MicroRNA-focused CRISPR/Cas9 screen identifies miR-142 as a key regulator of Epstein-Barr virus reactivation.

Reactivation from latency plays a significant role in maintaining persistent lifelong Epstein-Barr virus (EBV) infection. Mechanisms governing successful activation and progression of the EBV lytic phase are not fully understood. EBV expresses multiple viral microRNAs (miRNAs) and manipulates several cellular miRNAs to support viral infection. To gain insight into the host miRNAs regulating transitions from EBV latency into the lytic stage, we conducted a CRISPR/Cas9-based screen in EBV+ Burkitt lymphoma (BL) cells using anti-Ig antibodies to crosslink the B cell receptor (BCR) and induce reactivation. Using a gRNA library against >1500 annotated human miRNAs, we identified miR-142 as a key regulator of EBV reactivation. Genetic ablation of miR-142 enhanced levels of immediate early and early lytic gene products in infected BL cells. Ago2-PAR-CLIP experiments with reactivated cells revealed miR-142 targets related to Erk/MAPK signaling, including components directly downstream of the B cell receptor (BCR). Consistent with these findings, disruption of miR-142 enhanced SOS1 levels and Mek phosphorylation in response to surface Ig cross-linking. Effects could be rescued by inhibitors of Mek (cobimetinib) or Raf (dabrafenib). Taken together, these results show that miR-142 functionally regulates SOS1/Ras/Raf/Mek/Erk signaling initiated through the BCR and consequently, restricts EBV entry into the lytic cycle.

Epstein-Barr Virus miR-BHRF1-3 Targets the BZLF1 3'UTR and Regulates the Lytic Cycle.

Suppression of lytic viral gene expression is a key aspect of the Epstein-Barr virus (EBV) life cycle to facilitate the establishment of latent infection. Molecular mechanisms regulating transitions between EBV lytic replication and latency are not fully understood. Here, we investigated the impact of viral microRNAs on the EBV lytic cycle. Through functional assays, we found that miR-BHRF1-3 attenuates EBV lytic gene expression following reactivation. To understand the miRNA targets contributing to this activity, we performed Ago PAR-CLIP analysis on EBV-positive, reactivated Burkitt's lymphoma cells and identified multiple miR-BHRF1-3 interactions with viral transcripts. Using luciferase reporter assays, we confirmed a miRNA interaction site within the 3'UTR of BZLF1 which encodes the essential immediate early (IE) transactivator Zta. Comparison of >850 published EBV genomes identified sequence polymorphisms within the miR-BHRF1-3 locus that deleteriously affect miRNA expression and function. Molecular interactions between the homologous viral miRNA, miR-rL1-17, and IE transcripts encoded by rhesus lymphocryptovirus were further identified. Our data demonstrate that regulation of IE gene expression by a BHRF1 miRNA is conserved among lymphocryptoviruses, and further reveal virally-encoded genetic elements that orchestrate viral antigen expression during the lytic cycle. IMPORTANCE Epstein-Barr virus infection is predominantly latent in healthy individuals, while periodic cycles of reactivation are thought to facilitate persistent lifelong infection. Lytic infection has been linked to development of certain EBV-associated diseases. Here, we demonstrate that EBV miR-BHRF1-3 can suppress lytic replication by directly inhibiting Zta expression. Moreover, we identify nucleotide variants that impact the function of miR-BHRF1-3, which may contribute to specific EBV pathologies.

Epstein-Barr virus microRNAs regulate B cell receptor signal transduction and lytic reactivation.

MicroRNAs (miRNAs) are post-transcriptional regulatory RNAs that can modulate cell signaling and play key roles in cell state transitions. Epstein-Barr virus (EBV) expresses >40 viral miRNAs that manipulate both viral and cellular gene expression patterns and contribute to reprogramming of the host environment during infection. Here, we identified a subset of EBV miRNAs that desensitize cells to B cell receptor (BCR) stimuli, and attenuate the downstream activation of NF-kappaB or AP1-dependent transcription. Bioinformatics and pathway analysis of Ago PAR-CLIP datasets identified multiple EBV miRNA targets related to BCR signal transduction, including GRB2, SOS1, MALT1, RAC1, and INPP5D, which we validated in reporter assays. BCR signaling is critical for B cell activation, proliferation, and differentiation, and for EBV, is linked to reactivation. In functional assays, we demonstrate that EBV miR-BHRF1-2-5p contributes to the growth of latently infected B cells through GRB2 regulation. We further determined that activities of EBV miR-BHRF1-2-5p, EBV miR-BART2-5p, and a cellular miRNA, miR-17-5p, directly regulate virus reactivation triggered by BCR engagement. Our findings provide mechanistic insight into some of the key miRNA interactions impacting the proliferation of latently infected B cells and importantly, governing the latent to lytic switch.

Role of IL-15 Signaling in the Pathogenesis of Simian Immunodeficiency Virus Infection in Rhesus Macaques.

Although IL-15 has been implicated in the pathogenic hyperimmune activation that drives progressive HIV and SIV infection, as well as in the generation of HIV/SIV target cells, it also supports NK and T cell homeostasis and effector activity, potentially benefiting the host. To understand the role of IL-15 in SIV infection and pathogenesis, we treated two cohorts of SIVmac239-infected rhesus macaques (RM; Macaca mulatta), one with chronic infection, the other with primary infection, with a rhesusized, IL-15-neutralizing mAb (versus an IgG isotype control) for up to 10 wk (n = 7-9 RM per group). In both cohorts, anti-IL-15 was highly efficient at blocking IL-15 signaling in vivo, causing 1) profound depletion of NK cells in blood and tissues throughout the treatment period; 2) substantial, albeit transient, depletion of CD8+ effector memory T cells (TEM) (but not the naive and central memory subsets); and 3) CD4+ and CD8+ TEM hyperproliferation. In primary infection, reduced frequencies of SIV-specific effector T cells in an extralymphoid tissue site were also observed. Despite these effects, the kinetics and extent of SIV replication, CD4+ T cell depletion, and the onset of AIDS were comparable between anti-IL-15- and control-treated groups in both cohorts. However, RM treated with anti-IL-15 during primary infection manifested accelerated reactivation of RM rhadinovirus. Thus, IL-15 support of NK cell and TEM homeostasis does not play a demonstrable, nonredundant role in SIV replication or CD4+ T cell deletion dynamics but may contribute to immune control of oncogenic γ-herpesviruses.

Roles of Non-coding RNAs During Herpesvirus Infection.

Non-coding RNAs (ncRNAs) play essential roles in multiple aspects of the life cycles of herpesviruses and contribute to lifelong persistence of herpesviruses within their respective hosts. In this chapter, we discuss the types of ncRNAs produced by the different herpesvirus families during infection, some of the cellular ncRNAs manipulated by these viruses, and the overall contributions of ncRNAs to the viral life cycle, influence on the host environment, and pathogenesis.

An Epstein-Barr Virus MicroRNA Blocks Interleukin-1 (IL-1) Signaling by Targeting IL-1 Receptor 1.

Epstein-Barr virus (EBV) encodes >44 viral microRNAs (miRNAs) that are differentially expressed throughout infection, can be detected in Epstein-Barr virus (EBV)-positive tumors, and manipulate several biological processes, including cell proliferation, apoptosis, and immune responses. Here, we show that EBV BHRF1-2 miRNAs block NF-κB activation following treatment with proinflammatory cytokines, specifically interleukin-1ß (IL-1ß). Analysis of EBV PAR-CLIP miRNA targetome data sets combined with pathway analysis revealed multiple BHRF1-2 miRNA targets involved in interleukin signaling pathways. By further analyzing changes in cellular gene expression patterns, we identified the IL-1 receptor 1 (IL1R1) as a direct target of miR-BHRF1-2-5p. Targeting the IL1R1 3' untranslated region (UTR) by EBV miR-BHRF1-2-5p was confirmed using 3'-UTR luciferase reporter assays and Western blot assays. Manipulation of EBV BHRF1-2 miRNA activity in latently infected B cells altered steady-state cytokine levels and disrupted IL-1ß responsiveness. These studies demonstrate functionally relevant BHRF1-2 miRNA interactions during EBV infection, which is an important step in understanding their roles in pathogenesis.IMPORTANCE IL-1 signaling plays an important role in inflammation and early activation of host innate immune responses following virus infection. Here, we demonstrate that a viral miRNA downregulates the IL-1 receptor 1 during EBV infection, which consequently alters the responsiveness of cells to IL-1 stimuli and changes the cytokine expression levels within infected cell populations. We postulate that this viral miRNA activity not only disrupts IL-1 autocrine and paracrine signaling loops that can alert effector cells to sites of infection but also provides a survival advantage by dampening excessive inflammation that may be detrimental to the infected cell.

MicroRNA-17~92 plays a causative role in lymphomagenesis by coordinating multiple oncogenic pathways.

MicroRNAs (miRNAs) have been broadly implicated in cancer, but their exact function and mechanism in carcinogenesis remain poorly understood. Elevated miR-17~92 expression is frequently found in human cancers, mainly due to gene amplification and Myc-mediated transcriptional upregulation. Here we show that B cell-specific miR-17~92 transgenic mice developed lymphomas with high penetrance and that, conversely, Myc-driven lymphomagenesis stringently requires two intact alleles of miR-17~92. We experimentally identified miR-17~92 target genes by PAR-CLIP and validated select target genes in miR-17~92 transgenic mice. These analyses demonstrate that miR-17~92 drives lymphomagenesis by suppressing the expression of multiple negative regulators of the PI3K and NFκB pathways and by inhibiting the mitochondrial apoptosis pathway. Accordingly, miR-17~92-driven lymphoma cells exhibited constitutive activation of the PI3K and NFκB pathways and chemical inhibition of either pathway reduced tumour size and prolonged the survival of lymphoma-bearing mice. These findings establish miR-17~92 as a powerful cancer driver that coordinates the activation of multiple oncogenic pathways, and demonstrate for the first time that chemical inhibition of miRNA downstream pathways has therapeutic value in treating cancers caused by miRNA dysregulation.

Japanese Macaque Rhadinovirus Encodes a Viral MicroRNA Mimic of the miR-17 Family.

UNLABELLED: Japanese macaque (JM) rhadinovirus (JMRV) is a novel, gamma-2 herpesvirus that was recently isolated from JM with inflammatory demyelinating encephalomyelitis (JME). JME is a spontaneous and chronic disease with clinical characteristics and immunohistopathology comparable to those of multiple sclerosis in humans. Little is known about the molecular biology of JMRV. Here, we sought to identify and characterize the small RNAs expressed during lytic JMRV infection using deep sequencing. Fifteen novel viral microRNAs (miRNAs) were identified in JMRV-infected fibroblasts, all of which were readily detectable by 24 h postinfection and accumulated to high levels by 72 h. Sequence comparisons to human Kaposi's sarcoma-associated herpesvirus (KSHV) miRNAs revealed several viral miRNA homologs. To functionally characterize JMRV miRNAs, we screened for their effects on nuclear factor kappa B (NF-κB) signaling in the presence of two proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß). Multiple JMRV miRNAs suppressed cytokine-induced NF-κB activation. One of these miRNAs, miR-J8, has seed sequence homology to members of the cellular miR-17/20/106 and miR-373 families, which are key players in cell cycle regulation as well as inflammation. Using reporters, we show that miR-J8 can target 3' untranslated regions (UTRs) with miR-17-5p or miR-20a cognate sites. Our studies implicate JMRV miRNAs in the suppression of innate antiviral immune responses, which is an emerging feature of many viral miRNAs. IMPORTANCE: Gammaherpesviruses are associated with multiple diseases linked to immunosuppression and inflammation, including AIDS-related cancers and autoimmune diseases. JMRV is a recently identified herpesvirus that has been linked to JME, an inflammatory demyelinating disease in Japanese macaques that mimics multiple sclerosis. There are few large-animal models for gammaherpesvirus-associated pathogenesis. Here, we provide the first experimental evidence of JMRV miRNAs in vitro and demonstrate that one of these viral miRNAs can mimic the activity of the cellular miR-17/20/106 family. Our work provides unique insight into the roles of viral miRNAs during rhadinovirus infection and provides an important step toward understanding viral miRNA function in a nonhuman primate model system.

EBV BART MicroRNAs Target Multiple Pro-apoptotic Cellular Genes to Promote Epithelial Cell Survival.

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that can give rise to cancers of both B-cell and epithelial cell origin. In EBV-induced cancers of epithelial origin, including nasopharyngeal carcinomas (NPCs) and gastric carcinomas, the latent EBV genome expresses very high levels of a cluster of 22 viral pre-miRNAs, called the miR-BARTs, and these have previously been shown to confer a degree of resistance to pro-apoptotic drugs. Here, we present an analysis of the ability of individual miR-BART pre-miRNAs to confer an anti-apoptotic phenotype and report that five of the 22 miR-BARTs demonstrate this ability. We next used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to globally identify the mRNA targets bound by these miR-BARTs in latently infected epithelial cells. This led to the identification of ten mRNAs encoding pro-apoptotic mRNA targets, all of which could be confirmed as valid targets for the five anti-apoptotic miR-BARTs by indicator assays and by demonstrating that ectopic expression of physiological levels of the relevant miR-BART in the epithelial cell line AGS resulted in a significant repression of the target mRNA as well as the encoded protein product. Using RNA interference, we further demonstrated that knockdown of at least seven of these cellular miR-BART target transcripts phenocopies the anti-apoptotic activity seen upon expression of the relevant EBV miR-BART miRNA. Together, these observations validate previously published reports arguing that the miR-BARTs can exert an anti-apoptotic effect in EBV-infected epithelial cells and provide a mechanistic explanation for this activity. Moreover, these results identify and validate a substantial number of novel mRNA targets for the anti-apoptotic miR-BARTs.

MicroRNA target site identification by integrating sequence and binding information.

High-throughput sequencing has opened numerous possibilities for the identification of regulatory RNA-binding events. Cross-linking and immunoprecipitation of Argonaute proteins can pinpoint a microRNA (miRNA) target site within tens of bases but leaves the identity of the miRNA unresolved. A flexible computational framework, microMUMMIE, integrates sequence with cross-linking features and reliably identifies the miRNA family involved in each binding event. It considerably outperforms sequence-only approaches and quantifies the prevalence of noncanonical binding modes.

EBV Noncoding RNAs.

EBV expresses a number of viral noncoding RNAs (ncRNAs) during latent infection, many of which have known regulatory functions and can post-transcriptionally regulate viral and/or cellular gene expression. With recent advances in RNA sequencing technologies, the list of identified EBV ncRNAs continues to grow. EBV-encoded RNAs (EBERs) , the BamHI-A rightward transcripts (BARTs) , a small nucleolar RNA (snoRNA) , and viral microRNAs (miRNAs) are all expressed during EBV infection in a variety of cell types and tumors. Recently, additional novel EBV ncRNAs have been identified. Viral miRNAs, in particular, have been under extensive investigation since their initial identification over ten years ago. High-throughput studies to capture miRNA targets have revealed a number of miRNA-regulated viral and cellular transcripts that tie into important biological networks. Functions for many EBV ncRNAs are still unknown; however, roles for many EBV miRNAs in latency and in tumorigenesis have begun to emerge. Ongoing mechanistic studies to elucidate the functions of EBV ncRNAs should unravel additional roles for ncRNAs in the viral life cycle. In this chapter, we will discuss our current knowledge of the types of ncRNAs expressed by EBV, their potential roles in viral latency, and their potential involvement in viral pathogenesis.

Differential RISC association of endogenous human microRNAs predicts their inhibitory potential.

It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression. While miRNAs of closely similar sequence showed comparable levels of RISC association in the same cell line, these varied between different cell types. Moreover, the level of RISC association could be modulated by overexpression of complementary target mRNAs. Together, these data indicate that the level of RISC association of a given endogenous miRNA is regulated by the available RNA targetome and predicts miRNA function.

Evolutionary conservation of primate lymphocryptovirus microRNA targets.

Epstein-Barr virus (EBV) and rhesus lymphocryptovirus (rLCV) are closely related gammaherpesviruses in the lymphocryptovirus subgroup that express viral microRNAs (miRNAs) during latent infection. In addition to many host mRNAs, EBV miRNAs are known to target latent viral transcripts, specifically those encoding LMP1, BHRF1, and EBNA2. The mRNA targets of rLCV miRNAs have not been investigated. Using luciferase reporter assays, photoactivatable cross-linking and immunoprecipitation (PAR-CLIP), and deep sequencing, we demonstrate that posttranscriptional regulation of LMP1 expression is a conserved function of lymphocryptovirus miRNAs. Furthermore, the mRNAs encoding the rLCV EBNA2 and BHRF1 homologs are regulated by miRNAs in rLCV-infected B cells. Homologous to sites in the EBV LMP1 and BHRF1 3'-untranslated regions (UTRs), we also identified evolutionarily conserved binding sites for the cellular miR-17/20/106 family in the LMP1 and BHRF1 3'UTRs of several primate LCVs. Finally, we investigated the functional consequences of LMP1 targeting by individual EBV BART miRNAs and show that select viral miRNAs play a role in the previously observed modulation of NF-κB activation.

Replication of many human viruses is refractory to inhibition by endogenous cellular microRNAs.

The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. Importance: Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of pathogenic viruses is not enhanced in human cells engineered to be unable to produce miRNAs, indicating that viruses have evolved to be resistant to inhibition by miRNAs. This result is important, as it implies that manipulation of miRNA levels is not likely to prove useful in inhibiting virus replication. It also focuses attention on the question of how viruses have evolved to resist inhibition by miRNAs and whether virus mutants that have lost this resistance might prove useful, for example, in the development of attenuated virus vaccines.

Viruses, microRNAs, and host interactions.

One of the most significant recent advances in biomedical research has been the discovery of the approximately 22-nt-long class of noncoding RNAs designated microRNAs (miRNAs). These regulatory RNAs provide a unique level of posttranscriptional gene regulation that modulates a range of fundamental cellular processes. Several viruses, especially herpesviruses, also encode miRNAs, and over 200 viral miRNAs have now been identified. Current evidence indicates that viruses use these miRNAs to manipulate both cellular and viral gene expression. Furthermore, viral infection can exert a profound impact on the cellular miRNA expression profile, and several RNA viruses have been reported to interact directly with cellular miRNAs and/or to use these miRNAs to augment their replication potential. Here we discuss our current knowledge of viral miRNAs and virally influenced cellular miRNAs and their relationship to viral infection.

The viral and cellular microRNA targetome in lymphoblastoid cell lines.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis and/or lymphomagenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3'UTRs. 531 of these sites contained seed matches to viral miRNAs. 24 PAR-CLIP-identified miRNA:3'UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies.

MicroRNA-mediated control of Epstein-Barr virus infection and potential diagnostic and therapeutic implications.

Herpesviruses, such as Epstein-Barr virus (EBV), encode multiple viral microRNAs that are expressed throughout various infection stages. While much progress has been made in evaluating both the viral and host microRNAs (miRNAs) that are detected during infection as well as elucidating their molecular targets in vitro, our understanding of their contributions to pathogenesis in vivo, viral oncogenesis, and clinical implications for these small molecules remains limited. miRNAs are widely recognized as key regulators of global cellular processes, including apoptosis, cell differentiation, and development of immune responses. This review discusses the roles of miRNAs in EBV infection and current advances in miRNA-based diagnostic and therapeutic strategies potentially applicable toward EBV-associated diseases.

Effects of Early Antiretroviral Therapy on the Composition and Diversity of the Fecal Microbiome of SIV-infected Rhesus Macaques (Macaca mulatta).

HIV-infected people develop reproducible disruptions in their gastrointestinal microbiota. Despite the suppression of HIV viremia via long-term antiretroviral therapy (ART), alterations still occur in gut microbial diversity and the commensal microbiota. Mounting evidence suggests these microbial changes lead to the development of gut dysbiosis-persistent inflammation that damages the gut mucosa-and correlate with various immune defects. In this study, we examined how early ART intervention influences microbial diversity in SIV-infected rhesus macaques. Using 16S rRNA sequencing, we defined the fecal microbiome in macaques given daily ART beginning on either 3 or 7 d after SIV infection (dpi) and characterized changes in composition, α diversity, and ß diversity from before infection through 112 dpi. The dominant phyla in the fecal samples before infection were Bacteroidetes, Firmicutes, Spirochaetes, and Proteobacteria. After SIV infection and ART, the relative abundance of Firmicutes and Bacteroidetes did not change significantly. Significant reductions in α diversity occurred across time when ART was initiated at 3 dpi but not at 7 dpi. Principal coordinate analysis of samples revealed a divergence in ß diversity in both treatment groups after SIV infection, with significant differences depending on the timing of ART administration. These results indicate that although administration of ART at 3 or 7 dpi did not substantially alter fecal microbial composition, the timing of early ART measurably altered phylogenetic diversity.

Rapamycin limits CD4+ T cell proliferation in simian immunodeficiency virus-infected rhesus macaques on antiretroviral therapy.

Proliferation of latently infected CD4+ T cells with replication-competent proviruses is an important mechanism contributing to HIV persistence during antiretroviral therapy (ART). One approach to targeting this latent cell expansion is to inhibit mTOR, a regulatory kinase involved with cell growth, metabolism, and proliferation. Here, we determined the effects of chronic mTOR inhibition with rapamycin with or without T cell activation in SIV-infected rhesus macaques (RMs) on ART. Rapamycin perturbed the expression of multiple genes and signaling pathways important for cellular proliferation and substantially decreased the frequency of proliferating CD4+ memory T cells (TM cells) in blood and tissues. However, levels of cell-associated SIV DNA and SIV RNA were not markedly different between rapamycin-treated RMs and controls during ART. T cell activation with an anti-CD3LALA antibody induced increases in SIV RNA in plasma of RMs on rapamycin, consistent with SIV production. However, upon ART cessation, both rapamycin and CD3LALA-treated and control-treated RMs rebounded in less than 12 days, with no difference in the time to viral rebound or post-ART viral load set points. These results indicate that, while rapamycin can decrease the proliferation of CD4+ TM cells, chronic mTOR inhibition alone or in combination with T cell activation was not sufficient to disrupt the stability of the SIV reservoir.