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Principal Investigator: JOSHUA ZIMMERBERG
Organization: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
Fiscal Year: 2022
Award: $1,745,170
Funding agency: Eunice Kennedy Shriver National Institute of Child Health and Human Development
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.
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