Numerous host factors function as intrinsic antiviral effectors to attenuate viral replication. MARCH8 is an E3 ubiquitin ligase that has been identified as a host restriction factor that inhibits the replication of various viruses. This study elucidated the mechanism by which MARCH8 restricts respiratory syncytial virus (RSV) replication through selective degradation of the viral small hydrophobic (SH) protein. We demonstrated that MARCH8 directly interacts with RSV-SH and catalyzes its ubiquitination at lysine 13, leading to SH degradation via the ubiquitin-lysosomal pathway. Functionally, MARCH8 expression enhances RSV-induced apoptosis through SH degradation, ultimately reducing viral titers. Conversely, an RSV strain harboring the SH-K13R mutation exhibited prolonged SH protein stability and attenuated apoptosis in infected cells, even in the presence of MARCH8. Targeted depletion of MARCH8 enhances cellular survival and potentially increases viral persistence. These findings demonstrate that MARCH8 promotes the early elimination of virus-infected cells by abrogating the anti-apoptotic function of SH, thereby reducing viral transmission. Our study provides novel insights into the interplay between host restriction factors and viral evasion strategies, potentially providing new therapeutic approaches for RSV infections.

In this study, we show that the coronavirus (CoV) genome may encode many functional hydrophobic alpha-helical peptides (HAHPs) in overlapping reading frames of major coronaviral proteins throughout the entire viral genome. These HAHPs can theoretically be expressed from non-canonical sub-genomic (sg)RNAs that are synthesized in substantial amounts in infected cells. We selected and analyzed five and six HAHPs encoded in the S gene regions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively. Two and three HAHPs derived from SARS-CoV-2 and MERS-CoV, respectively, specifically interacted with both the SARS-CoV-2 and MERS-CoV S proteins and inhibited their membrane fusion activity. Furthermore, one of the SARS-CoV-2 HAHPs specifically inhibited viral RNA synthesis by accumulating at the site of viral RNA synthesis. Our data show that a group of HAHPs in the coronaviral genome potentially has a regulatory role in viral propagation.

N6-methyladenosine (m6A) epitranscriptomic modifications play crucial roles in regulating both host and viral gene expression. Here, we revealed a novel mechanism by which paramyxoviruses exploit host m6A machinery to simultaneously enhance viral replication and suppress host immunity. Our results demonstrated that the viral matrix protein (M) of bovine parainfluenza virus type 3 (BPIV3) binds to the methyltransferase domain of METTL3 in the nucleus and facilitates its translocation to the cytoplasm through an exportin-1-dependent pathway. This mechanism is conserved across multiple paramyxoviruses, including human parainfluenza virus type 3, Sendai virus, Nipah virus, and measles virus, suggesting an evolutionarily conserved viral strategy. The relocated METTL3 catalyzes m6A modification at specific sites within viral nucleocapsid protein (N) mRNA, significantly enhancing its stability and protein expression. Using reverse genetics, we generated recombinant viruses harbouring mutations at these m6A acceptor sites, which exhibited markedly attenuated viral replication, confirming the critical role of these epitranscriptomic marks in the viral life cycle. Rescue experiments demonstrated that the expression of exogenous N protein partially restored the viral titer and concomitant genome/antigenome synthesis in m6A site mutant, indicating that reduced N protein abundance represents a key mechanism underlying impaired viral replication. Furthermore, M protein-mediated depletion of nuclear METTL3 significantly reduces m6A modification of host IFN-β mRNA, resulting in diminished interferon expression and compromised antiviral responses. Supporting this mechanism, infection with viruses bearing nuclear export signal mutations that prevent METTL3 cytoplasmic translocation, maintained IFN-β mRNA m6A modification and resulted in significantly elevated IFN-β expression. These findings provide direct mechanistic evidence that paramyxoviruses utilize M-driven METTL3 relocalization as a sophisticated immune evasion strategy. Our study illuminates how paramyxoviruses strategically manipulate epitranscriptomic regulation to create an environment conducive to viral propagation, thereby advancing our understanding of virus-host interactions and identifying potential targets for antiviral therapeutics.

Introduction. Human bocavirus 1 (HBoV1) is a member of Bocaparvovirus primate1 and is associated with paediatric respiratory infections. Although genetic grouping has been described, it remains unclear whether temporal genetic changes occur among clinically circulating HBoV1 strains. Hypothesis/Gap Statement. HBoV1 shows limited genetic diversity, but temporal genetic changes may occur in circulating strains. Aim. To investigate whether temporal genetic changes are present in HBoV1 isolates collected in Fukushima, Japan, between 2018 and 2024. Methodology. Nasopharyngeal swabs were collected from paediatric inpatients with acute respiratory infections. Thirty-two HBoV1 isolates were obtained using air–liquid interface cultures of human bronchial/tracheal epithelial cells. Nearly complete genomes were sequenced by next-generation sequencing and analysed together with additional reference sequences (total n =100, including outgroups) using phylogenetic and time-scaled methods. Results. Phylogenetic analyses classified the 32 Fukushima isolates into Group 1 ( n =12) and Group 2 ( n =20). Group 2 predominated in 2018, Group 1 from 2019 to 2021 and Group 2 again from 2022 to 2024, indicating temporal shifts in circulating strains. The average number of nucleotide differences among HBoV1 genomes was 13.9 (maximum 55), with Groups 1 and 2 diverging around 2004–2005. Divergence was concentrated in the VP genes, and synonymous substitutions within VP genes overlapped the ORFx reading frame, leading to distinct amino acid differences in ORFx. Conclusion. Clinically circulating HBoV1 undergoes temporal genetic change, with group differentiation determined primarily by VP genes and uniquely by ORFx at the amino acid level. These findings suggest a potential biological role for ORFx in the HBoV1 life cycle. Understanding these temporal genetic shifts is important for enhancing the accuracy of molecular surveillance tools, particularly for distinguishing genetically diverse HBoV1 strains.