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. 2007 Dec;81(24):13544-51.
doi: 10.1128/JVI.01521-07. Epub 2007 Oct 3.

RNA editing of the human herpesvirus 8 kaposin transcript eliminates its transforming activity and is induced during lytic replication

Sharon Z Gandy  1 , Sarah D Linnstaedt, Sumitra Muralidhar, Kathleen A Cashman, Leonard J Rosenthal, John L Casey
Affiliations

RNA editing of the human herpesvirus 8 kaposin transcript eliminates its transforming activity and is induced during lytic replication

Sharon Z Gandy et al. J Virol. 2007 Dec.

Abstract

Human herpesvirus 8 is the etiologic agent associated with Kaposi's sarcoma and primary effusion lymphoma (PEL). The K12 RNA, which produces as many as three variants of the kaposin protein, as well as a microRNA, is the most abundant transcript expressed in latent Kaposi's sarcoma-associated herpesvirus infection, and yet it is also induced during lytic replication. The portion of the transcript that includes the microRNA and the kaposin A sequence has been shown to have tumorigenic potential. Genome coordinate 117990, which is within this transcript, has been found to be heterogeneous, primarily in RNAs but also among viral DNA sequences. This sequence heterogeneity affects an amino acid in kaposins A and C and the microRNA. The functional effects of this sequence heterogeneity have not been studied, and its origin has not been definitively settled; both RNA editing and heterogeneity at the level of the viral genome have been proposed. Here, we show that transcripts containing A at position 117990 are tumorigenic, while those with G at this position are not. Using a highly sensitive quantitative assay, we observed that, in PEL cells under conditions where more than 60% of cDNAs derived from K12 RNA transcripts have G at coordinate 117990, there is no detectable G in the viral DNA sequence at this position, only A. This result is consistent with RNA editing by one of the host RNA adenosine deaminases (ADARs). Indeed, we observed that purified human ADAR1 efficiently edits K12 RNA in vitro. Remarkably, the amount of editing correlated with the replicative state of the virus; editing levels were nearly 10-fold higher in cells treated to induce lytic viral replication. These results suggest that RNA editing controls the function of one segment of the kaposin transcript, such that it has transforming activity during latent replication and possibly another, as-yet-undetermined, function during lytic replication.

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Figures

FIG. 1.
FIG. 1.
Electropherograms showing direct sequencing of PCR and RT-PCR products. HHV-8 viral DNA and RNA were harvested from TPA-stimulated BCBL1 and BC3 cells and subjected to PCR and RT-PCR amplification using the primers SZ4 and SZ5 as described in Materials and Methods. Colors: G, black; A, green; C, blue; T/U, red. The sequence shown is in the same orientation as that of the kaposin coding sequence. A dashed line indicates the location of position 117990.
FIG. 2.
FIG. 2.
Sensitive quantitative assay for sequence heterogeneity at position 117990. (A) Schematic showing the primary K12 RNA transcript (thin horizontal line), the locations of position 117990, and the primers used for PCR amplification (left and right arrows). Primer K12F3 contains two changes (underlined) that create an XcmI restriction site in PCR products derived from templates with a G at position 117990. Incubation of PCR products containing an A at position 117990 with XcmI yields an undigested 119-bp DNA, whereas PCR products with a G at position 117990 are digested into 93- and 26-bp fragments. (B) The upper panels show the electrophoretic separation of 32P-labeled PCR products either digested with XcmI (+) or undigested (−). The 119- and 93-bp digestion products are indicated as 117990 A and 117990 G, respectively; the 26-bp product is not visible. The results are shown for products obtained from DNA and RNA harvested from BCBL1 and BC3 cells. The lower panel shows digested PCR products obtained from BCBL1 and BC3 cell DNA compared to that of a control template containing 99% A 117990 and 1% G 117990. Brightness and contrast were adjusted identically using the program ImageJ such that the 119-bp digestion product for the 1% G control sample is readily visible; quantification of this sample indicated 0.9% G.
FIG. 3.
FIG. 3.
Kaposin RNA is efficiently edited by ADAR1 in vitro. (A) Kaposin A RNA (1.2 fmol) was incubated with increasing amounts of purified human ADAR1 expressed in Sf9 cells. ADAR1 concentrations were, from left to right: 0, 1, 2, 4, 8, and 16 nM. RNAs were subjected to RT-PCR amplification and digestion with XcmI as described in the text. (B) Editing of kaposin RNA (K12) and two HDV RNAs (MDIII-2 and DC1S) by purified ADAR1. Reaction conditions were identical for all three RNAs.
FIG. 4.
FIG. 4.
The transforming ability of kaposin transcripts is lost when position 117,990 is changed to G. (A) Focus formation in Rat3 cells. Low-passage Rat3 cells were transfected with pCMV-Tag1 (vector), pCMV-Kap-Tag (Kap), or pCMV-Kap-Tag in which position 117990 was changed from an A to a G (Kap 117,990G). Foci were counted at 21 days posttransfection. (B) Tumorigenicity of kaposin variants in nu/nu mice. Rat3 cells were stably transfected with the kaposin transcript expression construct pCMV-Kap-Tag designed to produce RNAs containing either A at coordinate 117990 (▵) or G at coordinate 117990 (□) or with the empty vector pCMV-Tag1 (○). Cells were injected into nu/nu mice, and the tumor volume was measured beginning on day 14.
FIG. 5.
FIG. 5.
Editing at coordinate 117990 is strongly induced by induction of lytic replication. BC3 cells seeded at 2 × 105 per ml were either left untreated or treated with 25 nM TPA or 3 mM sodium butyrate. RNAs were harvested at 48 h and analyzed for sequence heterogeneity at position 117990 as described in the text.
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References

    1. Adang, L. A., C. H. Parsons, and D. H. Kedes. 2006. Asynchronous progression through the lytic cascade and variations in intracellular viral loads revealed by high-throughput single-cell analysis of Kaposi's sarcoma-associated herpesvirus infection. J. Virol. 80:10073-10082. - PMC - PubMed
    1. Arvanitakis, L., E. A. Mesri, R. G. Nador, J. W. Said, A. S. Asch, D. M. Knowles, and E. Cesarman. 1996. Establishment and characterization of a primary effusion (body cavity-based) lymphoma cell line (BC-3) harboring Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) in the absence of Epstein-Barr virus. Blood 88:2648-2654. - PubMed
    1. Bass, B. L. 2002. RNA editing by adenosine deaminases that act on RNA. Annu. Rev. Biochem. 71:817-846. - PMC - PubMed
    1. Bhalla, T., J. J. Rosenthal, M. Holmgren, and R. Reenan. 2004. Control of human potassium channel inactivation by editing of a small mRNA hairpin. Nat. Struct. Mol. Biol. 11:950-956. - PubMed
    1. Brinkmann, M. M., M. Pietrek, O. Dittrich-Breiholz, M. Kracht, and T. F. Schulz. 2007. Modulation of host gene expression by the K15 protein of Kaposi's sarcoma-associated herpesvirus. J. Virol. 81:42-58. - PMC - PubMed

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