Importin-7-dependent nuclear translocation of the Flavivirus core protein is required for infectious virus production.

Flaviviridae is a family of positive-stranded RNA viruses, including human pathogens, such as Japanese encephalitis virus (JEV), dengue virus (DENV), Zika virus (ZIKV), and West Nile virus (WNV). Nuclear localization of the viral core protein is conserved among Flaviviridae, and this feature may be targeted for developing broad-ranging anti-flavivirus drugs. However, the mechanism of core protein translocation to the nucleus and the importance of nuclear translocation in the viral life cycle remain unknown. We aimed to identify the molecular mechanism underlying core protein nuclear translocation. We identified importin-7 (IPO7), an importin-ß family protein, as a nuclear carrier for Flaviviridae core proteins. Nuclear import assays revealed that core protein was transported into the nucleus via IPO7, whereas IPO7 deletion by CRISPR/Cas9 impaired their nuclear translocation. To understand the importance of core protein nuclear translocation, we evaluated the production of infectious virus or single-round-infectious-particles in wild-type or IPO7-deficient cells; both processes were significantly impaired in IPO7-deficient cells, whereas intracellular infectious virus levels were equivalent in wild-type and IPO7-deficient cells. These results suggest that IPO7-mediated nuclear translocation of core proteins is involved in the release of infectious virus particles of flaviviruses.

Genes involved in the limited spread of SARS-CoV-2 in the lower respiratory airways of hamsters may be associated with adaptive evolution.

Novel respiratory viruses can cause a pandemic and then evolve to coexist with humans. The Omicron strain of severe acute respiratory syndrome coronavirus 2 has spread worldwide since its emergence in late 2021, and its sub-lineages are now established in human society. Compared to previous strains, Omicron is markedly less invasive in the lungs and causes less severe disease. One reason for this is that humans are acquiring immunity through previous infection and vaccination, but the nature of the virus itself is also changing. Using our newly established low-volume inoculation system, which reflects natural human infection, we show that the Omicron strain spreads less efficiently into the lungs of hamsters compared with an earlier Wuhan strain. Furthermore, by characterizing chimeric viruses with the Omicron gene in the Wuhan strain genetic background and vice versa, we found that viral genes downstream of ORF3a, but not the S gene, were responsible for the limited spread of the Omicron strain in the lower airways of the virus-infected hamsters. Moreover, molecular evolutionary analysis of SARS-CoV-2 revealed a positive selection of genes downstream of ORF3a (M and E genes). Our findings provide insight into the adaptive evolution of the virus in humans during the pandemic convergence phase.IMPORTANCEThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has spread worldwide since its emergence in late 2021, and its sub-lineages are established in human society. Compared to previous strains, the Omicron strain is less invasive in the lower respiratory tract, including the lungs, and causes less severe disease; however, the mechanistic basis for its restricted replication in the lower airways is poorly understood. In this study, using a newly established low-volume inoculation system that reflects natural human infection, we demonstrated that the Omicron strain spreads less efficiently into the lungs of hamsters compared with an earlier Wuhan strain and found that viral genes downstream of ORF3a are responsible for replication restriction in the lower respiratory tract of Omicron-infected hamsters. Furthermore, we detected a positive selection of genes downstream of ORF3a (especially the M and E genes) in SARS-CoV-2, suggesting that these genes may undergo adaptive changes in humans.

SARS-CoV-2 papain-like protease inhibits ISGylation of the viral nucleocapsid protein to evade host anti-viral immunity.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes mild-to-severe respiratory symptoms, including acute respiratory distress. Despite remarkable efforts to investigate the virological and pathological impacts of SARS-CoV-2, many of the characteristics of SARS-CoV-2 infection still remain unknown. The interferon-inducible ubiquitin-like protein ISG15 is covalently conjugated to several viral proteins to suppress their functions. It was reported that SARS-CoV-2 utilizes its papain-like protease (PLpro) to impede ISG15 conjugation, ISGylation. However, the role of ISGylation in SARS-CoV-2 infection remains unclear. We aimed to elucidate the role of ISGylation in SARS-CoV-2 replication. We observed that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation in cultured cells. Site-directed mutagenesis reveals that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation, alongside conserved lysine residue in MERS-CoV (K372) and SARS-CoV (K375). We also observed that the nucleocapsid-ISGylation results in the disruption of nucleocapsid oligomerization, thereby inhibiting viral replication. Knockdown of ISG15 mRNA enhanced SARS-CoV-2 replication in the SARS-CoV-2 reporter replicon cells, while exogenous expression of ISGylation components partially hampered SARS-CoV-2 replication. Taken together, these results suggest that SARS-CoV-2 PLpro inhibits ISGylation of the nucleocapsid protein to promote viral replication by evading ISGylation-mediated disruption of the nucleocapsid oligomerization.IMPORTANCEISG15 is an interferon-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation in many viruses. However, the role of ISGylation in SARS-CoV-2 infection remains largely unclear. Here, we demonstrated that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation. We also found that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation. We obtained evidence suggesting that nucleocapsid-ISGylation results in the disruption of nucleocapsid-oligomerization, thereby suppressing SARS-CoV-2 replication. We discovered that SARS-CoV-2 papain-like protease inhibits ISG15 conjugation of nucleocapsid protein via its de-conjugating enzyme activity. The present study may contribute to gaining new insight into the roles of ISGylation-mediated anti-viral function in SARS-CoV-2 infection and may lead to the development of more potent and selective inhibitors targeted to SARS-CoV-2 nucleocapsid protein.

Enterovirus 3A protein disrupts endoplasmic reticulum homeostasis through interaction with GBF1.

Enteroviruses are single-stranded, positive-sense RNA viruses causing endoplasmic reticulum (ER) stress to induce or modulate downstream signaling pathways known as the unfolded protein responses (UPR). However, viral and host factors involved in the UPR related to viral pathogenesis remain unclear. In the present study, we aimed to identify the major regulator of enterovirus-induced UPR and elucidate the underlying molecular mechanisms. We showed that host Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1), which supports enteroviruses replication, was a major regulator of the UPR caused by infection with enteroviruses. In addition, we found that severe UPR was induced by the expression of 3A proteins encoded in human pathogenic enteroviruses, such as enterovirus A71, coxsackievirus B3, poliovirus, and enterovirus D68. The N-terminal-conserved residues of 3A protein interact with the GBF1 and induce UPR through inhibition of ADP-ribosylation factor 1 (ARF1) activation via GBF1 sequestration. Remodeling and expansion of ER and accumulation of ER-resident proteins were observed in cells infected with enteroviruses. Finally, 3A induced apoptosis in cells infected with enteroviruses via activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/C/EBP homologous protein (CHOP) pathway of UPR. Pharmaceutical inhibition of PERK suppressed the cell death caused by infection with enteroviruses, suggesting the UPR pathway is a therapeutic target for treating diseases caused by infection with enteroviruses.IMPORTANCEInfection caused by several plus-stranded RNA viruses leads to dysregulated ER homeostasis in the host cells. The mechanisms underlying the disruption and impairment of ER homeostasis and its significance in pathogenesis upon enteroviral infection remain unclear. Our findings suggested that the 3A protein encoded in human pathogenic enteroviruses disrupts ER homeostasis by interacting with GBF1, a major regulator of UPR. Enterovirus-mediated infections drive ER into pathogenic conditions, where ER-resident proteins are accumulated. Furthermore, in such scenarios, the PERK/CHOP signaling pathway induced by an unresolved imbalance of ER homeostasis essentially drives apoptosis. Therefore, elucidating the mechanisms underlying the virus-induced disruption of ER homeostasis might be a potential target to mitigate the pathogenesis of enteroviruses.

JAK inhibition during the early phase of SARS-CoV-2 infection worsens kidney injury by suppressing endogenous antiviral activity in mice.

Coronavirus disease 2019 (COVID-19) induces respiratory dysfunction as well as kidney injury. Although the kidney is considered a target organ of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and affected by the COVID-19-induced cytokine storm, the mechanisms of renal reaction in SARS-CoV-2 infection are unknown. In this study, a murine COVID-19 model was induced by nasal infection with mouse-adapted SARS-CoV-2 (MA10). MA10 infection induced body weight loss along with lung inflammation in mice 4 days after infection. Serum creatinine levels and the urinary albumin/creatinine ratio increased on day 4 after MA10 infection. Measurement of the urinary neutrophil gelatinase-associated lipocalin/creatinine ratio and hematoxylin and eosin staining revealed tubular damage in MA10-infected murine kidneys, indicating kidney injury in the murine COVID-19 model. Interferon (IFN)-γ and interleukin-6 upregulation in the sera of MA10-infected mice, along with the absence of MA10 in the kidneys, implied that the kidneys were affected by the MA10 infection-induced cytokine storm rather than by direct MA10 infection of the kidneys. RNA-sequencing analysis revealed that antiviral genes, such as the IFN/Janus kinase (JAK) pathway, were upregulated in MA10-infected kidneys. Upon administration of the JAK inhibitor baricitinib on days 1-3 after MA10 infection, an antiviral pathway was suppressed, and MA10 was detected more frequently in the kidneys. Notably, JAK inhibition upregulated the hypoxia response and exaggerated kidney injury. These results suggest that endogenous antiviral activity protects against SARS-CoV-2-induced kidney injury in the early phase of infection, providing valuable insights into the pathogenesis of COVID-19-associated nephropathy.NEW & NOTEWORTHY Patients frequently present with acute kidney injury or abnormal urinary findings after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we investigated how the kidneys respond during SARS-CoV-2 infection using a murine coronavirus disease 2019 (COVID-19) model and showed that Janus kinase-mediated endogenous antiviral activity protects against kidney injury in the early phase of SARS-CoV-2 infection. These findings provide valuable insights into the renal pathophysiology of COVID-19.

Adjuvant-free parenterally injectable vaccine platform that harnesses previously induced IgG as an antigen delivery carrier.

Subunit vaccines are among the most useful vaccine modalities; however, their low immunogenicity necessitates the addition of adjuvants. Although adjuvants improve immune responses induced by vaccines, they often cause adverse reactions. To address this, we developed an adjuvant-free subunit vaccine platform that uses pre-existing antibodies generated from past infections or vaccinations as carriers for the delivery of vaccine antigens. Although we have confirmed the usefulness of this platform for nasal vaccines, its suitability as a parenterally injectable vaccine remains uncertain. Here, we verified the potential of our vaccine platform to harness pre-existing immunity for parenterally injectable vaccines. We generated RBD-HA by combining the receptor binding domain (RBD) derived from SARS-CoV-2 as a vaccine antigen with hemagglutinin (HA) sourced from influenza viruses to serve as the carrier protein. We revealed that subcutaneous vaccination with RBD-HA effectively triggered strong RBD-specific IgG responses in mice previously infected with the influenza A virus, even in the absence of adjuvants, and conferred protection to mice against SARS-CoV-2 upon challenge. Furthermore, we revealed that vaccination with RBD-HA did not induce an inflammatory response, such as inflammatory cytokine production, swelling, and recruitment of inflammatory immune cells, whereas conventional vaccines combined with adjuvants induced these adverse reactions. In addition, we demonstrated the remarkable versatility of this platform using a vaccine antigen derived from Streptococcus pneumoniae. These findings indicate the potential of this adjuvant-free vaccine platform to enhance the efficacy of parenterally injectable subunit vaccines and reduce adverse reactions.

Akaluc bioluminescence offers superior sensitivity to track in vivo dynamics of SARS-CoV-2 infection.

Monitoring in vivo viral dynamics can improve our understanding of pathogenicity and tissue tropism. Because the gene size of RNA viruses is typically small, NanoLuc is the primary choice for accommodation within viral genome. However, NanoLuc/Furimazine and also the conventional firefly luciferase/D-luciferin are known to exhibit relatively low tissue permeability and thus less sensitivity for visualization of deep tissue including lungs. Here, we demonstrated in vivo sufficient visualization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using the pair of a codon-optimized Akaluc and AkaLumine. We engineered the codon-optimized Akaluc gene possessing the similar GC ratio of SARS-CoV-2. Using the SARS-CoV-2 recombinants carrying the codon-optimized Akaluc, we visualized in vivo infection of respiratory organs, including the tissue-specific differences associated with particular variants. Additionally, we could evaluate the efficacy of antivirals by monitoring changes in Akaluc signals. Overall, we offer an effective technology for monitoring viral dynamics in live animals.

Characterization of a neutralizing antibody that recognizes a loop region adjacent to the receptor-binding interface of the SARS-CoV-2 spike receptor-binding domain.

Although the global crisis caused by the coronavirus disease 2019 (COVID-19) pandemic is over, the global epidemic of the disease continues. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of COVID-19, initiates infection via the binding of the receptor-binding domain (RBD) of its spike protein to the human angiotensin-converting enzyme II (ACE2) receptor, and this interaction has been the primary target for the development of COVID-19 therapeutics. Here, we identified neutralizing antibodies against SARS-CoV-2 by screening mouse monoclonal antibodies and characterized an antibody, CSW1-1805, that targets a narrow region at the RBD ridge of the spike protein. CSW1-1805 neutralized several variants in vitro and completely protected mice from SARS-CoV-2 infection. Cryo-EM and biochemical analyses revealed that this antibody recognizes the loop region adjacent to the ACE2-binding interface with the RBD in both a receptor-inaccessible "down" state and a receptor-accessible "up" state and could stabilize the RBD conformation in the up-state. CSW1-1805 also showed different binding orientations and complementarity determining region properties compared to other RBD ridge-targeting antibodies with similar binding epitopes. It is important to continuously characterize neutralizing antibodies to address new variants that continue to emerge. Our characterization of this antibody that recognizes the RBD ridge of the spike protein will aid in the development of future neutralizing antibodies.IMPORTANCESARS-CoV-2 cell entry is initiated by the interaction of the viral spike protein with the host cell receptor. Therefore, mechanistic findings regarding receptor recognition by the spike protein help uncover the molecular mechanism of SARS-CoV-2 infection and guide neutralizing antibody development. Here, we characterized a SARS-CoV-2 neutralizing antibody that recognizes an epitope, a loop region adjacent to the receptor-binding interface, that may be involved in the conformational transition of the receptor-binding domain (RBD) of the spike protein from a receptor-inaccessible "down" state into a receptor-accessible "up" state, and also stabilizes the RBD in the up-state. Our mechanistic findings provide new insights into SARS-CoV-2 receptor recognition and guidance for neutralizing antibody development.

Vero cell-adapted SARS-CoV-2 strain shows increased viral growth through furin-mediated efficient spike cleavage.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes several host proteases to cleave the spike (S) protein to enter host cells. SARS-CoV-2 S protein is cleaved into S1 and S2 subunits by furin, which is closely involved in the pathogenicity of SARS-CoV-2. However, the effects of the modulated protease cleavage activity due to S protein mutations on viral replication and pathogenesis remain unclear. Herein, we serially passaged two SARS-CoV-2 strains in Vero cells and characterized the cell-adapted SARS-CoV-2 strains in vitro and in vivo. The adapted strains showed high viral growth, effective S1/S2 cleavage of the S protein, and low pathogenicity compared with the wild-type strain. Furthermore, the viral growth and S1/S2 cleavage were enhanced by the combination of the Δ68-76 and H655Y mutations using recombinant SARS-CoV-2 strains generated by the circular polymerase extension reaction. The recombinant SARS-CoV-2 strain, which contained the mutation of the adapted strain, showed increased susceptibility to the furin inhibitor, suggesting that the adapted SARS-CoV-2 strain utilized furin more effectively than the wild-type strain. Pathogenicity was attenuated by infection with effectively cleaved recombinant SARS-CoV-2 strains, suggesting that the excessive cleavage of the S proteins decreases virulence. Finally, the high-growth-adapted SARS-CoV-2 strain could be used as the seed for a low-cost inactivated vaccine; immunization with this vaccine can effectively protect the host from SARS-CoV-2 variants. Our findings provide novel insights into the growth and pathogenicity of SARS-CoV-2 in the evolution of cell-cell transmission. IMPORTANCE: The efficacy of the S protein cleavage generally differs among the SARS-CoV-2 variants, resulting in distinct viral characteristics. The relationship between a mutation and the entry of SARS-CoV-2 into host cells remains unclear. In this study, we analyzed the sequence of high-growth Vero cell-adapted SARS-CoV-2 and factors determining the enhancement of the growth of the adapted virus and confirmed the characteristics of the adapted strain by analyzing the recombinant SARS-CoV-2 strain. We successfully identified mutations Δ68-76 and H655Y, which enhance viral growth and the S protein cleavage by furin. Using recombinant viruses enabled us to conduct a virus challenge experiment in vivo. The pathogenicity of SARS-CoV-2 introduced with the mutations Δ68-76, H655Y, P812L, and Q853L was attenuated in hamsters, indicating the possibility of the attenuation of excessive cleaved SARS-CoV-2. These findings provide novel insights into the infectivity and pathogenesis of SARS-CoV-2 strains, thereby significantly contributing to the field of virology.

Amino acid catabolite markers for early prognostication of pneumonia in patients with COVID-19.

Effective early-stage markers for predicting which patients are at risk of developing SARS-CoV-2 infection have not been fully investigated. Here, we performed comprehensive serum metabolome analysis of a total of 83 patients from two cohorts to determine that the acceleration of amino acid catabolism within 5 days from disease onset correlated with future disease severity. Increased levels of de-aminated amino acid catabolites involved in the de novo nucleotide synthesis pathway were identified as early prognostic markers that correlated with the initial viral load. We further employed mice models of SARS-CoV2-MA10 and influenza infection to demonstrate that such de-amination of amino acids and de novo synthesis of nucleotides were associated with the abnormal proliferation of airway and vascular tissue cells in the lungs during the early stages of infection. Consequently, it can be concluded that lung parenchymal tissue remodeling in the early stages of respiratory viral infections induces systemic metabolic remodeling and that the associated key amino acid catabolites are valid predictors for excessive inflammatory response in later disease stages.