Elimination of Aicardi–Goutières syndrome protein SAMHD1 activates cellular innate immunity and suppresses SARS-CoV-2 replication

The lack of antiviral innate immune responses during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is characterized by limited production of interferons (IFNs). One protein associated with Aicardi–Goutières syndrome, SAMHD1, has been shown to negatively regulate the IFN-1 signaling pathway. However, it is unclear whether elevated IFN signaling associated with genetic loss of SAMHD1 would affect SARS-CoV-2 replication. In this study, we established in vitro tissue culture model systems for SARS-CoV-2 and human coronavirus OC43 infections in which SAMHD1 protein expression was absent as a result of CRISPR–Cas9 gene KO or lentiviral viral protein X–mediated proteosomal degradation. We show that both SARS-CoV-2 and human coronavirus OC43 replications were suppressed in SAMHD1 KO 293T and differentiated THP-1 macrophage cell lines. Similarly, when SAMHD1 was degraded by virus-like particles in primary monocyte-derived macrophages, we observed lower levels of SARS-CoV-2 RNA. The loss of SAMHD1 in 293T and differentiated THP-1 cells resulted in upregulated gene expression of IFNs and innate immunity signaling proteins from several pathways, with STAT1 mRNA being the most prominently elevated ones. Furthermore, SARS-CoV-2 replication was significantly increased in both SAMHD1 WT and KO cells when expression and phosphorylation of STAT1 were downregulated by JAK inhibitor baricitinib, which over-rode the activated antiviral innate immunity in the KO cells. This further validates baricitinib as a treatment of SARS-CoV-2–infected patients primarily at the postviral clearance stage. Overall, our tissue culture model systems demonstrated that the elevated innate immune response and IFN activation upon genetic loss of SAMHD1 effectively suppresses SARS-CoV-2 replication.     

SARS-CoV-2 accessory protein ORF8 is secreted extracellularly as a glycoprotein homodimer

ORF8 is an accessory protein encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Consensus regarding the biological functions of ORF8 is lacking, largely because the fundamental characteristics of this protein in cells have not been determined. To clarify these features, we herein established an ORF8 expression system in 293T cells. Using this system, approximately 41% of the ORF8 expressed in 293T cells were secreted extracellularly as a glycoprotein homodimer with inter/intramolecular disulfide bonds. Intracellular ORF8 was sensitive to the glycosidase Endo H, whereas the secreted portion was Endo-H-resistant, suggesting that secretion occurs via a conventional pathway. Additionally, immunoblotting analysis showed that the total amounts of the major histocompatibility complex class Ι (MHC-I), angiotensin-converting enzyme 2 (ACE2), and SARS-CoV-2 spike (CoV-2 S) proteins coexpressed in cells were not changed by the increased ORF8 expression, although FACS analysis revealed that the expression of the cell surface MHC-I protein, but not that of ACE2 and CoV-2 S proteins, was reduced by ORF8 expression. Finally, we demonstrate by RNA-seq analysis that ORF8 had no significant stimulatory effects in human primary monocyte-derived macrophages (MDMs). Taken together, our results provide fundamental evidence that the ORF8 glycoprotein acts as a secreted homodimer, and its functions are likely associated with the intracellular transport and/or extracellular signaling in SARS-CoV-2 infection.     

SARS-CoV-2 ORF9b inhibits RIG-I-MAVS antiviral signaling by interrupting K63-linked ubiquitination of NEMO

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SARS-CoV-2 and the Nucleus

The ongoing COVID-19 pandemic is caused by an RNA virus, SARS-CoV-2. The genome of SARS-CoV-2 lacks a nuclear phase in its life cycle and is replicated in the cytoplasm. However, interfering with nuclear trafficking using pharmacological inhibitors greatly reduces virus infection and virus replication of other coronaviruses is blocked in enucleated cells, suggesting a critical role of the nucleus in virus infection. Here, we summarize the alternations of nuclear pathways caused by SARS-CoV-2, including nuclear translocation pathways, innate immune responses, mRNA metabolism, epigenetic mechanisms, DNA damage response, cytoskeleton regulation, and nuclear rupture. We consider how these alternations contribute to virus replication and discuss therapeutic treatments that target these pathways, focusing on small molecule drugs that are being used in clinical studies.     

ORF3a of SARS-CoV-2 promotes lysosomal exocytosis-mediated viral egress

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Humoral Immunity against SARS-CoV-2 and the Impact on COVID-19 Pathogenesis

It has been more than a year since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first emerged. Many studies have provided insights into the various aspects of the immune response in coronavirus disease 2019 (COVID-19). Especially for antibody treatment and vaccine development, humoral immunity to SARS-CoV-2 has been studied extensively, though there is still much that is unknown and controversial. Here, we introduce key discoveries on the humoral immune responses in COVID-19, including the immune dynamics of antibody responses and correlations with disease severity, neutralizing antibodies and their cross-reactivity, how long the antibody and memory B-cell responses last, aberrant autoreactive antibodies generated in COVID-19 patients, and the efficacy of currently available therapeutic antibodies and vaccines against circulating SARS-CoV-2 variants, and highlight gaps in the current knowledge.     

Functional landscape of SARS-CoV-2 cellular restriction

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Immune response and potential therapeutic strategies for the SARS-CoV-2 associated with the COVID-19 pandemic

Following onset of the first recorded case of Coronavirus disease 2019 (COVID-19) in December 2019, more than 269 million cases and over 5.3 million deaths have been confirmed worldwide. COVID-19 is a highly infectious pneumonia, caused by a novel virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, it poses a severe threat to human health across the globe, a trend that is likely to persist in the foreseeable future. This paper reviews SARS-CoV-2 immunity, the latest development of anti-SARS-CoV-2 drugs as well as exploring in detail, immune escape induced by SARS-CoV-2. We expect that the findings will provide a basis for COVID-19 prevention and treatment.     

Receptor-binding domain of SARS-CoV-2 spike protein efficiently inhibits SARS-CoV-2 infection and attachment to mouse lung

COVID-19, caused by a novel coronavirus, SARS-CoV-2, poses a serious global threat. It was first reported in 2019 in China and has now dramatically spread across the world. It is crucial to develop therapeutics to mitigate severe disease and viral spread. The receptor-binding domains (RBDs) in the spike protein of SARS-CoV and MERS-CoV have shown anti-viral activity in previous reports suggesting that this domain has high potential for development as therapeutics. To evaluate the potential antiviral activity of recombinant SARS-CoV-2 RBD proteins, we determined the RBD residues of SARS-CoV-2 using a homology search with RBD of SARS-CoV. For efficient expression and purification, the signal peptide of spike protein was identified and used to generate constructs expressing recombinant RBD proteins. Highly purified RBD protein fused with the Fc domain of human IgG showed potent anti-viral efficacy, which was better than that of a protein fused with a histidine tag. Intranasally pre-administrated RBD protein also inhibited the attachment of SARS-COV-2 to mouse lungs. These findings indicate that RBD protein could be used for the prevention and treatment of SARS-CoV-2 infection.     

Phase separation by the SARS-CoV-2 nucleocapsid protein: Consensus and open questions

In response to the recent SARS-CoV-2 pandemic, a number of labs across the world have reallocated their time and resources to better our understanding of the virus. For some viruses, including SARS-CoV-2, viral proteins can undergo phase separation: a biophysical process often related to the partitioning of protein and RNA into membraneless organelles in vivo. In this review, we discuss emerging observations of phase separation by the SARS-CoV-2 nucleocapsid (N) protein—an essential viral protein required for viral replication—and the possible in vivo functions that have been proposed for N-protein phase separation, including viral replication, viral genomic RNA packaging, and modulation of host-cell response to infection. Additionally, since a relatively large number of studies examining SARS-CoV-2 N-protein phase separation have been published in a short span of time, we take advantage of this situation to compare results from similar experiments across studies. Our evaluation highlights potential strengths and pitfalls of drawing conclusions from a single set of experiments, as well as the value of publishing overlapping scientific observations performed simultaneously by multiple labs.     

Activation of the alternative complement pathway by mannose-binding lectin via a C2-bypass pathway

MBL is a serum lectin that activates the lectin pathway of the complement system. MBL forms complexes with three types of MASPs. Upon binding to Salmonella serogroup C-specific oligosaccharide, MBL activates the alternative pathway via a C2-bypass pathway without involving MASP-2, C2 or C4. We demonstrate that mannan-bound MBL activates the alternative pathway via a C2-bypass pathway that requires MASP-2 and C4. Thus, depending on the ligands to which MBL binds, there may be two distinct MBL-mediated C2-bypass pathways.     

含分子内佐剂的SARS-CoV-2 RBD域重组蛋白制备及免疫效果评价北大核心CSCD

制备含破伤风毒素肽(tetanus toxin,TT)、促吞噬肽(tuftsin)和新型冠状病毒刺突蛋白(spike,S蛋白)受体结合域(receptor-binding domain,RBD)的融合蛋白,探讨分子内佐剂对RBD蛋白体液免疫和细胞免疫效果的影响。将破伤风毒素肽、促吞噬肽与S蛋白RBD区域通过柔性多肽串联,密码子优化后构建重组载体,原核表达纯化制备重组S-TT-tuftsin蛋白,与铝佐剂混合后免疫BALB/c小鼠,对其体液及细胞免疫效果进行评价。重组S-TT-tuftsin蛋白以包涵体形式表达,离子交换层析纯化后采用梯度透析进行复性,复性蛋白经Dot blotting鉴定,可与新冠亚单位疫苗(安徽智飞公司)免疫后人血清发生反应。小鼠免疫实验结果表明,免疫35 d时抗体水平到达平台期,含分子内佐剂重组蛋白(铝佐剂)免疫小鼠后血清ELISA抗体效价高达1︰66240,显著高于S-RBD蛋白(铝佐剂)免疫小鼠抗体效价(P<0.05)。同时,含分子内佐剂重组蛋白刺激小鼠产生更强的淋巴细胞增殖能力,刺激指数可达4.71±0.15,相较于S-RBD蛋白的刺激指数1.83±0.09具有显著性差异(P<0.0001)。分子内佐剂破伤风毒素肽和促吞噬肽可显著增强新冠S蛋白RBD域的体液免疫和细胞免疫效果,可为新冠亚单位疫苗和其他病毒亚单位疫苗的研制提供理论基础和参考。     

Effect of clinical isolate or cleavage site mutations in the SARS-CoV-2 spike protein on protein stability,cleavage, and cell–cell fusion

The trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) is the sole viral protein responsible for both viral binding to a host cell and the membrane fusion event needed for cell entry. In addition to facilitating fusion needed for viral entry, S can also drive cell–cell fusion, a pathogenic effect observed in the lungs of SARS-CoV-2–infected patients. While several studies have investigated S requirements involved in viral particle entry, examination of S stability and factors involved in S cell–cell fusion remain limited. A furin cleavage site at the border between the S1 and S2 subunits (S1/S2) has been identified, along with putative cathepsin L and transmembrane serine protease 2 cleavage sites within S2. We demonstrate that S must be processed at the S1/S2 border in order to mediate cell–cell fusion and that mutations at potential cleavage sites within the S2 subunit alter S processing at the S1/S2 border, thus preventing cell–cell fusion. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S-mediated cell–cell fusion. In addition, we examined S stability and protein cleavage kinetics in a variety of mammalian cell lines, including a bat cell line related to the likely reservoir species for SARS-CoV-2, and provide evidence that proteolytic processing alters the stability of the S trimer. This work therefore offers insight into S stability, proteolytic processing, and factors that mediate S cell–cell fusion, all of which help give a more comprehensive understanding of this high-profile therapeutic target.     

Inhibition of SARS-CoV-2 Infection by Human Defensin HNP1 and Retrocyclin RC-101

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is an enveloped virus responsible for the COVID-19 pandemic. The emergence of new potentially more transmissible and vaccine-resistant variants of SARS-CoV-2 is an ever-present threat. Thus, it remains essential to better understand innate immune mechanisms that can inhibit the virus. One component of the innate immune system with broad antipathogen, including antiviral, activity is a group of cationic immune peptides termed defensins. The ability of defensins to neutralize enveloped and non-enveloped viruses and to inactivate numerous bacterial toxins correlate with their ability to promote the unfolding of proteins with high conformational plasticity. We found that human neutrophil α-defensin HNP1 binds to SARS-CoV-2 Spike protein with submicromolar affinity that is more than 20 fold stronger than its binding to serum albumin. As such, HNP1, as well as a θ-defensin retrocyclin RC-101, both interfere with Spike-mediated membrane fusion, Spike-pseudotyped lentivirus infection, and authentic SARS-CoV-2 infection in cell culture. These effects correlate with the abilities of the defensins to destabilize and precipitate Spike protein and inhibit the interaction of Spike with the ACE2 receptor. Serum reduces the anti-SARS-CoV-2 activity of HNP1, though at high concentrations, HNP1 was able to inactivate the virus even in the presence of serum. Overall, our results suggest that defensins can negatively affect the native conformation of SARS-CoV-2 Spike, and that α- and θ-defensins may be valuable tools in developing SARS-CoV-2 infection prevention strategies.     

Comparative computational analysis of SARS-CoV-2 nucleocapsid protein epitopes in taxonomically related coronaviruses

Several research lines are currently ongoing to address the multitude of facets of the pandemic COVID-19. In line with the One-Health concept, extending the target of the studies to the animals which humans are continuously interacting with may favor a better understanding of the SARS-CoV-2 biology and pathogenetic mechanisms; thus, helping to adopt the most suitable containment measures. The last two decades have already faced severe manifestations of the coronavirus infection in both humans and animals, thus, circulating epitopes from previous outbreaks might confer partial protection from SARS-CoV-2 infections. In the present study, we provide an in-silico survey of the major nucleocapsid protein epitopes and compare them with the homologues of taxonomically-related coronaviruses with tropism for animal species that are closely inter-related with the human beings population all over the world. Protein sequence alignment provides evidence of high sequence homology for some of the investigated proteins. Moreover, structural epitope mapping by homology modelling revealed a potential immunogenic value also for specific sequences scoring a lower identity with SARS-CoV-2 nucleocapsid proteins. These evidence provide a molecular structural rationale for a potential role in conferring protection from SARS-CoV-2 infection and identifying potential candidates for the development of diagnostic tools and prophylactic-oriented strategies.     

Delineation of the complement receptor type 2-C3d complex by site-directed mutagenesis and molecular docking

The interactions between the complement receptor type 2 (CR2) and the C3 complement fragments C3d, C3dg, and iC3b are essential for the initiation of a normal immune response. A crystal-derived structure of the two N-terminal short consensus repeat (SCR1-2) domains of CR2 in complex with C3d has previously been elucidated. However, a number of biochemical and biophysical studies targeting both CR2 and C3d appear to be in conflict with these structural data. Previous mutagenesis and heteronuclear NMR spectroscopy studies directed toward the C3d-binding site on CR2 have indicated that the CR2-C3d cocrystal structure may represent an encounter/intermediate or nonphysiological complex. With regard to the CR2-binding site on C3d, mutagenesis studies by Isenman and coworkers [Isenman, D. E., Leung, E., Mackay, J. D., Bagby, S. & van den Elsen, J. M. H. (2010). Mutational analyses reveal that the staphylococcal immune evasion molecule Sbi and complement receptor 2 (CR2) share overlapping contact residues on C3d: Implications for the controversy regarding the CR2/C3d cocrystal structure. J. Immunol. 184, 1946-1955] have implicated an electronegative “concave” surface on C3d in the binding process. This surface is discrete from the CR2-C3d interface identified in the crystal structure.We generated a total of 18 mutations targeting the two (X-ray crystallographic- and mutagenesis-based) proposed CR2 SCR1-2 binding sites on C3d. Using ELISA analyses, we were able to assess binding of mutant forms of C3d to CR2. Mutations directed toward the concave surface of C3d result in substantially compromised CR2 binding. By contrast, targeting the CR2-C3d interface identified in the cocrystal structure and the surrounding area results in significantly lower levels of disruption in binding. Molecular modeling approaches used to investigate disparities between the biochemical data and the X-ray structure of the CR2-C3d cocrystal result in highest-scoring solutions in which CR2 SCR1-2 is docked within the concave surface of C3d.     

mRNA Vaccines: Possible Tools to Combat SARS-CoV-2

正Coronaviruses are large, enveloped, positive-strand RNA viruses. Several coronaviruses are pathogenic in humans,including severe acute respiratory syndrome coronavirus(SARS-CoV), Middle East respiratory syndrome coronavirus(MERS-CoV) and this novel virus, SARS-CoV-2,     

新型冠状病毒(SARS-CoV-2)蛋白靶位的生信分析

目前新型冠状病毒肺炎(COVID-19)疫情仍在全球肆虐,但尚无针对该病毒的治疗特效药.在此背景,以美国化学文摘社(Chemical Abstracts Service,CAS)提供的SARS-CoV-2病毒及宿主蛋白靶标为研究对象,运用基因功能富集、蛋白网络等方法进行生物信息分析.结果发现,人网格蛋白介导型内吞和依赖...