Abstract
Gammaherpesviruses, including the human pathogens Epstein–Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) are oncogenic viruses that establish lifelong infections in hosts and are associated with the development of lymphoproliferative diseases and lymphomas. Recent studies have shown that the majority of the mammalian genome is transcribed and gives rise to numerous long non-coding RNAs (lncRNAs). Likewise, the large double-stranded DNA virus genomes of herpesviruses undergo pervasive transcription, including the expression of many as yet uncharacterized lncRNAs. Murine gammaperherpesvirus 68 (MHV68, MuHV-4, γHV68) is a natural pathogen of rodents, and is genetically and pathogenically related to EBV and KSHV, providing a highly tractable model for studies of gammaherpesvirus biology and pathogenesis. Through the integrated use of parallel data sets from multiple sequencing platforms, we previously resolved transcripts throughout the MHV68 genome, including at least 144 novel transcript isoforms. Here, we sought to molecularly validate novel transcripts identified within the M3/M2 locus, which harbors genes that code for the chemokine binding protein M3, the latency B cell signaling protein M2, and 10 microRNAs (miRNAs). Using strand-specific northern blots, we validated the presence of M3-04, a 3.91 kb polyadenylated transcript that initiates at the M3 transcription start site and reads through the M3 open reading frame (ORF), the M3 poly(a) signal sequence, and the M2 ORF. This unexpected transcript was solely localized to the nucleus, strongly suggesting that it is not translated and instead may function as a lncRNA. Use of an MHV68 mutant lacking two M3-04-antisense pre-miRNA stem loops resulted in highly increased expression of M3-04 and increased virus replication in the lungs of infected mice, demonstrating a key role for these RNAs in regulation of lytic infection. Together these findings suggest the possibility of a tripartite regulatory relationship between the lncRNA M3-04, antisense miRNAs, and the latency gene M2.
Highlights
Our understanding and interpretation of mammalian genomes was almost exclusively focused on the 1% of the genome that is protein-coding
The human gammaherpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV) expresses at least two long non-coding RNA that are likely important players in specific phases of infection: the 1.08 kb polyadenylated nuclear (PAN) RNA accumulates to high levels during lytic infection [4,5,6,7], and the 10 kb antisense-to-latency transcript (ALT) lies antisense to the KSHV latency associated region [8,9]
We demonstrated that M3-04 is polyadenylated and localized to the nucleus, strongly suggesting that it functions as a long non-coding RNAs (lncRNAs)
Summary
Our understanding and interpretation of mammalian genomes was almost exclusively focused on the 1% of the genome that is protein-coding. While PacBio SMRT sequencing facilitates the identification of long-read transcript isoforms, individual full-length transcript isoforms can be validated by bioinformatically coupling parallel data sets from Illumina RNA-seq and deepCAGE platforms using their novel pipeline TRIMD (transcript resolution through integration of multi-platform data) This approach revealed almost 300 novel polyadenylated EBV transcripts, which were generated from complex promoter usage, intergenic transcription, alternative splicing, and readthrough transcription [12]. Through the coupling of PacBio long-read sequencing, with parallel data sets from Illumina RNA-seq and deepCAGE platforms, we resolved 258 MHV68 transcript isoforms, including transcripts for 55 truncated, spliced, or novel ORFs, and up to 25 potential non-coding RNAs [16]. We have further defined suppression of M3-04 transcription by antisense miRNAs, suggesting a tight regulatory relationship between M3-04, antisense miRNAs, and the overlapping latency gene M2
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