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Identification and Characterization of BmNPV m6A Sites and Their Possible Roles During Viral Infection
Xing Zhang1,2, Yaxin Zhang1, Jun Pan1
1School of Biology & Basic Medical Science, Soochow University, Suzhou, China.
Abstract:
Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens and causes serious economic losses in sericulture. At present, there is no epigenetic modification of BmNPV transcripts, especially of m6A, and this modification mediates diverse cellular and viral functions. This study showed that m6A modifications are widespread in BmNPV transcripts in virally infected cells and the identified m6A peaks with a conserved RRACH sequence. m6A sites predominantly appear in the coding sequences (CDS) and the 3'-end of CDS. About 37% of viral genes with m6A sites deleted from the viral genome did not produce any infectious virions in KOV-transfected cells. Among the viral genes related to replication and proliferation, ie-1 mRNA was identified with a higher m6A level than other viral genes. The m6A sites in the ie-1 mRNA may be negatively related to the protein expression. Viral replication was markedly inhibited in cells overexpressed with BmYTHDF3 in a dose-dependent manner, and a contrary effect was found in si-BmYTHDF3-transfected cells. Collectively, the identification of putative m6A modification in BmNPV transcripts provides a foundation for comprehensively understanding the viral infection, replication, and pathobiology in silkworms.
Insights
This study reveals widespread N6-methyladenosine (m6A) modifications in Bombyx mori nucleopolyhedrovirus (BmNPV) transcripts, impacting viral gene function and replication. These m6A modifications are crucial for producing infectious virions and regulating viral gene expression.
Area of Science:
- Virology
- Molecular Biology
- Epigenetics
Background:
- Bombyx mori nucleopolyhedrovirus (BmNPV) causes significant economic losses in sericulture.
- Epigenetic modifications, particularly N6-methyladenosine (m6A), are known to regulate viral functions but were uncharacterized in BmNPV transcripts.
- Understanding BmNPV transcript modifications is crucial for controlling silkworm diseases.
Purpose of the Study:
- To investigate the presence and characteristics of m6A modifications in BmNPV transcripts.
- To determine the functional significance of m6A modifications in viral gene expression and replication.
- To explore the role of host factors, like BmYTHDF3, in mediating m6A-dependent viral processes.
Main Methods:
- Bioinformatic analysis to identify m6A peaks in BmNPV transcripts from infected cells.
- Site-directed mutagenesis to delete identified m6A sites in the viral genome.
- Quantitative analysis of viral gene expression and infectious virion production.
- Manipulation of BmYTHDF3 expression levels (overexpression and knockdown) in infected cells.
Main Results:
- m6A modifications are widespread in BmNPV transcripts, with conserved RRACH motifs, primarily in coding sequences and 3'-ends.
- Deletion of m6A sites in approximately 37% of viral genes abolished infectious virion production.
- The immediate-early gene 1 (ie-1) mRNA exhibited high m6A levels, potentially negatively correlating with protein expression.
- Overexpression of BmYTHDF3 inhibited viral replication, while its knockdown enhanced it.
Conclusions:
- This study provides the first evidence of m6A modification in BmNPV transcripts, revealing its critical role in viral gene expression and replication.
- m6A modification is essential for the production of infectious BmNPV virions.
- The host factor BmYTHDF3 plays a significant role in regulating BmNPV replication through m6A-dependent mechanisms.
- These findings lay the groundwork for understanding BmNPV pathobiology and developing novel antiviral strategies.
