The BHLF1 Locus of Epstein-Barr Virus Contributes to Viral Latency and B-Cell Immortalization

Abstract

The Epstein-Barr virus (EBV) BHLF1 gene encodes an abundant linear and several circular RNAs believed to perform non-coding functions during virus replication, though an open reading frame is retained among an unknown percentage of EBV isolates. Evidence suggests that BHLF1 is also transcribed during latent infection, which prompted us to investigate the contribution of this locus to latency. Analysis of transcripts transiting BHLF1 revealed its transcription is widespread among B-cell lines supporting the latency I or III program of EBV protein expression, and to be more complex than originally presumed. EBV-negative Burkitt lymphoma cell lines infected with either wild-type or two different BHLF1 mutant EBVs were initially indistinguishable in supporting latency III. However, cells infected with BHLF1- virus ultimately transitioned to the more restrictive latency I, whereas cells infected with wild-type virus either sustained latency III or transitioned more slowly to latency I. Upon infection of primary B cells, which require latency III for growth in vitro, both BHLF1- viruses exhibited variably reduced immortalization potential relative to wild-type virus. Finally, in transfection experiments, efficient protein expression from an intact BHLF1 ORF required the EBV post-transcriptional regulator protein SM, whose expression is limited to the replicative cycle. Thus, one way in which BHLF1 may contribute to latency is through a mechanism, possibly mediated or regulated by a long non-coding RNA, that supports latency III critical for the establishment of EBV latency and lifelong persistence within its host, whereas any retained protein-dependent function of BHLF1 may be restricted to the replication cycle. IMPORTANCE Epstein-Barr virus (EBV) has significant oncogenic potential that is linked to its latent infection of B lymphocytes, during which virus replication is not supported. Establishment of latent infection, which is life long and can precede tumor development by years, requires the concerted actions of nearly a dozen EBV proteins and numerous small non-protein-coding RNAs. Elucidation of how these EBV products contribute to latency is crucial to understanding EBV's role in specific malignancies, and ultimately to clinical intervention. Historically, EBV genes that contribute to virus replication have been excluded from consideration of a role in latency, primarily because of the general incompatibility between virus production and cell survival. However, here we provide evidence that the genetic locus containing one such gene, BHLF1, indeed contributes to key aspects of EBV latency, including its ability to promote continuous growth of B lymphocytes, thus providing significant new insight into EBV biology and oncogenic potential.

Long non-coding RNAs in Epstein–Barr virus-related cancer

“…Previous studies have indicated that the BHLF1 gene encodes several circular and a large number of linear RNAs that play non-coding roles during virus replication [ 34 , 35 ]. However, Yetming et al recently demonstrated that BHLF1 is also transcribed during latency and may function mainly instead via its transcript as a lncRNA to contribute to viral latency and B cell immortalization [ 36 ]. However, the specific role of BHLF1 lncRNA in tumourigenesis is still not clear.…”

Role of Virally Encoded Circular RNAs in the Pathogenicity of Human Oncogenic Viruses

“…circBHLF1 is derived from the intra-exonic backsplicing of the BHLF1 gene (Toptan et al, 2018;Ungerleider et al, 2018). Increasing evidence has indicated that BHLF1 can be expressed in both latency and lytic cycles and may work as a non-coding gene for various EBV strains (Lin et al, 2010(Lin et al, , 2013Yetming et al, 2020). The expression level of circBHLF1 is largely consistent with the isogenic linear transcript and it can be detected in most of the latency and reactivation conditions examined.…”

Stress-Induced Epstein-Barr Virus Reactivation

“…In addition, B cells from three out of four donors infected with either deletion demonstrated impaired B cell immortalization when compared to wild-type EBV. Cells from one donor were transformed equally effectively regardless of which of the three viruses were used to infect the cells, although the authors noted that these B cells appeared to be more sensitive to transformation than B cells from other donors [ 108 ]. Notably, the above experiments on B cell transformation were performed in vitro.…”