Date Published: December 22, 2015
Publisher: Public Library of Science
Author(s): Xiaochen Lin, Ming-Han Tsai, Anatoliy Shumilov, Remy Poirey, Helmut Bannert, Jaap M. Middeldorp, Regina Feederle, Henri-Jacques Delecluse, Nancy Raab-Traub.
The Epstein-Barr virus (EBV) is a B lymphotropic virus that infects the majority of the human population. All EBV strains transform B lymphocytes, but some strains, such as M81, also induce spontaneous virus replication. EBV encodes 22 microRNAs (miRNAs) that form a cluster within the BART region of the virus and have been previously been found to stimulate tumor cell growth. Here we describe their functions in B cells infected by M81. We found that the BART miRNAs are downregulated in replicating cells, and that exposure of B cells in vitro or in vivo in humanized mice to a BART miRNA knockout virus resulted in an increased proportion of spontaneously replicating cells, relative to wild type virus. The BART miRNAs subcluster 1, and to a lesser extent subcluster 2, prevented expression of BZLF1, the key protein for initiation of lytic replication. Thus, multiple BART miRNAs cooperate to repress lytic replication. The BART miRNAs also downregulated pro- and anti-apoptotic mediators such as caspase 3 and LMP1, and their deletion did not sensitize B-cells to apoptosis. To the contrary, the majority of humanized mice infected with the BART miRNA knockout mutant developed tumors more rapidly, probably due to enhanced LMP1 expression, although deletion of the BART miRNAs did not modify the virus transforming abilities in vitro. This ability to slow cell growth could be confirmed in non-humanized immunocompromized mice. Injection of resting B cells exposed to a virus that lacks the BART miRNAs resulted in accelerated tumor growth, relative to wild type controls. Therefore, we found that the M81 BART miRNAs do not enhance B-cell tumorigenesis but rather repress it. The repressive effects of the BART miRNAs on potentially pathogenic viral functions in infected B cells are likely to facilitate long-term persistence of the virus in the infected host.
The Epstein-Barr virus (EBV) is a strongly B lymphotropic virus that infects the majority of the world human population and is associated with the development of malignant tumors, mainly lymphomas and carcinomas of the nasopharynx (NPC) and of the stomach . Shortly after infection, B cells start to divide and generate continuously growing cell lines, commonly termed lymphoblastoid cell lines (LCLs) . Infected cells express a set of latent proteins ascribed to subfamilies known as Epstein-Barr virus nuclear antigens (EBNA) and latent membrane proteins (LMP), most of which are essential or strongly potentiate the B cell transformation process . EBV also encodes 44 miRNAs that are divided into two clusters located around the BHRF1 gene (BHRF1 miRNAs) or within the introns of the BART gene (BART miRNAs) [3–5]. Viruses devoid of the BHRF1 miRNA locus are less transforming than their wild type counterparts [6–9]. Indeed, a recombinant virus that lacks this cluster retains only 1/20th of the wild type transforming capacity .
The study of spontaneous EBV lytic replication has been hampered by the propensity of the virus to enter latency in infected cells. LCLs initiate some degree of lytic replication after treatment with chemicals such as TPA or butyrate . Some non-lymphoid cell lines such as 293 cells can support lytic replication after transfection with BZLF1 . Infection of primary epithelial cells gives rise to spontaneous lytic replication but the efficiency of infection remains low and these cells are difficult to grow in large numbers [31,32]. Thus, tractable experimental systems have not been available for a long time. However, M81, a virus isolated from an NPC patient replicates strongly in primary B cells isolated from any individual tested so far . Furthermore, M81 is amenable to a genetic analysis after its cloning as a bacterial artificial chromosome . We addressed the function of the BART miRNAs by constructing viruses that evince partial or complete deletions of this locus, as well as a revertant thereof. We found that the BART miRNAs negatively regulate spontaneous lytic replication in B cells, as their excision from the M81 viral genome gives rise to an increase in spontaneous lytic replication in vitro and in vivo in humanized mice. This phenotypic trait disappears in the revertant virus or upon complementation. The BART miRNAs seem to target BZLF1 directly as its mRNA is recruited more efficiently to the RISC in cells infected by wild type virus than in LCLs generated with the BART miRNA knockout virus and expression of a luciferase gene fused to BZLF1 3’UTR is lower in LCLs generated with wild type virus relative to LCLs generated with the ΔAll virus. However, the difficulties to transfect primary LCLs with high efficiency somehow qualifies the latter result. Typically, miRNAs and their cognate targets are expressed in the same cells and the miRNA down-regulate protein expression in all cells that express them. In LCLs, the BART miRNAs are expressed in latently infected cells that do not express the BZLF1. Therefore, the BART miRNAs can only exert their function on BZLF1 in the minority of cells that initiate lytic replication in a given LCL. Thus, they do not directly control lytic replication but come into play only in cells that have already initiated BZLF1 synthesis. Such a scenario fits with the observation that the number of spontaneously replicating cells in LCLs infected with M81/ΔAll does not exceed 15%. The remaining 85% are devoid of BART miRNAs but nevertheless remain BZLF1-negative. However, in cells that have already initiated lytic replication through expression of BZLF1, the expression of the BART miRNAs apparently needs to be lower than in non-replicating cells. This, combined with the observation that individual replicating cells in LCLs infected with wild type or with M81/ΔAll express the BZLF1 protein at the same level suggests that the halved level of BART miRNAs present in replicating cells infected with wild type viruses is too low to efficiently down-regulate BZLF1 protein. This would mean that the expression of the BART miRNAs needs be lower than in latent cells, but does not need necessarily need to be completely extinguished.