A Viral microRNA Cluster Regulates the Expression of PTEN, p27 and of a bcl-2 Homolog

Abstract

The Epstein-Barr virus (EBV) infects and transforms B-lymphocytes with high efficiency. This process requires expression of the viral latent proteins and of the 3 miR-BHRF1 microRNAs. Here we show that B-cells infected by a virus that lacks these non-coding RNAs (Δ123) grew more slowly between day 5 and day 20, relative to wild type controls. This effect could be ascribed to a reduced S phase entry combined with a moderately increased apoptosis rate. Whilst the first phenotypic trait was consistent with an enhanced PTEN expression in B-cells infected with Δ123, the second could be explained by very low BHRF1 protein and RNA levels in the same cells. Indeed, B-cells infected either by a recombinant virus that lacks the BHRF1 protein, a viral bcl-2 homolog, or by Δ123 underwent a similar degree of apoptosis, whereas knockouts of both BHRF1 microRNAs and protein proved transformation-incompetent. We find that that the miR-BHRF1-3 seed regions, and to a lesser extent those of miR-BHRF1-2 mediate these stimulatory effects. After this critical period, B-cells infected with the Δ123 mutant recovered a normal growth rate and became more resistant to provoked apoptosis. This resulted from an enhanced BHRF1 protein expression relative to cells infected with wild type viruses and correlated with decreased p27 expression, two pro-oncogenic events. The upregulation of BHRF1 can be explained by the observation that large BHRF1 mRNAs are the source of BHRF1 protein but are destroyed following BHRF1 microRNA processing, in particular of miR-BHRF1-2. The BHRF1 microRNAs are unlikely to directly target p27 but their absence may facilitate the selection of B-cells that express low levels of this protein. Thus, the BHRF1 microRNAs allowed a time-restricted expression of the BHRF1 protein to innocuously expand the virus B-cell reservoir during the first weeks post-infection without increasing long-term immune pressure.

The Epstein-Barr virus miR-BHRF1 microRNAs regulate viral gene expression in cis

“…In contrast, Seto et al (2010), who blocked miRNA expression by scrambling the three miR-BHRF1 miRNA sequences in a way that would also inactivate the two potential splice donors present in miR-BHRF2*, actually saw an ~2-fold increase in BHRF1 mRNA expression. These results are consistent with the hypothesis that it is the mutation introduced into the miR-BHRF1-2 pre-miRNA stem-loop in the Δ123 EBV mutant, rather than loss of miR-BHRF1-2 or 1-2* expression per se , that results in the reduced level of BHRF1 mRNA and protein expression in the Δ123 LCLs reported by Bernhardt et al (2016).…”

“…Whether this novel splicing event has any effect on EBV gene expression is unclear, though it clearly has the potential to inhibit BHRF1 protein expression. In this context, we note that Bernhardt et al (2016) reported that the Δ123 EBV mutant used in this manuscript expresses a far lower level of the BHRF1 protein, and a 5- to 10-fold lower level of BHRF1 mRNA, in LCLs early after EBV infection. In contrast, Seto et al (2010), who blocked miRNA expression by scrambling the three miR-BHRF1 miRNA sequences in a way that would also inactivate the two potential splice donors present in miR-BHRF2*, actually saw an ~2-fold increase in BHRF1 mRNA expression.…”

“…So far, EBV-encoded miRNAs have been shown to play a role in immune evasion (Albanese et al, 2016; Tagawa et al, 2016; Xia et al, 2008), apoptosis (Kang et al, 2015; Lei et al, 2013), and inhibition of tumor suppressors (Bernhardt et al, 2016). Additionally, inactivation of the miR-BHRF1 miRNA cluster results in a reproducible and robust reduction in B cell transformation and LCL growth (Feederle et al, 2011a, 2011b; Haar et al, 2015; Seto et al, 2010), which suggested that the miR-BHRF1 miRNA cluster was promoting EBV transformation by downregulating the expression in trans of cellular mRNAs that inhibit this process.…”

TLR4-Mediated Inflammation Promotes KSHV-Induced Cellular Transformation and Tumorigenesis by Activating the STAT3 Pathway

“…miR-155 could promote the expansion of B cells in vivo (40), and play a critical role in immunity and inflammation in macrophages (41). Interestingly, PTEN, a negative regulator of PI3K-AKT pathway, is a target of miR-155 and BHRF1 miR-cluster of EBV, another malignant herpesvirus (42). …”

The functions of tumor suppressor PTEN in innate and adaptive immunity

“…During viral infection, virus-coding miRNAs can also target PTEN to regulate anti-viral immune responses. 50 …”