28 days postinfection (dpi) was more dominantly controlled by the B-cell-activating factor receptor (BAFF-R), a gene that is linked to but located outside the genetically mapped region containing Rfv3. Although one prototypical Rfv3 susceptible mouse strain, A/WySn, indeed contains a dysfunctional BAFF-R, two other Rfv3 susceptible strains, BALB/c and A.BY, express functional BAFF-R genes, determined on the basis of genotyping and B-cell immunophenotyping. Furthermore, transcomplementation studies in (C57BL/6 [B6] × BALB/c)F(1) and (B6 × A.BY)F(1) mice revealed that the B6 Apobec3 gene significantly influences recovery from FV viremia, cellular infection, and disease at 28 dpi. Finally, the Rfv3 phenotypes of prototypic B6, A.BY, A/WySn, and BALB/c mouse strains correlate with reported Apobec3 mRNA expression levels. Overall, these findings argue against the generality of BAFF-R polymorphisms as a dominant mechanism to explain the Rfv3 recovery phenotype and further strengthen the evidence that Apobec3 encodes Rfv3.
Subject(s)
Cytidine Deaminase/deficiency , Friend murine leukemia virus/physiology , Friend murine leukemia virus/pathogenicity , Virus Replication , Animals , B-Cell Activation Factor Receptor/genetics , B-Cell Activation Factor Receptor/metabolism , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , Genotype , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Retroviridae Infections , Tumor Virus InfectionsABSTRACT
Apobec3/Rfv3 is an innate immune factor that promotes the neutralizing Ab response against Friend retrovirus (FV) in infected mice. Based on its evolutionary relationship to activation-induced deaminase, Apobec3 might directly influence Ab class switching and affinity maturation independently of viral infection. Alternatively, the antiviral activity of Apobec3 may indirectly influence neutralizing Ab responses by reducing early FV-induced pathology in critical immune compartments. To distinguish between these possibilities, we immunized wild-type and Apobec3-deficient C57BL/6 (B6) mice with (4-hydroxy-3-nitrophenyl) acetyl (NP) hapten and evaluated the binding affinity of the resultant NP-specific Abs. These studies revealed similar affinity maturation of NP-specific IgG1 Abs between wild-type and Apobec3-deficient mice in the absence of FV infection. In contrast, hapten-specific Ab affinity maturation was significantly compromised in Apobec3-deficient mice infected with FV. In highly susceptible (B6 x A.BY)F(1) mice, the B6 Apobec3 gene protected multiple cell types in the bone marrow and spleen from acute FV infection, including erythroid, B, T, and myeloid cells. In addition, B6 Apobec3 deficiency was associated with elevated Ig levels, but decreased induction of splenic germinal center B cells and plasmablasts during acute FV infection. These data suggest that Apobec3 indirectly influences FV-specific neutralizing Ab responses by reducing virus-induced immune dysfunction. These findings raise the possibility that enabling Apobec3 activity during acute infection with human pathogenic retroviruses, such as HIV-1, may similarly facilitate stronger virus-specific neutralizing Ab responses.
Subject(s)
Antibodies, Neutralizing/immunology , Antibody Affinity/immunology , Cytidine Deaminase/immunology , Friend murine leukemia virus/immunology , Animals , Antibodies, Viral/immunology , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , Female , Flow Cytometry , Humans , Immunization , Leukemia, Experimental/genetics , Leukemia, Experimental/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Nitrophenols/administration & dosage , Nitrophenols/immunology , Phenylacetates/administration & dosage , Phenylacetates/immunology , Retroviridae Infections/genetics , Retroviridae Infections/immunology , Reverse Transcriptase Polymerase Chain Reaction , Spleen/immunology , Spleen/metabolism , Spleen/pathology , Tumor Virus Infections/genetics , Tumor Virus Infections/immunologyABSTRACT
Coronavirus disease 2019 (COVID-19) is frequently associated with neurological deficits, but how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces these effects remains unclear. Here, we show that astrocytes are readily infected by SARS-CoV-2, but surprisingly, neuropilin-1, not angiotensin-converting enzyme 2 (ACE2), serves as the principal receptor mediating cell entry. Infection is further positively modulated by the two-pore segment channel 2 (TPC2) protein that regulates membrane trafficking and endocytosis. Astrocyte infection produces a pathological response closely resembling reactive astrogliosis characterized by elevated type I interferon (IFN) production, increased inflammation, and the decreased expression of transporters of water, ions, choline, and neurotransmitters. These combined events initiated within astrocytes produce a hostile microenvironment that promotes the dysfunction and death of uninfected bystander neurons. IMPORTANCE SARS-CoV-2 infection primarily targets the lung but may also damage other organs, including the brain, heart, kidney, and intestine. Central nervous system (CNS) pathologies include loss of smell and taste, headache, delirium, acute psychosis, seizures, and stroke. Pathological loss of gray matter occurs in SARS-CoV-2 infection, but it is unclear whether this is due to direct viral infection, indirect effects associated with systemic inflammation, or both. Here, we used induced pluripotent stem cell (iPSC)-derived brain organoids and primary human astrocytes from the cerebral cortex to study direct SARS-CoV-2 infection. Our findings support a model where SARS-CoV-2 infection of astrocytes produces a panoply of changes in the expression of genes regulating innate immune signaling and inflammatory responses. The deregulation of these genes in astrocytes produces a microenvironment within the CNS that ultimately disrupts normal neuron function, promoting neuronal cell death and CNS deficits.
Subject(s)
COVID-19 , Humans , SARS-CoV-2/physiology , Astrocytes , Neuropilin-1 , Brain , Inflammation , Neurons , OrganoidsABSTRACT
The spread of SARS-CoV-2 has led to a devastating pandemic, with infections resulting in a range of symptoms collectively known as COVID-19. The full repertoire of human tissues and organs susceptible to infection is an area of active investigation, and some studies have implicated the reproductive system. The effects of COVID-19 on human reproduction remain poorly understood, and particularly the impact on early embryogenesis and establishment of a pregnancy are not known. In this work, we explore the susceptibility of early human embryos to SARS-CoV-2 infection. By using RNA-seq and immunofluorescence, we note that ACE2 and TMPRSS2, two canonical cell entry factors for SARS-CoV-2, are co-expressed in cells of the trophectoderm in blastocyst-stage preimplantation embryos. For the purpose of viral entry studies, we used fluorescent reporter virions pseudotyped with Spike (S) glycoprotein from SARS-CoV-2, and we observe robust infection of trophectoderm cells. This permissiveness could be attenuated with blocking antibodies targeting S or ACE2. When exposing human blastocysts to the live, fully infectious SARS-CoV-2, we detected cases of infection that compromised embryo health. Therefore, we identify a new human target tissue for SARS-CoV-2 with potential medical implications for reproductive health during the COVID-19 pandemic and its aftermath.