Zhiqiang Bai

Regulation of NF-κB inhibitor IκBα and viral replication by a KSHV microRNA

Kaposis sarcoma-associated herpesvirus (KSHV) is causally linked to several acquired immune deficiency syndrome-related malignancies, including Kaposis sarcoma, primary effusion lymphoma (PEL) and a subset of multicentric Castlemans disease. Control of viral lytic replication is essential for KSHV latency, evasion of the host immune system and induction of tumours. Here, we show that deletion of a 14 microRNA (miRNA) cluster from the KSHV genome significantly enhances viral lytic replication as a result of reduced NF-κB activity. The miRNA cluster regulates the NF-κB pathway by reducing expression of IκBα protein, an inhibitor of NF-κB complexes. Computational and miRNA seed mutagenesis analyses were used to identify KSHV miR-K1, which directly regulates the IκBα protein level by targeting the 3′UTR of its transcript. Expression of miR-K1 is sufficient to rescue NF-κB activity and inhibit viral lytic replication, whereas inhibition of miR-K1 in KSHV-infected PEL cells has the opposite effect. Thus, KSHV encodes an miRNA to control viral replication by activating the NF-κB pathway. These results demonstrate an important role for KSHV miRNAs in regulating viral latency and lytic replication by manipulating the host survival pathway.

KSHV MicroRNAs Mediate Cellular Transformation and Tumorigenesis by Redundantly Targeting Cell Growth and Survival Pathways

Kaposis sarcoma-associated herpesvirus (KSHV) is causally linked to several human cancers, including Kaposis sarcoma, primary effusion lymphoma and multicentric Castlemans disease, malignancies commonly found in HIV-infected patients. While KSHV encodes diverse functional products, its mechanism of oncogenesis remains unknown. In this study, we determined the roles KSHV microRNAs (miRs) in cellular transformation and tumorigenesis using a recently developed KSHV-induced cellular transformation system of primary rat mesenchymal precursor cells. A mutant with a cluster of 10 precursor miRs (pre-miRs) deleted failed to transform primary cells, and instead, caused cell cycle arrest and apoptosis. Remarkably, the oncogenicity of the mutant virus was fully restored by genetic complementation with the miR cluster or several individual pre-miRs, which rescued cell cycle progression and inhibited apoptosis in part by redundantly targeting IκBα and the NF-κB pathway. Genomic analysis identified common targets of KSHV miRs in diverse pathways with several cancer-related pathways preferentially targeted. These works define for the first time an essential viral determinant for KSHV-induced oncogenesis and identify NF-κB as a critical pathway targeted by the viral miRs. Our results illustrate a common theme of shared functions with hierarchical order among the KSHV miRs.

A Kaposi's Sarcoma-Associated Herpesvirus MicroRNA and Its Variants Target the Transforming Growth Factor β Pathway To Promote Cell Survival

ABSTRACT Transforming growth factor β (TGF-β) signaling regulates cell growth and survival. Dysregulation of the TGF-β pathway is common in viral infection and cancer. Latent infection by Kaposis sarcoma-associated herpesvirus (KSHV) is required for the development of several AIDS-related malignancies, including Kaposis sarcoma and primary effusion lymphoma (PEL). KSHV encodes more than two dozen microRNAs (miRs) derived from 12 pre-miRs with largely unknown functions. In this study, we show that miR variants processed from pre-miR-K10 are expressed in KSHV-infected PEL cells and endothelial cells, while cellular miR-142-3p and its variant miR-142-3p_-1_5, which share the same seed sequence with miR-K10a_ +1_5, are expressed only in PEL cells and not in uninfected and KSHV-infected TIME cells. KSHV miR-K10 variants inhibit TGF-β signaling by targeting TGF-β type II receptor (TβRII). Computational and reporter mutagenesis analyses identified three functional target sites in the TβRII 3′ untranslated region (3′UTR). Expression of miR-K10 variants is sufficient to inhibit TGF-β-induced cell apoptosis. A suppressor of the miRs sensitizes latent KSHV-infected PEL cells to TGF-β and induces apoptosis. These results indicate that miR-K10 variants manipulate the TGF-β pathway to confer cells with resistance to the growth-inhibitory effect of TGF-β. Thus, KSHV miRs might target the tumor-suppressive TGF-β pathway to promote viral latency and contribute to malignant cellular transformation.

HIV-1 Tat Promotes Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) vIL-6-Induced Angiogenesis and Tumorigenesis by Regulating PI3K/PTEN/AKT/GSK-3β Signaling Pathway

Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is etiologically associated with KS, the most common AIDS-related malignancy. KS is characterized by vast angiogenesis and hyperproliferative spindle cells. We have previously reported that HIV-1 Tat can trigger KSHV reactivation and accelerate Kaposin A-induced tumorigenesis. Here, we explored Tat promotion of KSHV vIL-6-induced angiogenesis and tumorigenesis. Tat promotes vIL-6-induced cell proliferation, cellular transformation, vascular tube formation and VEGF production in culture. Tat enhances vIL-6-induced angiogenesis and tumorigenesis of fibroblasts and human endothelial cells in a chicken chorioallantoic membrane (CAM) model. In an allograft model, Tat promotes vIL-6-induced tumorigenesis and expression of CD31, CD34, SMA, VEGF, b-FGF, and cyclin D1. Mechanistic studies indicated Tat activates PI3K and AKT, and inactivates PTEN and GSK-3β in vIL-6 expressing cells. LY294002, a specific inhibitor of PI3K, effectively impaired Tat’s promotion of vIL-6-induced tumorigenesis. Together, these results provide the first evidence that Tat might contribute to KS pathogenesis by synergizing with vIL-6, and identify PI3K/AKT pathway as a potential therapeutic target in AIDS-related KS patients.

A KSHV microRNA Directly Targets G Protein-Coupled Receptor Kinase 2 to Promote the Migration and Invasion of Endothelial Cells by Inducing CXCR2 and Activating AKT Signaling

Kaposis sarcoma (KS) is a highly disseminated angiogenic tumor of endothelial cells linked to infection by Kaposis sarcoma-associated herpesvirus (KSHV). KSHV encodes more than two dozens of miRNAs but their roles in KSHV-induced tumor dissemination and metastasis remain unknown. Here, we found that ectopic expression of miR-K12-3 (miR-K3) promoted endothelial cell migration and invasion. Bioinformatics and luciferase reporter analyses showed that miR-K3 directly targeted G protein-coupled receptor (GPCR) kinase 2 (GRK2, official gene symbol ADRBK1). Importantly, overexpression of GRK2 reversed miR-K3 induction of cell migration and invasion. Furthermore, the chemokine receptor CXCR2, which was negatively regulated by GRK2, was upregulated in miR-K3-transduced endothelial cells. Knock down of CXCR2 abolished miR-K3-induced cell migration and invasion. Moreover, miR-K3 downregulation of GRK2 relieved its direct inhibitory effect on AKT. Both CXCR2 induction and the release of AKT from GRK2 were required for miR-K3 maximum activation of AKT and induction of cell migration and invasion. Finally, deletion of miR-K3 from the KSHV genome abrogated its effect on the GRK2/CXCR2/AKT pathway and KSHV-induced migration and invasion. Our data provide the first-line evidence that, by repressing GRK2, miR-K3 facilitates cell migration and invasion via activation of CXCR2/AKT signaling, which likely contribute to the dissemination of KSHV-induced tumors.

Regulation of herpesvirus lifecycle by viral microRNAs.

Human herpes viruses have latency and lytic replication phases in their lifecycle. Proper regulation of herpes viral lifecycle is essential for the evasion of host immune surveillance and development of their related diseases. Recent advancements indicate a role of a novel class of viral non-coding RNAs, microRNAs (miRNAs), in the fine-tuning of herpes viral lifecycle. So far, herpes viral miRNAs have been shown to promote viral latency by inhibiting viral lytic replication either through direct targeting of key viral replication genes or through manipulation of host pathways that regulate viral lifecycle. The oncogenic Kaposis sarcoma-associated herpes virus (KSHV) has adapted both strategies to control viral latency. Our recent study has identified a KSHV miRNA that inhibits viral lytic replication by upregulating the NF-κB pathway.

Genomewide mapping and screening of Kaposi's sarcoma-associated herpesvirus (KSHV) 3' untranslated regions identify bicistronic and polycistronic viral transcripts as frequent targets of KSHV microRNAs.

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) encodes over 90 genes and 25 microRNAs (miRNAs). The KSHV life cycle is tightly regulated to ensure persistent infection in the host. In particular, miRNAs, which primarily exert their effects by binding to the 3′ untranslated regions (3′UTRs) of target transcripts, have recently emerged as key regulators of KSHV life cycle. Although studies with RNA cross-linking immunoprecipitation approach have identified numerous targets of KSHV miRNAs, few of these targets are of viral origin because most KSHV 3′UTRs have not been characterized. Thus, the extents of viral genes targeted by KSHV miRNAs remain elusive. Here, we report the mapping of the 3′UTRs of 74 KSHV genes and the effects of KSHV miRNAs on the control of these 3′UTR-mediated gene expressions. This analysis reveals new bicistronic and polycistronic transcripts of KSHV genes. Due to the 5′-distal open reading frames (ORFs), KSHV bicistronic or polycistronic transcripts have significantly longer 3′UTRs than do KSHV monocistronic transcripts. Furthermore, screening of the 3′UTR reporters has identified 28 potential new targets of KSHV miRNAs, of which 11 (39%) are bicistronic or polycistronic transcripts. Reporter mutagenesis demonstrates that miR-K3 specifically targets ORF31-33 transcripts at the lytic locus via two binding sites in the ORF33 coding region, whereas miR-K10a-3p and miR-K10b-3p and their variants target ORF71-73 transcripts at the latent locus through distinct binding sites in both 5′-distal ORFs and intergenic regions. Our results indicate that KSHV miRNAs frequently target the 5′-distal coding regions of bicistronic or polycistronic transcripts and highlight the unique features of KSHV miRNAs in regulating gene expression and life cycle.

Cellular MicroRNAs 498 and 320d Regulate Herpes Simplex Virus 1 Induction of Kaposi’s Sarcoma-Associated Herpesvirus Lytic Replication by Targeting RTA

Kaposi’s sarcoma-associated herpesvirus (KSHV) infection was necessary but not sufficient for KS development without other cofactors. We have previously reported that herpes simplex virus (HSV)-1 was an important cofactor that reactivated KSHV from latency by inducing the expression of KSHV replication and transcription activator (RTA), the lytic switch protein. Here, we further investigated the possible cellular microRNAs (miRNAs) involved in regulation of RTA during HSV-1-induced KSHV replication. The differential profiles of miRNAs expression between Mock- and HSV-1-infected body cavity-based lymphoma (BCBL-1) cells were identified by miRNA microarray analysis. Bioinformatics and luciferase reporter analyses showed that two of the HSV-1-downregulated cellular miRNAs, miR-498 and miR-320d, directly targeted the 3′ untranslated region (UTR) of KSHV RTA. As a result, overexpression of these two miRNAs significantly inhibited HSV-1-induced KSHV replication, whereas repression of these miRNAs with specific suppressors enhanced HSV-1-mediated KSHV replication. In addition, miR-498 or miR-320d alone, without HSV-1 infection, regulated KSHV replication in BCBL-1 cells. Finally, bioinformatics Gene Ontology (GO) analysis indicated that targets of HSV-1-regulated miRNAs were enriched for proteins, whose roles were involved in protein binding, enzyme activity, biological regulation, and several potential signaling pathways including transforming growth factor (TGF)-β were likely to participate in HSV-1-induced KSHV replication. Collectively, these novel findings demonstrated that host-encoded miR-498 and miR-320d regulated HSV-1 induction of KSHV lytic replication by targeting RTA, which provided further insights into the molecular mechanisms controlling KSHV lytic replication.

MicroRNAs control herpesviral dormancy

Infections by herpesviruses are widespread in humans, and are the causes for several important diseases. Gammaherpesvirus Kaposis sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposis sarcoma (KS), a highly inflammatory and angiogenic cancer commonly found in AIDS patients.1 KSHV is also associated with primary effusion lymphoma and a subset of multicentric Castleman’s disease, two rare lymphoproliferative malignancies.

Identification and function of MicroRNAs encoded by herpesviruses

MicroRNAs (miRNAs) play important roles in eukaryotes, plants and some viruses. It is increasingly clear that miRNAs-encoded by viruses can affect the viral life cycle and host physiology. Viral miRNAs could repress the innate and adaptive host immunity, modulate cellular signaling pathways, and regulate the expression of cellular and viral genes. These functions facilitate viral acute and persistent infections, and have profound effects on the host cell survival and disease progression. Here, we discuss the miRNAs encoded by herpesviruses, and their regulatory roles involved in virus-host interactions.