New Noncoding Lytic Transcripts Derived from the Epstein-Barr Virus Latency Origin of Replication, oriP, Are Hyperedited, Bind the Paraspeckle Protein, NONO/p54nrb, and Support Viral Lytic Transcription.
We have previously shown that the Epstein-Barr virus (EBV) likely encodes hundreds of viral long noncoding RNAs (vlncRNAs) that are expressed during reactivation. Here we show that the EBV latency origin of replication (oriP) is transcribed bi-directionally during reactivation and that both leftward (oriPtLs) and rightward (oriPtRs) transcripts are largely localized in the nucleus. While the oriPtLs are most likely noncoding, at least some of the oriPtRs contain the BCRF1/vIL10 open reading frame. Nonetheless, oriPtR transcripts with long 5' untranslated regions may partially serve noncoding functions. Both oriPtL and oriPtR transcripts are expressed with late kinetics, and their expression is inhibited by phosphonoacetic acid. RNA sequencing (RNA-seq) analysis showed that oriPtLs and oriPtRs exhibited extensive "hyperediting" at their Family of Repeat (FR) regions. RNA secondary structure prediction revealed that the FR region of both oriPtLs and oriPtRs may form large evolutionarily conserved and thermodynamically stable hairpins. The double-stranded RNA-binding protein and RNA-editing enzyme ADAR was found to bind to oriPtLs, likely facilitating editing of the FR hairpin. Further, the multifunctional paraspeckle protein, NONO, was found to bind to oriPt transcripts, suggesting that oriPts interact with the paraspeckle-based innate antiviral immune pathway. Knockdown and ectopic expression of oriPtLs showed that it contributes to global viral lytic gene expression and viral DNA replication. Together, these results show that these new vlncRNAs interact with cellular innate immune pathways and that they help facilitate progression of the viral lytic cascade.


OBJECTIVE
Recent studies have revealed that the complexity of lytic herpesviral transcriptomes is significantly greater than previously appreciated with hundreds of viral long noncoding RNAs (vlncRNAs) being recently discovered. Work on cellular lncRNAs over the past several years has just begun to give us an initial appreciation for the array of functions they play in complex formation and regulatory processes in the cell. The newly identified herpesvirus lncRNAs are similarly likely to play a variety of different functions, although these functions are likely tailored to specific needs of the viral infection cycles. Here we describe novel transcripts derived from the EBV latency origin of replication. We show that they are hyperedited, that they interact with a relatively newly appreciated antiviral pathway, and that they play a role in facilitating viral lytic gene expression. These investigations are a starting point to unraveling the complex arena of vlncRNA function in herpesvirus lytic replication.
Department of Structural and Cellular Biology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA.
Subing
S
Cao
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
Walter
W
Moss
Department of Pathology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA.
Tina
T
O'Grady
Department of Pathology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA.
Monica
M
Concha
Department of Pathology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA.
Michael J
http://orcid.org/0000-0001-9930-3578
MJ
Strong
Department of Pathology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA.
Xia
X
Wang
Department of Cellular and Molecular Biology Graduate Program, Tulane University, New Orleans, Louisiana, USA.
Yi
Y
Yu
Department of Pathology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA Tulane Cancer Center, New Orleans, Louisiana, USA.
Melody
M
Baddoo
Department of Computer Science, Xavier University of Louisiana, New Orleans, Louisiana, USA.
Kun
K
Zhang
Department of Pathology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA.
Claire
C
Fewell
Department of Pathology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA Tulane Cancer Center, New Orleans, Louisiana, USA.
Zhen
Z
Lin
Department of Structural and Cellular Biology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA Tulane Cancer Center, New Orleans, Louisiana, USA.
Yan
Y
Dong
Department of Pathology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA Tulane Cancer Center, New Orleans, Louisiana, USA erik@tulane.edu.
Erik K
EK
Flemington
D002460:Cell Line; D004268:DNA-Binding Proteins; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D007113:Immunity, Innate; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism*; D050810:Octamer Transcription Factors / Q000378:metabolism*; D011485:Protein Binding; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D018741:Replication Origin; D014158:Transcription, Genetic; D014779:Virus Replication
D004268:DNA-Binding Proteins; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D050810:Octamer Transcription Factors; D022661:RNA, Untranslated; D016601:RNA-Binding Proteins