Double Stranded DNA Viruses

Double-stranded DNA viruses include viruses of the Polyomaviridae and Herpesviridae families. Their genomes are circular dsDNA of *5 kbp in the case of polyoma virus and linear dsDNA of * 125-250 kbp dependent upon the specific herpesvirus. Transcription of viral genes in both cases occurs in the nucleus by the cellular RNA polymerase II machinery. ADAR-mediated RNA editing has been described for viral RNA transcripts of mouse polyoma virus (Liu et al. 1994; Kumar and Carmichael 1997; Gu et al. 2009) and two herpesviruses, Kaposi's sarcoma-associated herpesvirus (Gandy et al. 2007) and Epstein-Barr virus (Iizasa et al. 2010).

Polyomaviridae: Mouse polyoma virus, mPyV, is a small DNA virus in which the naked virion capsid encloses a single *5 kbp molecule of circular dsDNA complexed with cellular histones to form a minichromosome. In mouse cells permissive for productive infection, the early and late regions of the viral genome are expressed bidirectionally, with early and late mRNAs transcribed from opposite strands of the DNA genome. Spliced early mRNAs encode three regulatory proteins that include large T antigen important for DNA replication. Spliced late mRNAs are expressed efficiently only after DNA replication and encode three capsid proteins VP1, VP2 and VP3 (Benjamin 2001; Knipe et al. 2007). Evidence has been presented that early RNA gene expression is regulated by sense-antisense interactions that result in extensive A-I editing of the early strand RNA transcripts at late times after infection (Liu and Carmichael 1993; Liu et al. 1994; Kumar and Carmichael 1997). Formation of dsRNA occurs in the regions of sense-antisense overlap of the early and late viral transcripts that includes overlap of the poly-adenylation signals (Gu et al. 2009). Extensive A-G (I) sequence changes are seen in mPyV early strand RNAs present in the nucleus at late times after infection (Kumar and Carmichael 1997), consistent with the hyperediting action of an ADAR. Given the nuclear localization of mPyV transcription, either ADAR1 or ADAR2 both of which are nuclear enzymes could presumably be responsible for the biased hyperediting in the 3'-overlap region of mPyV RNAs. While transient knockdown of ADAR1 in NIH3T3 cells caused a defect in early-to-late switch suggesting mPyV infection is sensitive to ADAR1 protein levels (Gu et al. 2009), the availability of MEFs genetically null in Adarl, Adarl p150 and Adar2 should permit the unequivocal identification of which ADAR edits mPyV RNA in the nucleus (Higuchi et al. 2000; Hartner et al. 2004, 2009; Wang et al. 2004; XuFeng et al. 2009; Ward et al. 2011). ADAR-catalyzed A-I editing of cellular mRNAs containing dsRNA structures in their 3'-UTRs, including inverted Alu repeats in the case of cellular mRNAs in human cells, may affect localization of the RNAs (Hundley and Bass 2010). However, for mPyV, it is not yet fully clear whether it is the formation of the dsRNA in the 3'-region of overlapping polyadenylation signals or the A-I editing per se of the dsRNA that is the critical determinant for regulation of mPyV RNA expression.

Herpesviridae: Kaposi's sarcoma-associated herpesvirus, KSHV or human herpesvirus 8 (HHV-8), is associated with Kaposi's tumors seen in immunosup-pressed patients including, for example, AIDS patients (Knipe et al. 2007; Mesri et al. 2010). In the case of KSHV, a viral transcript is edited in a manner that affects both a protein coding sequence and a microRNA (Gandy et al. 2007). During lytic infection most KSHV viral genes are transcribed in cascades with temporal regulation, whereas during latency only a few viral genes are expressed and among the most abundant is the K12 kaposin transcript. The K12 transcript encodes three kaposin proteins (A, B, C) and a microRNA (miR-K10) and has oncogenic potential (Damania 2004). A-I editing occurs at genome posi-tion117990 in the K12 transcript, and in the kaposin A ORF, changes serine at position 38 to glycine. The nt substitution also changes position 2 at the 5'end of miR-K10, potentially altering targeting. Editing levels are increased nearly 10-fold following treatment with phorbol ester or sodium butyrate to activate lytic virus replication. Transcripts containing an A at 117990 are tumorigenic, while those with a G corresponding to edited RNAs with I are not tumorigenic as measured by focus formation in Rat3 cells and tumor production in nude mice. ADAR1, at least the p110 form expressed using baculovirus and purified from Sf9 insect cells, efficiently edits the K12 transcript (Gandy et al. 2007).

Epstein-Barr virus, EBV or human herpes virus 4 (HHV-4), is a lymphotropic herpesvirus that can infect and transform a range of human B cells and is associated with latent infections and diseases including infectious mononucleosis and Burkitt's lymphoma (Knipe et al. 2007). EBV encodes more than 20 mi-croRNAs and among them is the BART6 miRNA (Pfeffer et al. 2004; Skalsky and Cullen 2010). In the case of EBV, four viral miRNAs including BART6 miRNA primary transcripts are edited in latently EBV-infected cells (Iizasa et al. 2010). Primary miRNA transcripts are processed by the Drosha and Dicer endonucleases that act together with dsRNA binding proteins to generate the mature 20-22-nt miRNAs that function in silencing of gene expression (Filipowicz et al. 2008; Skalsky and Cullen 2010). The BART6 viral miRNA targets the Dicer nuclease at multiple sites in the 3'-UTR of Dicer mRNA. A-I editing of BART6 dramatically reduces loading of miRs onto the RISC silencing complex and inhibits silencing activity. The editing analysis of EBV primary miRNAs in EBV-infected human lymphoblastoid, Daudi Burkitt lymphoma and nasopharyngeal carcinoma cell lines suggest that EBV miR-BART6 RNAs play important roles in the regulation of viral replication and latency, and that A-I editing of BART6 may be an adaptive selection to counteract the targeting of Dicer by miR-BART6 (Iizasa et al. 2010).

Poxviridae and Adenoviridae: Finally, gene products of two viruses with linear dsDNA genomes, vaccinia virus of the Poxviridae that multiplies in the cytoplasm and adenovirus 5 of the Adenoviridae that multiplies in the nucleus, have been demonstrated to antagonize ADAR1 enzymatic activity. The vaccinia virus E3L protein and the adenovirus VAI RNA inhibit A-I editing activity of ADAR1 (Lei et al. 1998; Liu et al. 2001). However, it is not known whether vaccinia virus or adenovirus viral RNA is edited by an ADAR. Interestingly, the Z-DNA binding domain present in the N-terminal region of ADAR1 p150 was originally described as a poxvirus E3L homology domain (Patterson and Samuel 1995). The Z-DNA binding domain present in the N-terminal region of vaccinia virus E3L plays a role in viral pathogenesis; mutations that decrease Z-DNA binding correlate with decreased viral pathogenicity in the mouse model (Kim et al. 2003). The E3L protein also binds dsRNA and mediates IFN resistance, promotes vaccinia virus growth and impairs virus-mediated apoptosis. Loss of PKR expression in HeLa cells complements the vaccinia virus E3L deletion mutant phenotype by restoration of viral protein synthesis and largely abolishes virus-induced apoptosis (Zhang et al. 2008). ADAR1 suppresses activation of PKR (Nie et al. 2007; Toth et al. 2009) as earlier discussed. Adenovirus VAI RNA, in addition to antagonizing PKR activation (Kitajewski et al. 1986), also antagonizes the activity of ADAR1 (Lei et al. 1998; Taylor et al. 2005).

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