Membrane Remodeling in Viral Infection and Viral Assembly

病毒感染和病毒组装中的膜重塑

基本信息

批准号：
10700691
负责人：
JOSHUA ZIMMERBERG
金额：
$ 174.52万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10700691
关键词：
2019-nCoV ACE2 Adhesives Affect Amino Acid Sequence Amino Acid Substitution Anabolism Antibodies Antibody Response Antigens Antiviral Response Arboviruses Area Avidity Binding Biological Assay C-terminal COVID-19 pandemic Capsid Capsid Proteins Cathepsins Cell membrane Cell surface Cells China Cryo-electron tomography Cytoplasm Cytoplasmic Tail Diffusion Early Diagnosis Encapsulated Endosomes Engineering Environment Environmental Hazards Equilibrium Event Flow Cytometry Freezing Genes Genetic Genome Genomics Geographic Locations Glycoproteins Hepatitis D Immune system Immunity India Individual Infection Inflammation Lateral Lead Light Lipids Luminescent Proteins Measurement Mediating Membrane Membrane Fusion Molecular Molecular Conformation Motion Mutate Mutation Mutation Analysis N-terminal Nature Negative Staining Organelles Pathogenicity Peptide Hydrolases Peptides Phenotype Physiological Plasma Cells Plasmids Probability Property Proteins Protomer RNA Viruses RNA vaccine Receptor Cell Reporter Genes Reporting Retroviridae Role SARS-CoV-2 B.1.1.7 SARS-CoV-2 B.1.617.2 SARS-CoV-2 variant Signaling Protein Site Speed Stains Structure Surface TMPRSS2 gene Temperature Testing Thermodynamics Time Ultracentrifugation Variant Viral Viral Genome Viral Load result Virion Virus Virus Assembly Virus Diseases acquired immunity cell type experience extracellular fitness flexibility genetic variant immunogenic infection rate interest member neutralizing antibody particle receptor receptor binding variants of concern virus envelope

项目摘要

We demonstrated, using four independent techniquesnegative stain TEM, flow cytometry, NTA, and cryo-EMthat PVs bearing the Delta variant spike or the closely related Delta sublineage Delta AY.4.2 spike aggregate, whereas PVs bearing other spike variants and Bald PVs do not aggregate. Since the PVs were prepared in parallel, handled under identical conditions, and factors that could promote aggregation such as pH changes, freeze-thaw cycles, and high-speed ultracentrifugation were avoided, the observed aggregation of Delta PVs most likely reflects a unique property of the Delta and Delta AY.4.2 spikes. The observation that Delta and Delta AY.4.2 PVs continued to aggregate in solution while stored at 4 deg. C suggests that aggregation occurs after budding from the producer cell; however, interaction between PVs could also initiate during biosynthesis and budding from the producer cell. Unlike SARS-CoV-2, which buds into the ERGIC compartment during final assembly, retroviruses, including MLV, generally bud directly from the plasma membrane (but not always). Thus, it is not known when or if Delta variant SARS-CoV-2 aggregates and this is currently being investigated. Delta SARS-CoV-2 could aggregate while budding into the ERGIC, during egress through alkalized lysosomal organelles, after egress in the extracellular milieu, or even on the target cell plasma membrane. The ability to aggregate may depend on the concentration of viral particles in each environment. The fact that Delta PVs continue to aggregate while stored at 4 deg. C is consistent with a mass action mechanism for Delta PV aggregation. Measurements of the rate of aggregation of Delta PVs at temperatures ranging up to physiological temperature could shed light on the thermodynamic properties of Delta aggregation, and advance understanding of the mechanism of aggregation. Furthermore, to produce PVs, a 19 aa C-terminal truncated version of each variant spike was expressed, which has been shown to increase the amount of spike incorporated into the PV envelope and PV infectivity. Truncations in the cytoplasmic tail could modify properties of the spike ectodomain structure and function. Though each variant spike possessed the same truncation, it is not impossible that the truncation uniquely affected the Delta spike ectodomain, conferring the aggregation property. Examples of Delta PVs with apparent spike tip interactions were observed by negative staining but these interactions did not extend to lateral aggregation of spike proteins on the surface of a PV. Ongoing cryo-electron tomography studies will reveal the nature of the interactions between aggregated PVs. Spike-mediated aggregation differs from antibody-driven aggregation of virions expected from polyvalent neutralizing antibodies, as those binding constants are expected to be much stronger. Thus, virions would be tightly packed and not likely to disaggregate at the cell surface. The spike tip interactions are more likely to come apart upon surface binding and receptor competition for the RBD at the tip of the spike. Analysis of the mutations present in the Delta and Delta AY.4.2 spikes compared to other variants may provide clues as to their unique property to aggregate. Delta and Delta AY.4.2 amino acid sequences are similar, except for four additional substitutions in Delta AY.4.2 (T95I, Y145H, A222V, and K458R). T95I is also present on the Omicron BA.1 spike and residue Y145 is also substituted in Omicron BA.1 (Y145D). A222V and K458R are unique to Delta AY.4.2. As the presence of these mutations in Delta AY.4.2 do not abrogate or enhance the aggregation of Delta AY.4.2 compared to Delta, they appear to have no effect on aggregation. Most of the other mutations on the Delta and Delta AY.4.2 spikes are shared with other variants. The D614G mutation is present in all variants, substitution L452R located in the RBD is present in the Kappa variant (B.1.617.1), and a similar substitution L452Q occurs in the Lambda variant (C.37). A second RBD substitution at T478K is also found in Omicron BA.1 and BA.2. The mutation P681R is present in Kappa, and P681H exists in Alpha, Omicron BA.1, BA.2, and Mu (B.1.621). Finally, substitution mutation D950N is also shared with Mu. Because the non-Delta variants studied here do not aggregate, it is unlikely that any of their Delta-shared mutations can be aggregation-dominant. There are, however, three residues, E156, F157, and R158, in the NTD of Delta and Delta AY.4.2 that are uniquely and identically mutated: substitution E156G, and deletions at F157 and R158. It is possible that these three mutations in the NTD are sufficient to bestow the aggregation property alone, or in the context of the other Delta mutations. The clustering of Delta PVs could account for the faster and larger initial infection observed in entry assays with the Delta PVs. Because the number of spike trimersis larger on an aggregate comprising multiple PVs, and the cell surface contact area is larger for any collision between the aggregate and a target cell, the effective on-rate for aggregate binding should be larger, resulting in faster binding. Furthermore, the avidity of the aggregate to the target cell would be enhanced manyfold due to the multiple potential binding partners on a single contacting surface. Moreover, the increased dwell time at that contact area will allow for diffusional and conformational motions of proteins and lipids to increase the chance of membrane fusion, as these factors are important for avoiding hemifusion and promoting full fusion. All these factors should lead to the relatively higher initial rate of PV entry into target cells from aggregated PVs. Whether or not aggregates could enable the simultaneous delivery of multiple copies of entry reporter genes to target cells is not clear, since the PVs need not display ACE2 and thus may not fuse to each other, even in an endosome; thus each virus in an aggregate may have to independently fuse to the endosomal membrane to place its genes to that cells cytoplasm. Implicitly, there would be more overall binding events for unaggregated PVs, each at another site. However, if the probability for PV entry was low due to unbinding, then the factors discussed above to increase PV avidity would tend to increase overall fusion and its rate. In summary, an ultrastructural analysis of retrovirus pseudotyped viral particles bearing SARS-CoV-2 spike variants led to a serendipitous discovery of significant aggregation when the Delta variant spike was expressed, but not upon expression of three other variant spikes. Viral aggregation can impart fitness benefits by protecting virions from environmental hazards and by effecting simultaneous delivery of multiple viral genomes, or collective infection. Notably, collective infection can favor initial infection in some contexts. Likely, the size and number of virions per aggregate is important to increasing infectivitytoo large and it would effectively reduce infectious units below a threshold, too few virions in an aggregate and the benefit of collective infection is not gained. The unique property of the Delta spike to aggregate PVs may underlie the faster infection by Delta PVs. Furthermore, spike mediated aggregation could be part of the molecular mechanism by which Delta variant SARS-CoV-2 achieves increased transmissibility and faster infection with a higher viral load. The continued aggregation of PVs over time indicates that clustering may be mediated by interactions between spike tips, which in turn may indicate an adhesivity of the viral surface recognized by the immune system thus altering the balance of host antiviral response towards inflammation.

我们使用四种独立技术染色TEM，流式细胞术，NTA和CRYO-EMTHAT PV带有Delta变体Spike或密切相关的Delta Sublineage delta ay.4.4.2.4.2尖峰聚集，而PVS pvs pvs bancike spike variants and Bald pvs bald bald pvs bald bald bald pvs a botregne becregne becregne becregne becrenecrene becregne becregne becregne becregne becregne becregecrene becregne copregne be na becregne。由于PV是在相同条件下并行处理的，并且可以避免使用pH变化，冻融周期和高速超速离心等因素，因此观察到的Delta PVS的聚集很可能反映出Delta和Delta ay.4.4.4.4.4.4.4.4.4.4.4.4.4.2。 Delta和Delta Ay.4.2 PVS继续在溶液中聚集的观察结果，储存在4度。 c表明聚集是从生产者细胞萌芽之后发生的。但是，在生物合成和生产者细胞中发芽期间，PV之间的相互作用也可以启动。与SARS-COV-2（在最后组装过程中芽到Ergic隔室）不同，包括MLV在内的逆转录病毒通常直接从质膜（但并非总是）芽。因此，尚不清楚何时还是是否何时或是否是否正在研究这一点。 Delta SARS-COV-2在通过碱化的溶酶体细胞器，出口到细胞外环境中，甚至在靶细胞质膜上，在出口到ERGIC时可以聚集。聚集的能力可能取决于每个环境中病毒颗粒的浓度。 Delta PVS继续聚集的事实，储存在4度。 C与Delta PV聚集的质量作用机制一致。在温度到生理温度不等的温度下，Delta PV的聚合速率的测量可能会揭示Delta聚集的热力学特性，并提高对聚集机理的理解。此外，为了产生PV，表达了每个变体尖峰的19个A C末端截短版本，已显示出来增加了掺入PV信封和PV感染性中的尖峰量。细胞质尾部的截断可以改变尖峰外生域结构和功能的性质。尽管每个变体尖峰都具有相同的截断，但截断并非不可能唯一地影响了三角洲尖峰外域域，从而赋予了聚集特性。通过负染色观察到具有明显的尖峰相互作用的三角洲PV的实例，但这些相互作用并未扩展到PV表面上尖峰蛋白的横向聚集。正在进行的冷冻电子断层扫描研究将揭示聚合PV之间相互作用的性质。尖峰介导的聚集与多价中和抗体预期的病毒体的抗体驱动的聚集不同，因为那些结合常数预计会更强大。因此，病毒体将被紧密填充，不太可能在细胞表面进行分解。在尖峰尖端的RBD表面结合和受体竞争上，尖峰尖端相互作用更有可能分开。与其他变体相比，对三角洲和三角洲AY.4.2尖峰中存在的突变的分析可能会提供有关其独特属性的线索。 Delta和Delta Ay.4.2氨基酸序列相似，除了Delta Ay.4.2（T95I，Y145H，A2222V和K458R）的其他四个额外取代。 T95i也存在于Omicron BA.1尖峰上，残基Y145也被取代在Omicron BA.1（Y145D）中。 A222V和K458R是Delta Ay.4.2独有的。由于与三角洲相比，这些突变在三角洲AY.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.2上似乎对聚集没有影响。 Delta和Delta Ay.4.2尖峰与其他变体共享。 D614g突变存在于所有变体中，rbd中的取代L452R存在于Kappa变体中（B.1.617.1），并且在lambda变体中出现类似的替代L452Q（c.37）。在Omicron BA.1和BA.2中也发现了T478K的第二个RBD替代。突变P681R存在于Kappa中，而P681H存在于Alpha，Omicron BA.1，BA.2和MU（B.1.621）中。最后，替代突变D950N也与MU共享。由于此处研究的非戴尔塔变体没有汇总，因此任何其三角洲共享突变都不可能是聚集的。但是，在Delta和Delta Ay.4.2中有三个残基E156，F157和R158，它们唯一且相同突变：替换E156G，以及在F157和R158处的缺失。 NTD中的这三个突变可能足以单独赋予聚合特性，或者在其他三角洲突变的背景下。 Delta PVS的聚类可以解释使用Delta PVS的入口分析中观察到的更快，更大的初始感染。由于在包含多个PV的聚集体上较大的尖峰三层数，并且对于骨料和靶细胞之间的任何碰撞，细胞表面接触面积较大，因此骨料结合的有效速率应更大，从而导致更快的结合。此外，由于单个接触表面上有多个潜在的结合伙伴，骨料与目标细胞的亲和力将得到增强。此外，该接触区域的停留时间增加将允许蛋白质和脂质的扩散和构象运动增加膜融合的机会，因为这些因素对于避免半分解和促进完全融合至关重要。所有这些因素都应导致从聚合PVS进入靶细胞的PV进入靶细胞的初始初始速率相对较高。骨料是否可以同时递送到目标细胞的多个输入报告基因副本，因为PVS不需要显示ACE2，因此即使在内体中也可能不会互相融合。因此，骨料中的每种病毒可能必须独立融合到内体膜上，以将其基因放在细胞细胞质上。隐含地，在另一个站点中，无聚集的PV将有更多的总体结合事件。但是，如果由于未连接而导致PV进入的概率很低，那么上面讨论的以增加PV亲和力的因素将倾向于增加总体融合及其速率。总而言之，对带有SARS-COV-2峰值变体的逆转录病毒型病毒颗粒进行超微结构分析，当表达了三角变体峰值时，导致了显着聚集的偶然性发现，但并非表达其他三个变体尖峰。病毒聚集可以通过保护病毒粒子免受环境危害并同时递送多种病毒基因组或集体感染来赋予健身益处。值得注意的是，集体感染可以在某些情况下有利于初始感染。每个骨料病毒群的大小和数量对于增加感染性很重要，并且有效地将感染单元降低到阈值以下，骨料中的病毒体太少，并且没有获得集体感染的好处。 Delta Spike汇总PVS的独特属性可能是Delta PVS更快的感染。此外，尖峰介导的聚集可能是分子机制的一部分，通过该机制，Delta变体SARS-COV-2实现了越来越多的传播性和更快的感染，而病毒载量较高。随着时间的推移，PV的持续聚集表明，峰值尖端之间的相互作用可以介导聚类，这反过来又表明了免疫系统识别的病毒表面的粘附性，从而改变了宿主抗病毒对炎症的平衡。