A new intracellular targeting motif in the cytoplasmic tail of the spike protein may act as a target to inhibit SARS-CoV-2 assembly.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a threat to global public health, underscoring the urgent need for the development of preventive and therapeutic measures. The spike (S) protein of SARS-CoV-2, which mediates receptor binding and subsequent membrane fusion to promote viral entry, is a major target for current drug development and vaccine design. The S protein comprises a large N-terminal extracellular domain, a transmembrane domain, and a short cytoplasmic tail (CT) at the C-terminus. CT truncation of the S protein has been previously reported to promote the infectivity of SARS-CoV and SARS-CoV-2 pseudoviruses. However, the underlying molecular mechanism has not been precisely elucidated. In addition, the CT of various viral membrane glycoproteins play an essential role in the assembly of virions, yet the role of the S protein CT in SARS-CoV-2 infection remains unclear. In this study, through constructing a series of mutations of the CT of the S protein and analyzing their impact on the packaging of the SARS-CoV-2 pseudovirus and live SARS-CoV-2 virus, we identified V1264L1265 as a new intracellular targeting motif in the CT of the S protein, that regulates the transport and subcellular localization of the spike protein through the interactions with cytoskeleton and vesicular transport-related proteins, ARPC3, SCAMP3, and TUBB8, thereby modulating SARS-CoV-2 pseudovirus and live SARS-CoV-2 virion assembly. Either disrupting the V1264L1265 motif or reducing the expression of ARPC3, SCAMP3, and TUBB8 significantly repressed the assembly of the live SARS-CoV-2 virion, raising the possibility that the V1264L1265 motif and the host responsive pathways involved could be new drug targets for the treatment of SARS-CoV-2 infection. Our results extend the understanding of the role played by the S protein CT in the assembly of pseudoviruses and live SARS-CoV-2 virions, which will facilitate the application of pseudoviruses to the study of SARS-CoV-2 and provide potential strategies for the treatment of SARS-CoV-2 infection.

The E484K Substitution in a SARS-CoV-2 Spike Protein Subunit Vaccine Resulted in Limited Cross-Reactive Neutralizing Antibody Responses in Mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially emerging variants, poses an increased threat to global public health. The significant reduction in neutralization activity against the variants such as B.1.351 in the serum of convalescent patients and vaccinated people calls for the design of new potent vaccines targeting the emerging variant. However, since most vaccines approved and in clinical trials are based on the sequence of the original SARS-CoV-2 strain, the immunogenicity and protective efficacy of vaccines based on the B.1.351 variant remain largely unknown. In this study, we evaluated the immunogenicity, induced neutralization activity, and protective efficacy of wild-type spike protein nanoparticle (S-2P) and mutant spike protein nanoparticle (S-4M-2P) carrying characteristic mutations of B.1.351 variant in mice. Although there was no significant difference in the induction of spike-specific IgG responses in S-2P- and S-4M-2P-immunized mice, neutralizing antibodies elicited by S-4M-2P exhibited noteworthy, narrower breadth of reactivity with SARS-CoV-2 variants compared with neutralizing antibodies elicited by S-2P. Furthermore, the decrease of induced neutralizing antibody breadth at least partly resulted from the amino acid substitution at position 484. Moreover, S-4M-2P vaccination conferred insufficient protection against live SARS-CoV-2 virus infection, while S-2P vaccination gave definite protection against SARS-CoV-2 challenge in mice. Together, our study provides direct evidence that the E484K substitution in a SARS-CoV-2 subunit protein vaccine limited the cross-reactive neutralizing antibody breadth in mice and, more importantly, draws attention to the unfavorable impact of this mutation in spike protein of SARS-CoV-2 variants on the induction of potent neutralizing antibody responses.