Modular characterization of SARS-CoV-2 nucleocapsid protein domain functions in nucleocapsid-like assembly.

SARS-CoV-2 and its variants, with the Omicron subvariant XBB currently prevailing the global infections, continue to pose threats on public health worldwide. This non-segmented positive-stranded RNA virus encodes the multi-functional nucleocapsid protein (N) that plays key roles in viral infection, replication, genome packaging and budding. N protein consists of two structural domains, NTD and CTD, and three intrinsically disordered regions (IDRs) including the NIDR, the serine/arginine rich motif (SRIDR), and the CIDR. Previous studies revealed functions of N protein in RNA binding, oligomerization, and liquid-liquid phase separation (LLPS), however, characterizations of individual domains and their dissected contributions to N protein functions remain incomplete. In particular, little is known about N protein assembly that may play essential roles in viral replication and genome packing. Here, we present a modular approach to dissect functional roles of individual domains in SARS-CoV-2 N protein that reveals inhibitory or augmented modulations of protein assembly and LLPS in the presence of viral RNAs. Intriguingly, full-length N protein (NFL) assembles into ring-like architecture whereas the truncated SRIDR-CTD-CIDR (N182-419) promotes filamentous assembly. Moreover, LLPS droplets of NFL and N182-419 are significantly enlarged in the presence of viral RNAs, and we observed filamentous structures in the N182-419 droplets using correlative light and electron microscopy (CLEM), suggesting that the formation of LLPS droplets may promote higher-order assembly of N protein for transcription, replication and packaging. Together this study expands our understanding of the multiple functions of N protein in SARS-CoV-2.

A self-assembled trimeric protein vaccine induces protective immunity against Omicron variant.

The recently emerged Omicron (B.1.1.529) variant has rapidly surpassed Delta to become the predominant circulating SARS-CoV-2 variant, given the higher transmissibility rate and immune escape ability, resulting in breakthrough infections in vaccinated individuals. A new generation of SARS-CoV-2 vaccines targeting the Omicron variant are urgently needed. Here, we developed a subunit vaccine named RBD-HR/trimer by directly linking the sequence of RBD derived from the Delta variant (containing L452R and T478K) and HR1 and HR2 in SARS-CoV-2 S2 subunit in a tandem manner, which can self-assemble into a trimer. In multiple animal models, vaccination of RBD-HR/trimer formulated with MF59-like oil-in-water adjuvant elicited sustained humoral immune response with high levels of broad-spectrum neutralizing antibodies against Omicron variants, also inducing a strong T cell immune response in vivo. In addition, our RBD-HR/trimer vaccine showed a strong boosting effect against Omicron variants after two doses of mRNA vaccines, featuring its capacity to be used in a prime-boost regimen. In mice and non-human primates, RBD-HR/trimer vaccination could confer a complete protection against live virus challenge of Omicron and Delta variants. The results qualified RBD-HR/trimer vaccine as a promising next-generation vaccine candidate for prevention of SARS-CoV-2, which deserved further evaluation in clinical trials.

SARS-CoV-2 impairs the disassembly of stress granules and promotes ALS-associated amyloid aggregation.

The nucleocapsid (N) protein of SARS-CoV-2 has been reported to have a high ability of liquid-liquid phase separation, which enables its incorporation into stress granules (SGs) of host cells. However, whether SG invasion by N protein occurs in the scenario of SARS-CoV-2 infection is unknow, neither do we know its consequence. Here, we used SARS-CoV-2 to infect mammalian cells and observed the incorporation of N protein into SGs, which resulted in markedly impaired self-disassembly but stimulated cell cellular clearance of SGs. NMR experiments further showed that N protein binds to the SG-related amyloid proteins via non-specific transient interactions, which not only expedites the phase transition of these proteins to aberrant amyloid aggregation in vitro, but also promotes the aggregation of FUS with ALS-associated P525L mutation in cells. In addition, we found that ACE2 is not necessary for the infection of SARS-CoV-2 to mammalian cells. Our work indicates that SARS-CoV-2 infection can impair the disassembly of host SGs and promote the aggregation of SG-related amyloid proteins, which may lead to an increased risk of neurodegeneration.

Structures of signaling complexes of lipid receptors S1PR1 and S1PR5 reveal mechanisms of activation and drug recognition.

Sphingosine-1-phosphate (S1P) is an important bioactive lipid molecule in cell membrane metabolism and binds to G protein-coupled S1P receptors (S1PRs) to regulate embryonic development, physiological homeostasis, and pathogenic processes in various organs. S1PRs are lipid-sensing receptors and are therapeutic targets for drug development, including potential treatment of COVID-19. Herein, we present five cryo-electron microscopy structures of S1PRs bound to diverse drug agonists and the heterotrimeric Gi protein. Our structural and functional assays demonstrate the different binding modes of chemically distinct agonists of S1PRs, reveal the mechanical switch that activates these receptors, and provide a framework for understanding ligand selectivity and G protein coupling.

Full-length three-dimensional structure of the influenza A virus M1 protein and its organization into a matrix layer.

Matrix proteins are encoded by many enveloped viruses, including influenza viruses, herpes viruses, and coronaviruses. Underneath the viral envelope of influenza virus, matrix protein 1 (M1) forms an oligomeric layer critical for particle stability and pH-dependent RNA genome release. However, high-resolution structures of full-length monomeric M1 and the matrix layer have not been available, impeding antiviral targeting and understanding of the pH-dependent transitions involved in cell entry. Here, purification and extensive mutagenesis revealed protein-protein interfaces required for the formation of multilayered helical M1 oligomers similar to those observed in virions exposed to the low pH of cell entry. However, single-layered helical oligomers with biochemical and ultrastructural similarity to those found in infectious virions before cell entry were observed upon mutation of a single amino acid. The highly ordered structure of the single-layered oligomers and their likeness to the matrix layer of intact virions prompted structural analysis by cryo-electron microscopy (cryo-EM). The resulting 3.4-Å-resolution structure revealed the molecular details of M1 folding and its organization within the single-shelled matrix. The solution of the full-length M1 structure, the identification of critical assembly interfaces, and the development of M1 assembly assays with purified proteins are crucial advances for antiviral targeting of influenza viruses.