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miRNA Biogenesis

Location and Conservation of HCMV miRNAs

Mapping of HCMV miRNAs

HCMV miRNA Conservation

Genomic Arrangement of HCMV miRNAs .

HCMV miRNA Expression

Kinetic Classes of HCMV miRNAs

Tissue-Specific HCMV miRNA Expression Latent Versus Lytic Infection

Potential Function of HCMV miRNAs

Future Directions

References

Abstract MicroRNAs (miRNAs) are approximately 22 nucleotide RNAs that mediate the posttranscriptional regulation of gene expression. miRNAs regulate diverse cellular processes such as development, differentiation, cell cycling, apoptosis, and immune responses. More than 400 miRNAs have been identified in humans and it is predicted that over 30% of human gene transcripts are regulated via miRNAs. Since 2004, many viral miRNAs have been described in several families of viruses. More than half of currently known viral miRNAs are encoded by viruses of the human Herepsviridae and 14 miRNAs have been found to be encoded by Human cytomegalovirus (HCMV). Thus far, HCMV is the only betaherpesvirus in which miRNAs have been described and these miRNAs possess many characteristics, including their genomic arrangement and temporal/spatial expression, which distinguish them from the other known herpesvirus miRNAs described. As a herpesvirus,

Program in Infectious Diseases and Immunity, School of Public Health, 140 Warren Hall, University of California, Berkeley, CA 94720, USA liu_fy @ uclink4 .berkeley. edu

T.E. Shenk and M.F. Stinski (eds.), Human Cytomegalovirus. 21

Current Topics in Microbiology and Immunology 325. © Springer-Verlag Berlin Heidelberg 2008

HCMV establishes infection for the life of the host characterized by latent infection with periodic reactivation for production and spread of infectious progeny. This multifaceted life cycle of the herpesvirus requires an abundance of gene products and regulatory elements that makes cytomegalovirus genomes one of the most complex among human viruses. The defining characteristics of the cytomegalovirus and the minimal impact on genome size afforded by miRNAs inform the logic of virus-encoded miRNAs.

Abbreviations miRNA: MicroRNA; HCMV: Human cytomegalovirus; EBV: Epstein Bar Virus; HSV: Herpes simplex virus; miRISC: MicroRNA-induced silencing complex; MHV 68: Murine gammaherpesvirus 68; RRV: Rhesus macaque rhadinovirus; rLCV: Rhesus lymphocryptovirus; MDV: Marek's disease virus

Introduction

In 1993, the discovery of an approximately 22-nucleotide RNA (lin-4) responsible for the posttranscriptional regulation of LIN-14 protein levels in caenorhabditis elegans represented the beginnings of a paradigm shift in molecular biology (Lee et al. 1993; Wightman et al. 1993). While lin-4 was discovered in 1993, it was not until 1999 that it was shown to inhibit protein synthesis after the initiation of translation (Olsen and Ambros 1999). Lin-4 is the founding member of a family of small RNAs, termed microRNAs (miRNA), that now number over 400 in humans (Bentwich et al. 2005). Not 15 years after lin-4's discovery, it has been predicted that over 30% of human gene products are subject to miRNA-mediated regulation (Lewis et al. 2005).

miRNAs are endogenously encoded approximately 22-nt RNAs that are responsible for the temporal and spatial regulation of gene products involved in diverse cellular processes including development, apoptosis, differentiation, cell cycle regulation, and immune response (Ambros 2004; Taganov et al. 2006; O'Connell et al. 2007; Rodriguez et al. 2007; Taganov et al. 2007). Functionally, miRNAs mediate gene silencing by guiding the miRNA-induced gene silencing complex (miRISC) to target mRNAs (Tang 2005). Targeting of mRNA by miRISC leads to transla-tional inhibition or cleavage of the targeted mRNA. The specificity of most animal miRNA-target mRNA complexes is determined by complementarity of a seed region, namely nucleotides 2-7 of the miRNA (Brennecke et al. 2005). When recognition of mRNA by miRNA is mediated primarily by the seed region, transla-tional inhibition is typically the end result, while extensive sequence complementarity between the miRNA and target mRNA results in cleavage of the target mRNA (Ambros 2004). Complementary sequences in the target mRNA usually reside in the 3' UTR (Lewis et al. 2005). Due to the size of the seed region, miRNAs are predicted to target as many as ten mRNAs. Additionally, multiple miRNAs may target the same mRNA with the 3'-UTRs containing target sites for multiple miRNAs, and in situ adenosine to inosine substitutions in mature miRNAs can also alter targeting (Wightman et al. 1993; Doench and Sharp 2004; Kawahara et al. 2007). Finally, the miRNA sequence outside the seed region has been found to play a role in subcellular localization of miRNAs (Hwang et al. 2007).

Eleven years after the discovery of miRNAs, the first virally encoded miRNAs were reported (Pfeffer et al. 2004). As most algorithms for the identification of miRNAs rely on conservation of sequence, viral miRNA prediction was particularly difficult due to the absence of significant homology to known miRNAs. By sequencing of a small RNA library from a Burkitt's lymphoma cell line latently infected with Epstein Bar Virus (EBV), Tuschl and colleagues identified five miRNAs that originated from the EBV (Pfeffer et al. 2004). Subsequently, many groups, including our own, have contributed to the identification of miRNAs encoded by other human herpesviruses. Additionally, many nonhuman herpesvirus miRNAs have been identified, providing animal models in which to study the function of virally encoded miRNAs. Thus far, 106 of the 108 mature viral miRNAs species in the miRNA registry are encoded by herpesviruses (Griffiths-Jones 2004, 2006; Griffiths-Jones et al. 2006). Currently, it is known that HCMV expresses 14 mature miRNAs from 11 precursor miRNAs (Fig. 1).

A herpesvirus is a large dsDNA virus that replicates in the nucleus of the host cell, and after an initial lytic replication cycle establishes latent infection for the life of the host. Reactivation from latency and initiation of secondary lytic replication occurs periodically. Betaherpesviruses, which include HCMV and herpesvirus-6 and -7, can replicate in a wide variety of cell types, but exhibit strict species specificity. The complex life cyle of herpes viruses illustrates the need for distinctive gene regulation mechanisms that viral miRNAs provide. Utilization of miRNA offers the virus, which possesses limited coding capacity, a means to alter gene expression with relatively minimal impact on genome size; maintenance of latent infection requires limited, nonimmunogenic gene expression; tissue tropism implies an array of gene products suited to the exploitation of specific host-cell types. As such, it is not surprising that the first virally encoded miRNAs were discovered in a herpesvirus, EBV.

miRNA Biogenesis

The progress made in understanding miRNA biogenesis (Fig. 2) stands in stark contrast to the limited understanding of miRNA-mediated regulatory networks. While many fundamental questions about regulation of miRNA biogenesis still need to be addressed, significant progress has been made concerning the process of miRNA maturation.

Many of the subtleties and details concerning miRNA biogenesis are beyond the scope of this review. Rather, it is our aim to provide an overview of the biogenesis process as an aid to understanding HCMV miRNA. There is little evidence to suggest that viral miRNA biogenesis differs from that of cellular miRNAs. Recently, however, aspects of miRNA biogenesis such as substrate

Fig. 1 HCMV miRNAs. The eleven HCMV pre-miRNAs found in the miRNA registry and their predicted foldback structures are presented. Mature miRNA sequences are highlighted. Information regarding evidence for the indicated miRNA its expression, position and potential targets are given

specificity and Dicer activity have been found to differ during stages of development (Eis et al. 2005; Kim 2005; Lund and Dahlberg 2006). Finally, unique features of the murine gammaherpesvirus-68 (MHV68) miRNAs such as transcription by pol III and short hairpin structure suggest distinct mechanisms of biogenesis

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