Dual isoform sequencing reveals complex transcriptomic and epitranscriptomic landscapes of a prototype baculovirus

Area of Science:

• Virology
• Molecular Biology
• Genomics

Background:

• Baculoviruses, such as Autographa californica multiple nucleopolyhedrovirus, are important tools in biotechnology and research.
• Understanding baculovirus gene expression at the transcriptomic level is crucial for optimizing their use.
• Previous transcriptomic studies were limited by short-read sequencing technologies.

Purpose of the Study:

• To comprehensively characterize the transcriptome of Autographa californica multiple nucleopolyhedrovirus using long-read sequencing (LRS).
• To identify novel viral transcripts, isoforms, and regulatory elements.
• To investigate RNA modifications within the viral transcriptome.

Main Methods:

• Utilized two LRS technologies: MinION (Oxford Nanopore Technologies) and Sequel (Pacific Biosciences).
• Analyzed full-length RNA molecules to distinguish transcript isoforms, mono- and polycistronic RNAs, and overlapping transcripts.
• Bioinformatic analysis to identify novel transcripts, open reading frames (ORFs), non-coding RNAs, and untranslated regions (UTRs).

Main Results:

• Detected a total of 875 transcript species, with 759 being novel and 116 previously annotated.
• Identified 41 novel putative protein-coding transcripts, 14 monocistronic, 99 polygenic RNAs, 101 non-coding RNAs, and 504 UTR isoforms.
• Discovered novel replication origin-associated transcripts, upstream ORFs, cis-regulatory sequences, and poly(A) sites. Detected RNA methylation in 99 viral genes and RNA hyper-editing in a specific transcript isoform.

Conclusions:

• LRS provides unprecedented resolution for characterizing complex viral transcriptomes.
• This study significantly expands the known transcriptional landscape of Autographa californica multiple nucleopolyhedrovirus.
• The findings reveal novel regulatory mechanisms and RNA modifications, offering new avenues for baculovirus research and engineering.