Virological characteristics of the SARS-CoV-2 Omicron BA.2 spike.

Soon after the emergence and global spread of the SARS-CoV-2 Omicron lineage BA.1, another Omicron lineage, BA.2, began outcompeting BA.1. The results of statistical analysis showed that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralization experiments revealed that immunity induced by COVID vaccines widely administered to human populations is not effective against BA.2, similar to BA.1, and that the antigenicity of BA.2 is notably different from that of BA.1. Cell culture experiments showed that the BA.2 spike confers higher replication efficacy in human nasal epithelial cells and is more efficient in mediating syncytia formation than the BA.1 spike. Furthermore, infection experiments using hamsters indicated that the BA.2 spike-bearing virus is more pathogenic than the BA.1 spike-bearing virus. Altogether, the results of our multiscale investigations suggest that the risk of BA.2 to global health is potentially higher than that of BA.1.

Enhanced fusogenicity and pathogenicity of SARS-CoV-2 Delta P681R mutation.

During the current coronavirus disease 2019 (COVID-19) pandemic, a variety of mutations have accumulated in the viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and, at the time of writing, four variants of concern are considered to be potentially hazardous to human society1. The recently emerged B.1.617.2/Delta variant of concern is closely associated with the COVID-19 surge that occurred in India in the spring of 2021 (ref. 2). However, the virological properties of B.1.617.2/Delta remain unclear. Here we show that the B.1.617.2/Delta variant is highly fusogenic and notably more pathogenic than prototypic SARS-CoV-2 in infected hamsters. The P681R mutation in the spike protein, which is highly conserved in this lineage, facilitates cleavage of the spike protein and enhances viral fusogenicity. Moreover, we demonstrate that the P681R-bearing virus exhibits higher pathogenicity compared with its parental virus. Our data suggest that the P681R mutation is a hallmark of the virological phenotype of the B.1.617.2/Delta variant and is associated with enhanced pathogenicity.

Host ZCCHC3 blocks HIV-1 infection and production through a dual mechanism.

Most mammalian cells prevent viral infection and proliferation by expressing various restriction factors and sensors that activate the immune system. Several host restriction factors that inhibit human immunodeficiency virus type 1 (HIV-1) have been identified, but most of them are antagonized by viral proteins. Here, we describe CCHC-type zinc-finger-containing protein 3 (ZCCHC3) as a novel HIV-1 restriction factor that suppresses the production of HIV-1 and other retroviruses, but does not appear to be directly antagonized by viral proteins. It acts by binding to Gag nucleocapsid (GagNC) via zinc-finger motifs, which inhibits viral genome recruitment and results in genome-deficient virion production. ZCCHC3 also binds to the long terminal repeat on the viral genome via the middle-folded domain, sequestering the viral genome to P-bodies, which leads to decreased viral replication and production. This distinct, dual-acting antiviral mechanism makes upregulation of ZCCHC3 a novel potential therapeutic strategy.

Generation of a Porcine Cell Line Stably Expressing Pig TMPRSS2 for Efficient Isolation of Swine Influenza Virus.

Pigs are important animals for meat production but can carry several zoonotic diseases, including the Japanese encephalitis virus, Nipah virus, and influenza viruses. Several Orthomyxoviridae and Coronavirinae respiratory viruses require cleavage of envelope proteins to acquire viral infectivity and consequently, need a host protease or the addition of exogenous trypsin for efficient propagation. Host TMPRSS2 is a key protease responsible for viral cleavage. Stable expression of human TMPRSS2 in African green monkey-derived Vero cells can enhance the porcine epidemic diarrhea virus. However, considering the narrow host tropism of viruses, a porcine cell line expressing pig TMPRSS2 could be optimal for replicating pig-derived viruses. Herein, we generated and evaluated a pig-derived PK-15 cell line stably expressing pig TMPRSS2. This cell line markedly (>1000-fold) and specifically enhanced the growth of influenza viruses. Furthermore, we demonstrated the usefulness of a PK-15 cell line lacking the Stat2 gene with a stable expression of pig TMPRSS2 for efficient virus isolation from clinical samples in the presence of type I interferons. Therefore, PK-15 cells expressing pig TMPRSS2 could be a valuable and promising tool for virus isolation, vaccine production, and virological studies of TMPRSS2-dependent viruses.

Generation of a bovine cell line for gene engineering using an HIV-1-based lentiviral vector.

Human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors are indispensable tools for gene engineering in mammalian cells. Conversely, lentiviral vector transduction is severely inhibited in bovine cells. Previous studies demonstrated that this inhibition is caused by the anti-lentiviral host factor tripartite motif containing 5 (TRIM5), which targets incoming HIV-1 virions by interacting with the viral capsid. In this study, we investigated several methods for overcoming the limited applicability of lentiviral vectors in bovine cells. First, we demonstrated that the SPRY domain of bovine TRIM5 is the major determinant of anti-viral activity. Second, we found that mutations that allow the capsid to evade rhesus macaque TRIM5α minimally rescued HIV-1 infectivity in bovine-derived MDBK cells. Third, we found that cyclosporine A, which relieves the inhibition of HIV-1 infection in monkey cells, significantly rescued the impaired HIV-1 infectivity in MDBK cells. Lastly, we successfully generated a bovine cell line lacking intact TRIM5 using the CRISPR/Cas9 technique. This TRIM5 knockout cell line displayed significantly higher susceptibility to an HIV-1-based lentiviral vector. In conclusion, our findings provide a promising gene engineering strategy for bovine cells, thereby contributing to innovations in agriculture and improvements in animal health.

The SARS-CoV-2 Lambda variant exhibits enhanced infectivity and immune resistance.

SARS-CoV-2 Lambda, a variant of interest, has spread in some South American countries; however, its virological features and evolutionary traits remain unclear. In this study, we use pseudoviruses and reveal that the spike protein of the Lambda variant is more infectious than that of other variants due to the T76I and L452Q mutations. The RSYLTPGD246-253N mutation, a unique 7-amino acid deletion in the N-terminal domain of the Lambda spike protein, is responsible for evasion from neutralizing antibodies and further augments antibody-mediated enhancement of infection. Although this mutation generates a nascent N-linked glycosylation site, the additional N-linked glycan is dispensable for the virological property conferred by this mutation. Since the Lambda variant has dominantly spread according to the increasing frequency of the isolates harboring the RSYLTPGD246-253N mutation, our data suggest that the RSYLTPGD246-253N mutation is closely associated with the substantial spread of the Lambda variant in South America.

The SARS-CoV-2 spike S375F mutation characterizes the Omicron BA.1 variant.

Recent studies have revealed the unique virological characteristics of Omicron, particularly those of its spike protein, such as less cleavage efficacy in cells, reduced ACE2 binding affinity, and poor fusogenicity. However, it remains unclear which mutation(s) determine these three virological characteristics of Omicron spike. Here, we show that these characteristics of the Omicron spike protein are determined by its receptor-binding domain. Of interest, molecular phylogenetic analysis revealed that acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidated that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue of another protomer in the spike trimer, conferring the attenuated cleavage efficiency and fusogenicity of Omicron spike. Our data shed light on the evolutionary events underlying the emergence of Omicron at the molecular level.