Showing papers by "Bryan R. Cullen published in 2010"

Viruses, microRNAs, and host interactions.

Abstract: One of the most significant recent advances in biomedical research has been the discovery of the ∼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.

Virally Induced Cellular MicroRNA miR-155 Plays a Key Role in B-Cell Immortalization by Epstein-Barr Virus

Abstract: Infection of resting primary human B cells by Epstein-Barr virus (EBV) results in their transformation into indefinitely proliferating lymphoblastoid cell lines (LCLs). LCL formation serves as a model for lymphomagenesis, and LCLs are phenotypically similar to EBV-positive diffuse large B-cell lymphomas (DLBCLs), which represent a common AIDS-associated malignancy. B-cell infection by EBV induces the expression of several cellular microRNAs (miRNAs), most notably miR-155, which is overexpressed in many tumors and can induce B-cell lymphomas when overexpressed in animals. Here, we demonstrate that miR-155 is the most highly expressed miRNA in LCLs and that the selective inhibition of miR-155 function specifically inhibits the growth of both LCLs and the DLBCL cell line IBL-1. Cells lacking miR-155 are inefficient in progressing through S phase and spontaneously undergo apoptosis. In contrast, three other B-cell lymphoma lines, including two EBV-positive Burkitt's lymphoma cell lines, grew normally in the absence of miR-155 function. These data identify the induction of cellular miR-155 expression by EBV as critical for the growth of both laboratory-generated LCLs and naturally occurring DLBCLs and suggest that targeted inhibition of miR-155 function could represent a novel approach to the treatment of DLBCL in vivo.

Identification of microRNAs expressed in two mosquito vectors, Aedes albopictus and Culex quinquefasciatus

Abstract: MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression in a variety of organisms, including insects, vertebrates, and plants. miRNAs play important roles in cell development and differentiation as well as in the cellular response to stress and infection. To date, there are limited reports of miRNA identification in mosquitoes, insects that act as essential vectors for the transmission of many human pathogens, including flaviviruses. West Nile virus (WNV) and dengue virus, members of the Flaviviridae family, are primarily transmitted by Aedes and Culex mosquitoes. Using high-throughput deep sequencing, we examined the miRNA repertoire in Ae. albopictus cells and Cx. quinquefasciatus mosquitoes. We identified a total of 65 miRNAs in the Ae. albopictus C7/10 cell line and 77 miRNAs in Cx. quinquefasciatus mosquitoes, the majority of which are conserved in other insects such as Drosophila melanogaster and Anopheles gambiae. The most highly expressed miRNA in both mosquito species was miR-184, a miRNA conserved from insects to vertebrates. Several previously reported Anopheles miRNAs, including miR-1890 and miR-1891, were also found in Culex and Aedes, and appear to be restricted to mosquitoes. We identified seven novel miRNAs, arising from nine different precursors, in C7/10 cells and Cx. quinquefasciatus mosquitoes, two of which have predicted orthologs in An. gambiae. Several of these novel miRNAs reside within a ~350 nt long cluster present in both Aedes and Culex. miRNA expression was confirmed by primer extension analysis. To determine whether flavivirus infection affects miRNA expression, we infected female Culex mosquitoes with WNV. Two miRNAs, miR-92 and miR-989, showed significant changes in expression levels following WNV infection. Aedes and Culex mosquitoes are important flavivirus vectors. Recent advances in both mosquito genomics and high-throughput sequencing technologies enabled us to interrogate the miRNA profile in these two species. Here, we provide evidence for over 60 conserved and seven novel mosquito miRNAs, expanding upon our current understanding of insect miRNAs. Undoubtedly, some of the miRNAs identified will have roles not only in mosquito development, but also in mediating viral infection in the mosquito host.

A Human Herpesvirus MicroRNA Inhibits p21 Expression and Attenuates p21-Mediated Cell Cycle Arrest

Abstract: The oncogenic human gammaherpesvirus Kaposi9s sarcoma-associated herpesvirus (KSHV) expresses 12 viral microRNAs (miRNAs) in latently infected cells. Here, we report that cellular mRNAs encoding the cellular cyclin-dependent kinase inhibitor p21, a key inducer of cell cycle arrest, are direct targets for KSHV miR-K1. Ectopically expressed KSHV miR-K1 specifically inhibited the expression of endogenous p21 in KSHV-negative cells and strongly attenuated the cell cycle arrest that normally occurs upon p53 activation, yet miR-K1 did not prevent the induction of other p53-responsive genes. Stable knockdown of miR-K1 in latently KSHV-infected human primary effusion lymphoma (PEL) B cells revealed a derepression of p21 expression and enhanced cell cycle arrest following activation of p53. Our data demonstrate that miR-K1 represses the expression of p21, a protein with known tumor suppressor functions, and suggest that this KSHV miRNA is likely to contribute to the oncogenic potential of this opportunistic viral pathogen.

In-Depth Analysis of Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Expression Provides Insights into the Mammalian MicroRNA-Processing Machinery

Abstract: We have used deep sequencing to analyze the pattern of viral microRNA (miRNA) expression observed in the B-cell line BC-3, which is latently infected with Kaposi's sarcoma-associated herpesvirus (KSHV). We recovered 14.6 × 106 total miRNA cDNA reads, of which a remarkable 92% were of KSHV origin. We detected 11 KSHV miRNAs as well as all 11 predicted miRNA* or passenger strands from the miRNA duplex intermediate. One previously reported KSHV miRNA, miR-K9, was found to be mutationally inactivated. This analysis revealed that the 5′ ends of 10 of the 11 KSHV miRNAs were essentially invariant, with significantly more variation being observed at the 3′ end, a result which is consistent with the proposal that the 5′-proximal region of miRNAs is critical for target mRNA recognition. However, one KSHV miRNA, miR-K10-3p, was detected in two isoforms differing by 1 nucleotide (nt) at the 5′ end that were present at comparable levels, and these two related KSHV miRNAs are therefore likely to target at least partially distinct mRNA populations. Finally, we also report the first detection of miRNA offset RNAs (moRs) in vertebrate somatic cells. moRs, which derive from primary miRNA (pri-miRNA) sequences that immediately flank the mature miRNA and miRNA* strands, were identified flanking one or both sides of nine of the KSHV miRNAs. These data provide new insights into the pattern of miRNA processing in mammalian cells and indicate that this process is highly conserved during animal evolution.

Five Questions about Viruses and MicroRNAs

Abstract: MicroRNAs (miRNAs) are ∼22-nt regulatory RNAs expressed by all multicellular eukaryotes [1]. Humans encode >700 miRNAs and similar numbers are likely to exist in other mammalian species. Almost all cellular miRNAs are initially transcribed by RNA polymerase II (Pol II) as part of a long, capped, polyadenylated primary miRNA (pri-miRNA) precursor. The miRNA forms part of one arm of an RNA stem-loop that consists of an ∼32-bp imperfect stem flanked by unstructured RNA sequences. This stem-loop is recognized by the nuclear RNase III enzyme Drosha, which cleaves the stem to liberate an ∼60-nt pre-miRNA hairpin. The pre-miRNA is then transported to the cytoplasm where it is cleaved by a second RNase III enzyme, called Dicer, which removes the terminal loop to generate the miRNA duplex intermediate. One strand of this duplex is incorporated into the RNA-induced silencing complex (RISC), where it acts as a guide RNA to direct RISC to complementary mRNA species [1]. Depending on the level of complementarity, RISC can either cleave bound mRNAs and/or inhibit their translation. Inhibition of mRNA translation generally requires full complementarity of the mRNA to nucleotides 2 through 7 or 8 from the miRNA 5′ end—the miRNA seed region. The primary, and possibly sole, function of mammalian miRNAs is therefore to act as specific post-transcriptional inhibitors of mRNA function.

Influenza A Virus Expresses High Levels of an Unusual Class of Small Viral Leader RNAs in Infected Cells

Abstract: Evidence has recently accumulated suggesting that small noncoding RNAs, and particularly microRNAs, have the po- tential to strongly affect the replication and pathogenic potential of a range of human virus species. Here, we report the use of deep sequencing to comprehensively analyze small viral RNAs (18 to 27 nucleotides (nt)) produced during infection by influenza A virus. Although influenza A virus differs from most other RNA viruses in that it replicates its genome in the nucleus and is therefore exposed to the nuclear microRNA processing factors Drosha and DGCR8, we did not observe any microRNAs encoded by influenza virus genes. However, influenza virus infection did induce the expression of very high levels—over 100,000 copies per cell by 8 h postinfection—of a population of 18- to 27-nt small viral leader RNAs (leRNAs) that originated from the precise 5=ends of all eight influenza virus genomic RNA (vRNA) segments. Like the vRNAs themselves, our data indicate that the leRNAs also bear a 5=-terminal triphosphate and are therefore not capable of functioning as microRNAs. Instead, the high-level produc- tion of leRNAs may imply a role in another aspect of the viral life cycle, such as regulation of the switch from viral mRNA tran- scription to genomic RNA synthesis. IMPORTANCE Influenza A virus is an important human pathogen that has the potential to give rise to serious pandemics. Here, we demonstrate that influenza A virus induces the expression of very high levels of small viral leader RNAs (leRNAs) within hours of infection. These RNAs are unusual in that they bear a 5=triphosphate and originate from the very 5=ends of the eight viral genomic RNA (vRNA) segments. Their high expression may imply an important role in the viral life cycle that could potentially serve as a novel target for antiviral drugs.

Identification of Viral MicroRNAs Expressed in Human Sacral Ganglia Latently Infected with Herpes Simplex Virus 2

Abstract: Deep sequencing of small RNAs isolated from human sacral ganglia latently infected with herpes simplex virus 2 (HSV-2) was used to identify HSV-2 microRNAs (miRNAs) expressed during latent infection. This effort resulted in the identification of five distinct HSV-2 miRNA species, two of which, miR-H3/miR-I and miR-H4/miR-II, have been previously reported. Three novel HSV-2 miRNAs were also identified, and two of these, miR-H7 and miR-H9, are derived from the latency-associated transcript (LAT) and are located antisense to the viral transcript encoding transactivator ICP0. A third novel HSV-2 miRNA, miR-H10, is encoded within the unique long (U(L)) region of the genome, 3' to the U(L)15 open reading frame, and is presumably excised from a novel, latent HSV-2 transcript distinct from LAT.

MicroRNA antagonism of the picornaviral life cycle: alternative mechanisms of interference.

Abstract: In addition to modulating the function and stability of cellular mRNAs, microRNAs can profoundly affect the life cycles of viruses bearing sequence complementary targets, a finding recently exploited to ameliorate toxicities of vaccines and oncolytic viruses. To elucidate the mechanisms underlying microRNA-mediated antiviral activity, we modified the 3′ untranslated region (3′UTR) of Coxsackievirus A21 to incorporate targets with varying degrees of homology to endogenous microRNAs. We show that microRNAs can interrupt the picornavirus life-cycle at multiple levels, including catalytic degradation of the viral RNA genome, suppression of cap-independent mRNA translation, and interference with genome encapsidation. In addition, we have examined the extent to which endogenous microRNAs can suppress viral replication in vivo and how viruses can overcome this inhibition by microRNA saturation in mouse cancer models.

Epstein-Barr virus (EBV)-associated miRNAs are important for the maintenance of EBV transformed B cells

Abstract: The human oncogenic gamma-herpesvirus, Epstein-Barr virus (EBV), infects approximately 95% of the adult human population and causes B-cell lymphomas in immune-compromised individuals, such as AIDS patients. EBV transforms primary human B cells into indefinitely proliferating lymphoblastoid cell lines (LCLs) in vitro, which represents an important model system for the transformation of B cells by EBV. EBV encodes 25 viral miRNA, but their role in virus replication and pathogenesis is unclear. Three of these miRNAs, miR-BHRF1-1, miR-BHRF1-2, and miR-BHRF1-3, are expressed during the latency III stage of EBV, the EBV gene expression pattern observed in AIDS-associated B cell lymphomas and LCLs. We have performed functional studies examining the roles of the three BHRF1 miRNAs during the transformation of primary B cells by EBV in vitro as well as in the maintenance of the transformed state in established LCL cultures. Our preliminary data suggest that at least one EBV miRNA is necessary for LCL outgrowth. EBV is also known to up-regulate a number of cellular microRNAs, including miR-155, and our preliminary data also suggest a role of miR-155 in the maintenance of LCLs. We are currently examining the mechanisms by which EBV and cellular microRNAs contribute to the growth and maintenance of LCLs. Together, our data point to a role for EBV-encoded and EBV-induced miRNAs in EBV-induced cell transformation.