The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the world is still expanding. Thus, there is an urgent need to better understand this novel virus and find a way to control its spread. Like other coronaviruses, the nucleocapsid (N) protein is one of the most crucial structural components of SARS-CoV-2. This protein shares 90% homology with the severe acute respiratory syndrome coronavirus N protein, implying functional significance. Based on the evolutionary conservation of the N protein in coronavirus, we reviewed the currently available knowledge regarding the SARS-CoV-2 N protein in terms of structure, biological functions, and clinical application as a drug target or vaccine candidate.

Influenza A virus matrix protein (M1) is the most abundant conservative protein that regulates the replication, assembly and budding of the viral particles upon infection. Several host cell factors have been determined to interact with M1 possibly in regulating influenza virus replication. By yeast two-hybrid screening, the isomerase cyclophilin A (CypA) was identified to interact with the M1 protein. CypA specifically interacted with M1 both in vitro and in vivo. The mutagenesis results showed CypA bound to the functional middle (M) domain of M1. The depletion of endogenous CypA by RNA interference resulted in the increase of influenza virus infectivity while overexpression of CypA caused decreasing the infectivity in affected cells. The immunofluorescence assays indicated that overexpressed CypA deduced the infectivity and inhibited the translocation of M1 protein into the nucleus while did not affect nucleoprotein entering the nucleus. Further studies indicated that overexpression of CypA significantly increased M1 self-association. Western blot with purified virions confirmed that CypA was encapsidated within the virus particle. These results together indicated that CypA interacted with the M1 protein and affected the early stage of the viral replication.

The Bunyavirales order is one of the largest groups of segmented negative-sense single-stranded RNA viruses, which includes many pathogenic strains that cause severe human diseases. The RNA segments of the bunyavirus genome are separately encapsidated by multiple copies of nucleoprotein (N), and both termini of each N-encapsidated genomic RNA segment bind to one copy of the viral L polymerase protein. The viral genomic RNA, N and L protein together form the ribonucleoprotein (RNP) complex that constitutes the molecular machinery for viral genome replication and transcription. Recently, breakthroughs have been achieved in understanding the architecture of bunyavirus RNPs with the determination of the atomic structures of the N and L proteins from various members of this order. In this review, we discuss the structures and functions of these bunyavirus RNP components, as well as viral genome replication and transcription mechanisms.

Cyclophilin A (CypA) is a typical member of the cyclophilin family of peptidyl-prolyl isomerases and is involved in the replication of several viruses. Previous studies indicate that CypA interacts with influenza virus M1 protein and impairs the early stage of the viral replication. To further understand the molecular mechanism by which CypA impairs influenza virus replication, a 293T cell line depleted for endogenous CypA was established. The results indicated that CypA inhibited the initiation of virus replication. In addition, the infectivity of influenza virus increased in the absence of CypA. Further studies indicated that CypA had no effect on the stages of virus genome replication or transcription and also did not impair the nuclear export of the viral mRNA. However, CypA decreased the viral protein level. Additional studies indicated that CypA enhanced the degradation of M1 through the ubiquitin/proteasome-dependent pathway. Our results suggest that CypA restricts influenza virus replication through accelerating degradation of the M1 protein.

As COVID-19 continues to spread rapidly worldwide and variants continue to emerge, the development and deployment of safe and effective vaccines are urgently needed. Here, we developed an mRNA vaccine based on the trimeric receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein fused to ferritin-formed nanoparticles (TF-RBD). Compared to the trimeric form of the RBD mRNA vaccine (T-RBD), TF-RBD delivered intramuscularly elicited robust and durable humoral immunity as well as a Th1-biased cellular response. After further challenge with live SARS-CoV-2, immunization with a two-shot low-dose regimen of TF-RBD provided adequate protection in hACE2-transduced mice. In addition, the mRNA template of TF-RBD was easily and quickly engineered into a variant vaccine to address SARS-CoV-2 mutations. The TF-RBD multivalent vaccine produced broad-spectrum neutralizing antibodies against Alpha (B.1.1.7) and Beta (B.1.351) variants. This mRNA vaccine based on the encoded self-assembled nanoparticle-based trimer RBD provides a reference for the design of mRNA vaccines targeting SARS-CoV-2.

The nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex. During the replication of influenza virus, the vRNP complex undergoes nuclear-cytoplasmic shuttling, during which NP serves as one of the determinants. To date, many phosphorylation sites on NP have been identified, but the biological functions of many of these phosphorylation sites remain unknown. In the present study, the functions of the phosphorylation sites S9, Y10, and Y296 were characterized. These residues are highly conserved, and their phosphorylation was essential for virus growth in cell culture and in a mouse model by regulating the activity of the viral polymerase and the nuclear-cytoplasmic shuttling of NP. The phosphorylation and dephosphorylation of S9 and Y10 controlled nuclear import of NP by affecting the binding affinity between NP and different isoforms of importin-α. In addition, the phosphorylation of Y296 caused nuclear retention of NP by reducing the interaction between NP and CRM1. Furthermore, tyrosine phosphorylation of NP during the early stage of virus infection was ablated when Y296 was mutated to F. However, at later stages of infection, it was weakened by the Y10F mutation. Taken together, the present data indicate that the phosphorylation and dephosphorylation of NP control the shuttling of NP between the nucleus and the cytoplasm during virus replication. IMPORTANCE It is well known that phosphorylation regulates the functions of viral proteins and the life cycle of influenza A virus. As NP is the most abundant protein in the vRNP complex of influenza A virus, several phosphorylation sites on this protein have been identified. However, the functions of these phosphorylation sites were unknown. The present study demonstrates that the phosphorylation status of these sites on NP can mediate its nuclear-cytoplasmic shuttling, which drives the trafficking of vRNP complexes in infected cells. The present data suggest that the phosphorylated residues of NP are multistep controllers of the virus life cycle and new targets for the design of anti-influenza drugs.