MicroRNA-155 (miR-155) regulates antitumor immune responses. However, its specific functions within distinct immune cell types have not been delineated in conditional KO mouse models. In this study, we investigated the role of miR-155 specifically within T cells during the immune response to syngeneic tumors. We found that miR-155 expression within T cells is required to limit syngeneic tumor growth and promote IFNγ production by T cells within the tumor microenvironment. Consequently, we found that miR-155 expression by T cells is necessary for proper tumor-associated macrophage expression of IFNγ-inducible genes. We also found that immune checkpoint-blocking (ICB) antibodies against programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) restored antitumor immunity in miR-155 T cell-conditional KO mice. We noted that these ICB antibodies rescued the levels of IFNγ-expressing T cells, expression of multiple activation and effector genes expressed by tumor-infiltrating CD8 and CD4 T cells, and tumor-associated macrophage activation. Moreover, the ICB approach partially restored expression of several derepressed miR-155 targets in tumor-infiltrating, miR-155-deficient CD8 T cells, suggesting that miR-155 and ICB regulate overlapping pathways to promote antitumor immunity. Taken together, our findings highlight the multifaceted role of miR-155 in T cells, in which it promotes antitumor immunity. These results suggest that the augmentation of miR-155 expression could be used to improve anticancer immunotherapies.

Clinically available drugs active against Epstein-Barr virus (EBV) and other human herpesviruses are limited to those targeting viral DNA replication. To identify compounds directed against other steps in the viral life cycle, we searched for drugs active against the EBV SM protein, which is essential for infectious virus production. SM has a highly gene-specific mode of action and preferentially enhances expression of several late lytic cycle EBV genes. Here we demonstrate that spironolactone, a mineralocorticoid receptor antagonist approved for clinical use, inhibits SM function and infectious EBV production. Expression of EBV viral capsid antigen is highly SM dependent, and spironolactone inhibits viral capsid antigen synthesis and capsid formation, blocking EBV virion production at a step subsequent to viral DNA replication. In addition, spironolactone inhibits expression of other SM-dependent genes necessary for infectious virion formation. We further demonstrate that molecules structurally related to spironolactone with similar antimineralocorticoid blocking activity do not inhibit EBV production. These findings pave the way for development of antiherpesvirus drugs with new mechanisms of action directed against SM and homologous essential proteins in other herpesviruses. . EBV infects the majority of humans worldwide and establishes a persistent, lifelong latent infection in memory B cells. Occasional reactivation from latency occurs, and EBV enters a lytic phase of replication, during which a tightly regulated series of lytic genes is expressed, culminating in lysis of the host cell and release of infectious viral progeny. All herpesviruses share these abilities to establish asymptomatic latent infection and reactivate intermittently.All herpesviruses also express a regulatory protein early in lytic replication that is essential for efficient gene expression and infectious virion production. The EBV member of this family, which includes herpes simplex virus ICP27, cytomegalovirus (CMV) UL69, and Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57, is known as SM (2). SM acts posttranscriptionally to enhance accumulation of many lytic EBV transcripts. SM activity is highly gene-specific and preferentially enhances expression of several late lytic transcripts, including those encoding the major viral capsid antigen (VCA) and gp350, which is required for infection of B lymphocytes (3-5). SM and its homologs in other herpesviruses may also enhance transcription and translation of specific viral genes (6).Currently, all available antiherpesvirus drugs target viral DNA polymerases and are usually highly effective, but toxicity and development of resistance limits their use (7). To discover potential novel therapeutic compounds, we applied a cell-based screening assay to identify compounds that would specifically target SM function (8). Using this assay, spironolactone (SPR), a drug in clinical use for over 50 y, was identified as inhibiting SM function and EBV virion production. SPR is an aldosterone antagonis...

Epstein-Barr virus (EBV) SM protein is an essential lytic cycle protein with multiple posttranscriptional mechanisms of action. SM binds RNA and increases accumulation of specific EBV transcripts. Previous studies using microarrays and PCR have shown that SM-null mutants fail to accumulate several lytic cycle mRNAs and proteins at wild-type levels. However, the complete effect of SM on the EBV transcriptome has been incompletely characterized. Here we precisely identify the effects of SM on all EBV transcripts by high-throughput RNA sequencing, quantitative PCR (qPCR), and Northern blotting. The effect of SM on EBV mRNAs was highly skewed and was most evident on 13 late genes, demonstrating why SM is essential for infectious EBV production. EBV DNA replication was also partially impaired in SM mutants, suggesting additional roles for SM in EBV DNA replication. While it has been suggested that SM specificity is based on recognition of either RNA sequence motifs or other sequence properties, no such unifying property of SM-responsive targets was discernible. The binding affinity of mRNAs for SM also did not correlate with SM responsiveness. These data suggest that while target RNA binding by SM may be required for its effect, specific activation by SM is due to differences in inherent properties of individual transcripts. We therefore propose a new model for the mechanism of action and specificity of SM and its homologs in other herpesviruses: that they bind many RNAs but only enhance accumulation of those that are intrinsically unstable and poorly expressed. IMPORTANCEThis study examines the mechanism of action of EBV SM protein, which is essential for EBV replication and infectious virus production. Since SM protein is not similar to any cellular protein and has homologs in all other human herpesviruses, it has potential importance as a therapeutic target. Here we establish which EBV RNAs are most highly upregulated by SM, allowing us to understand why it is essential for EBV replication. By comparing and characterizing these RNA transcripts, we conclude that the mechanism of specific activity is unlikely to be based simply on preferential recognition of a target motif. Rather, SM binding to its target RNA may be necessary but not sufficient for enhancing accumulation of the RNA. Preferential effects of SM on its most responsive RNA targets may depend on other inherent characteristics of these specific mRNAs that require SM for efficient expression, such as RNA stability. E pstein-Barr virus (EBV) SM protein is an essential regulatory protein expressed by EBV during the lytic phase of replication (for reviews, see references 1 and 2). SM protein is detectable after immediate early gene expression and prior to expression of other early gene products (3, 4). When the SM gene is insertionally inactivated in recombinant EBV genomes, the resultant virus is incapable of completing the lytic cycle and producing infectious virions (5, 6). The exact reasons that SM is essential for lytic replication and virus pro...

Epstein-Barr virus (EBV) is linked to the development of both lymphoid and epithelial malignancies worldwide. The M81 strain of EBV, isolated from a Chinese patient with nasopharyngeal carcinoma (NPC), demonstrates spontaneous lytic replication and high-titer virus production in comparison to the prototype B95-8 EBV strain. Genetic comparisons of M81 and B95-8 EBVs were previously been performed in order to determine if the hyperlytic property of M81 is associated with sequence differences in essential lytic genes. EBV SM is an RNA-binding protein expressed during early lytic replication that is essential for virus production. We compared the functions of M81 SM and B95-8 SM and demonstrate that polymorphisms in SM do not contribute to the lytic phenotype of M81 EBV. However, the expression level of the EBV DNA polymerase protein was much higher in M81- than in B95-8-infected cells. The relative deficiency in the expression of B95-8 DNA polymerase was related to the B95-8 genome deletion, which truncates the BALF5 3' untranslated region (UTR). Similarly, the insertion of bacmid DNA into the widely used recombinant B95-8 bacmid creates an inefficient BALF5 3' UTR. We further showed that the while SM is required for and facilitates the efficient expression of both M81 and B95-8 mRNAs regardless of the 3' UTR, the BALF5 3' UTR sequence is important for BALF5 protein translation. These data indicate that the enhanced lytic replication and virus production of M81 compared to those of B95-8 are partly due to the robust translation of EBV DNA polymerase required for viral DNA replication due to a more efficient BALF5 3' UTR in M81. Epstein-Barr virus (EBV) infects more than 90% of the human population, but the incidence of EBV-associated tumors varies greatly in different parts of the world. Thus, understanding the connection between genetic polymorphisms from patient isolates of EBV, gene expression phenotypes, and disease is important and may help in developing antiviral therapy. This study examines potential causes of the enhanced lytic replicative properties of M81 EBV isolated from a nasopharyngeal carcinoma (NPC) patient and provides new evidence for the role of the BALF5 gene 3' UTR sequence in DNA polymerase protein expression during lytic replication. Variation in the gene structure of the DNA polymerase gene may therefore contribute to lytic virus reactivation and pathogenesis.

The proinflammatory microRNA-155 (miR-155) is highly expressed in the serum and CNS lesions of patients with multiple sclerosis (MS). Global knockout (KO) of miR-155 in mice confers resistance to a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), by reducing the encephalogenic potential of CNS-infiltrating Th17 T cells. However, cell-intrinsic roles for miR-155 during EAE have not been formally determined. In this study, we use single-cell RNA sequencing and cell-specific conditional miR-155 KOs to determine the importance of miR-155 expression in distinct immune cell populations. Time-course single-cell sequencing revealed reductions in T cells, macrophages, and dendritic cells (DCs) in global miR-155 KO mice compared with wild-type controls at day 21 after EAE induction. Deletion of miR-155 in T cells, driven by CD4 Cre, significantly reduced disease severity similar to global miR-155 KOs. CD11c Cre-mediated deletion of miR-155 in DCs also resulted in a modest yet significant reduction in the development of EAE, with both T cell– and DC-specific KOs showing a reduction in Th17 T cell infiltration into the CNS. Although miR-155 is highly expressed in infiltrating macrophages during EAE, deletion of miR-155 using LysM Cre did not impact disease severity. Taken together, these data show that although miR-155 is highly expressed in most infiltrating immune cells, miR-155 has distinct roles and requirements depending on the cell type, and we have demonstrated this using the gold standard conditional KO approach. This provides insights into which functionally relevant cell types should be targeted by the next generation of miRNA therapeutics.