Showing papers by "Bryan R. Cullen published in 2006"
TL;DR: The pathogenic lymphocryptovirus Epstein–Barr virus (EBV) is shown to express at least 17 distinct microRNAs (miRNAs) in latently infected cells, seven of which are closely related to EBV miRNAs.
Abstract: The pathogenic lymphocryptovirus Epstein-Barr virus (EBV) is shown to express at least 17 distinct microRNAs (miRNAs) in latently infected cells. These are arranged in two clusters: 14 miRNAs are located in the introns of the viral BART gene while three are located adjacent to BHRF1. The BART miRNAs are expressed at high levels in latently infected epithelial cells and at lower, albeit detectable, levels in B cells. In contrast to the tissue-specific expression pattern of the BART miRNAs, the BHRF1 miRNAs are found at high levels in B cells undergoing stage III latency but are essentially undetectable in B cells or epithelial cells undergoing stage I or II latency. Induction of lytic EBV replication was found to enhance the expression of many, but not all, of these viral miRNAs. Rhesus lymphocryptovirus, which is separated from EBV by > or =13 million years of evolution, expresses at least 16 distinct miRNAs, seven of which are closely related to EBV miRNAs. Thus, lymphocryptovirus miRNAs are under positive selection and are likely to play important roles in the viral life cycle. Moreover, the differential regulation of EBV miRNA expression implies distinct roles during infection of different human tissues.
557 citations
TL;DR: The potential functions of the virally encoded miRNAs recently identified in several pathogenic human viruses are discussed and it is proposed that cellular mi RNAs may have had a substantial effect on viral evolution and may continue to influence the in vivo tissue tropism of viruses.
Abstract: The discovery of RNA interference and cellular microRNAs (miRNAs) has not only affected how biological research is conducted but also revealed an entirely new level of post-transcriptional gene regulation. Here, I discuss the potential functions of the virally encoded miRNAs recently identified in several pathogenic human viruses and propose that cellular miRNAs may have had a substantial effect on viral evolution and may continue to influence the in vivo tissue tropism of viruses. Our increasing knowledge of the role and importance of virally encoded miRNAs will probably offer new insights into how viruses that establish latent infections, such as herpesviruses, avoid elimination by the host innate or adaptive immune system. Research into viral miRNA function might also suggest new approaches for treating some virally induced diseases.
420 citations
TL;DR: The data suggest that the APOBEC3 protein family may have evolved, at least in part, to defend the integrity of the human genome against endogenous retrotransposons.
Abstract: Long interspersed element (LINE) 1 retrotransposons are major genomic parasites that represent ≈17% of the human genome. The LINE-1 ORF2 protein is also responsible for the mobility of Alu elements, which constitute a further ≈11% of genomic DNA. Representative members of each element class remain mobile, and deleterious retrotransposition events can induce spontaneous genetic diseases. Here, we demonstrate that APOBEC3A and APOBEC3B, two members of the APOBEC3 family of human innate antiretroviral resistance factors, can enter the nucleus, where LINE-1 and Alu reverse transcription occurs, and specifically inhibit both LINE-1 and Alu retrotransposition. These data suggest that the APOBEC3 protein family may have evolved, at least in part, to defend the integrity of the human genome against endogenous retrotransposons.
368 citations
TL;DR: The results argue that APOBEC3A inhibits IAP retrotransposition via a novel mechanism that is distinct from, and in this case more effective than, the DNA editing mechanism characteristic of APOBec3G and APOB EC3B.
Abstract: While the ability of APOBEC3G to reduce the replication of a range of exogenous retroviruses is now well established, recent evidence has suggested that APOBEC3G can also inhibit the replication of endogenous retrotransposons that bear long terminal repeats. Here, we extend this earlier work by showing that two other members of the human APOBEC3 protein family, APOBEC3B and APOBEC3A, can reduce retrotransposition by the intracisternal A-particle (IAP) retrotransposon in human cells by 20-fold to up to 100-fold, respectively. This compares to an � 4-fold inhibition in IAP retrotransposition induced by APOBEC3G. While both APOBEC3G and APOBEC3B specifically interact with the IAP Gag protein in co-expressing cells, and induce extensive editing of IAP reverse transcripts, APOBEC3A fails to package detectably into IAP virus-like particles and does not edit IAP reverse transcripts. These data, which identify human APOBEC3A as a highly potent inhibitor of LTR-retrotransposon function, are the first to ascribe a biological activity to APOBEC3A. Moreover, these results argue that APOBEC3A inhibits IAP retrotransposition via a novel mechanism that is distinct from, and in this case more effective than, the DNA editing mechanism characteristic of APOBEC3G and APOBEC3B.
282 citations
TL;DR: Metazoan organisms are subject to invasion by a wide range of microbial pathogens and have, as a result, evolved a range of defensive measures, including antibodies and cytotoxic T cells.
Abstract: Metazoan organisms are subject to invasion by a wide range of microbial pathogens and have, as a result, evolved a range of defensive measures. While scientific attention historically has focused on adaptive immune responses, such as antibodies and cytotoxic T cells, it has of late become
274 citations
TL;DR: The potential pitfalls associated with RNAi are reviewed, and experimental approaches that can be used to maximize the specificity of RNAi or to confirm that the data obtained are indeed valid are suggested.
Abstract: RNA interference (RNAi) provides a powerful technique for the derivation and analysis of loss-of-function phenotypes in vertebrate cells, where alternative approaches are either arduous or frequently ineffective RNAi, however, is not always totally specific, and confirming the specificity, and hence the validity, of data obtained using RNAi therefore forms an essential component of experiments that rely on this technique Here I briefly review the potential pitfalls associated with RNAi, and then suggest experimental approaches that can be used to maximize the specificity of RNAi or to confirm that the data obtained are indeed valid
194 citations
TL;DR: It is concluded that the popular hypothesis in favor of whether RNA interference forms part of the mammalian innate immune response remains far from proven and may not be valid.
Abstract: RNA interference constitutes a key component of the innate immune response to viral infection in both plants and invertebrate animals and has been postulated to have a similar protective function in mammals. This perspective reviews the available data addressing whether RNA interference forms part of the mammalian innate immune response and concludes that the popular hypothesis in favor of that possibility remains far from proven and may not be valid.
169 citations
TL;DR: It is shown that the primary miR-K5 sequence present in BCBL-1 results in diminished processing by Drosha both in vivo and in vitro, the first report of a naturally occurring sequence polymorphism in an miRNA precursor that results in reduced processing and therefore lower levels of mature miRNA expression and function.
Abstract: MicroRNAs (miRNAs) are a class of ∼22-nucleotide noncoding RNAs that inhibit the expression of specific target genes at the posttranscriptional level. Recently, 11 miRNAs encoded by the pathogenic human herpesvirus Kaposi9s sarcoma-associated herpesvirus (KSHV) were cloned from latently infected cells. While the expression of these miRNAs has been confirmed by Northern analysis, their ability to inhibit target gene expression has not been demonstrated. We have devised a novel assay for miRNA function that uses lentiviral indicator vectors carrying two perfectly complementary target sites for each given miRNA in the 3′ untranslated region of the Renilla luciferase gene. This assay allowed us to demonstrate the activity of each viral miRNA upon cotransduction of cells with the Renilla luciferase indicator vector together with a firefly luciferase control vector. In KSHV-infected BC-1 and BCBL-1 cells, but not uninfected control cells, Renilla luciferase expression was selectively reduced up to 10-fold. Interestingly, one of the viral miRNAs (miR-K5) exhibited much higher activity in BC-1 cells than in BCBL-1 cells. Sequence analysis of both viral genomes revealed a single nucleotide polymorphism in the miR-K5 precursor stem-loop, which inhibits the expression of mature miR-K5 in BCBL-1 cells. We show that the primary miR-K5 sequence present in BCBL-1 results in diminished processing by Drosha both in vivo and in vitro. This is the first report of a naturally occurring sequence polymorphism in an miRNA precursor that results in reduced processing and therefore lower levels of mature miRNA expression and function.
144 citations
TL;DR: Three latent transcripts are defined, derived from two distinct KSHV latent promoters, that function as both K SHV primary microRNA precursors and as kaposin pre-mRNAs that undergo two entirely distinct fates, depending on whether the viral polyadenylation site located at position 122070 is ignored or recognized.
Abstract: Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 11 distinct microRNAs, all of which are found clustered within the major latency-associated region of the KSHV genome in the same transcriptional orientation. Because the KSHV microRNAs are all expressed in latently infected cells and are largely unaffected by induction of lytic replication, it appeared probable that they would be processed out of KSHV transcripts that are derived from a latent promoter(s) present in this region. Here, we define three latent transcripts, derived from two distinct KSHV latent promoters, that function as both KSHV primary microRNA precursors and as kaposin pre-mRNAs. These activities require the readthrough of a leaky viral polyadenylation signal located at nucleotide 122070 in the KSHV genome. In contrast, recognition of this polyadenylation signal gives rise to previously identified mRNAs that encode the KSHV open reading frames (ORFs) 71, 72 and 73 proteins as well as a novel unspliced KSHV mRNA that encodes only ORF72 and ORF71. Thus, transcripts initiating at the two latent promoters present in the KSHV latency-associated region can undergo two entirely distinct fates, i.e., processing to give a kaposin mRNA and viral microRNAs on the one hand or expression as KSHV ORF71, ORF72, or ORF73 mRNAs on the other, depending on whether the viral polyadenylation site located at position 122070 is ignored or recognized, respectively.
140 citations
TL;DR: A set of design criteria, experimental steps and expression vectors that can facilitate the effective knock-down of almost any vertebrate gene product in cultured cells or in experimental animals are described.
Abstract: Over the last years, RNA interference (RNAi) has become a widely used technique that permits the knock-down, and hence functional analysis, of individual genes in vertebrate cells. However, the high failure rate of the RNA molecules used in RNAi experiments continues to be a problem. In this paper, I describe a set of design criteria, experimental steps and expression vectors that can facilitate the effective knock-down of almost any vertebrate gene product in cultured cells or in experimental animals.Gene Therapy (2006) 13, 503-508. doi:10.1038/sj.gt.3302656; published online 29 September 2005.
84 citations
TL;DR: It is concluded that HPV31, and possibly human papillomaviruses in general, does not express viral microRNAs.
Abstract: It has recently become clear that several pathogenic DNA viruses express virally encoded microRNAs in infected cells. In particular, numerous microRNAs have been identified in a range of human and animal herpesviruses, and individual microRNAs have also been identified in members of the polyoma- and adenovirus families. Although their functions remain largely unknown, it seems likely that these viral microRNAs play an important role in viral replication in vivo. Here we present an analysis of the microRNAs expressed in human cells during the latent and productive phases of the human papillomavirus genotype 31 (HPV31) replication cycle. Although over 500 cellular microRNAs were cloned and identified, not a single HPV31-specific microRNA was obtained. We therefore concluded that HPV31, and possibly human papillomaviruses in general, does not express viral microRNAs.
TL;DR: The up-regulation of the antiapoptotic HIAP-1/CIAP-2 gene as a consistent phenotype of HTLV-1-transformed and ATL-derived cultures and its stimulation by the viral oncoprotein Tax are described.
TL;DR: This chapter describes methods that allow the overexpression of human miRNAs and also discusses how primary mi RNAs (pri-miRNAs), the much longer precursors of mature miRNas, are processed in human cells, as well as in vitro.
Abstract: MicroRNAs (miRNAs) are approx 22-nucleotide (nt)-long, single-stranded, endogenous, noncoding RNAs that are widely expressed in multicellular organisms. This chapter describes methods that allow the overexpression of human miRNAs and also discusses how primary miRNAs (pri-miRNAs), the much longer precursors of mature miRNAs, are processed in human cells, as well as in vitro.
TL;DR: It is demonstrated that Mason-Pfizer monkey virus (MPMV) is resistant to inhibition by the APOBEC3G protein expressed in its normal host, the rhesus macaque, but highly susceptible to inhibitionBy murine APOBec3 (mA3), which is a potent barrier to the successful infection of mammalian species by retroviruses.
Abstract: The APOBEC3 protein family can constitute a potent barrier to the successful infection of mammalian species by retroviruses. Therefore, any retrovirus that has evolved the ability to replicate in a given animal must have developed mechanisms that allow it to avoid or inhibit the APOBEC3 proteins expressed in that animal. Here, we demonstrate that Mason-Pfizer monkey virus (MPMV) is resistant to inhibition by the APOBEC3G protein expressed in its normal host, the rhesus macaque, but highly susceptible to inhibition by murine APOBEC3 (mA3). MPMV virion particles fail to package rhesus APOBEC3G (rA3G), and MPMV Gag binds rA3G poorly in coexpressing cells. In contrast, MPMV virions package mA3 efficiently and MPMV Gag-mA3 complexes are readily detected. Moreover, mA3, but not rA3G, partially colocalizes with MPMV Gag in the cytoplasm of coexpressing cells. Previously, we have demonstrated that murine leukemia virus also escapes inhibition by APOBEC3 proteins by avoiding virion incorporation of its cognate APOBEC3 protein, mA3, yet is inhibited by primate APOBEC3G proteins, which it packages effectively (B. P. Doehle, A. Schafer, H. L. Wiegand, H. P. Bogerd, and B. R. Cullen, J. Virol. 79:8201-8207, 2005). The finding that two essentially unrelated beta- and gammaretroviruses use similar mechanisms to escape inhibition by the APOBEC3 proteins found in their normal host species suggests that the selective exclusion of APOBEC3 proteins from virion particles may be a general mechanism used by simple mammalian retroviruses.
TL;DR: What is currently known about the expression and function of the 12 miRNAs that are expressed by the pathogenic gamma herpesv virus Kaposi's sarcoma-associated herpesvirus in latently infected human B cells is discussed.
Abstract: microRNAs (miRNAs) are widely used by animal and plant cells to posttranscriptionally regulate cellular gene expression, but this regulatory mechanism can also be used by pathogenic viruses for the same purpose. It is now well established that numerous miRNAs are expressed by a wide range of pathogenic herpesviruses, although their mRNA targets and role in the viral life cycle remain unclear. Here, we discuss what is currently known about the expression and function of the 12 miRNAs that are expressed by the pathogenic gamma herpesvirus Kaposi's sarcoma-associated herpesvirus in latently infected human B cells.
01 Jan 2006
TL;DR: A large family of approximately 22 nucleotide long, non-coding RNAs processed from stemloop secondary structures are studied for their role in negatively regulating the expression of their target genes in plants and animals.
Abstract: MicroRNAs are a large family of approximately 22 nucleotide long, non-coding RNAs processed from stemloop secondary structures. Current evidence indicates that miRNAs negatively regulate the expression of their target genes in plants and animals.