Dataset Information

Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus recruits uracil DNA glycosylase 2 at the terminal repeats and is important for latent persistence of the virus.

ABSTRACT: Latency-associated nuclear antigen (LANA) of KSHV is expressed in all forms of Kaposi's sarcoma-associated herpesvirus (KSHV)-mediated tumors and is important for TR-mediated replication and persistence of the virus. LANA does not exhibit any enzymatic activity by itself but is critical for replication and maintenance of the viral genome. To identify LANA binding proteins, we used a LANA binding sequence 1 DNA affinity column and determined the identities of a number of proteins associated with LANA. One of the identified proteins was uracil DNA glycosylase 2 (UNG2). UNG2 is important for removing uracil residues yielded after either misincorporation of dUTP during replication or deamination of cytosine. The specificity of the 'LANA-UNG2 interaction was confirmed by using a scrambled DNA sequence affinity column. Interaction of LANA and UNG2 was further confirmed by in vitro binding and coimmunoprecipitation assays. Colocalization of these proteins was also detected in primary effusion lymphoma (PEL) cells, as well as in a cotransfected KSHV-negative cell line. UNG2 binds to the carboxyl terminus of LANA and retains its enzymatic activity in the complex. However, no major effect on TR-mediated DNA replication was observed when a UNG2-deficient (UNG(-/-)) cell line was used. Infection of UNG(-/-) and wild-type mouse embryonic fibroblasts with KSHV did not reveal any difference; however, UNG(-/-) cells produced a significantly reduced number of virion particles after induction. Interestingly, depletion of UNG2 in PEL cells with short hairpin RNA reduced the number of viral genome copies and produced infection-deficient virus.

SUBMITTER: Verma SC

PROVIDER: S-EPMC1642147 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus recruits uracil DNA glycosylase 2 at the terminal repeats and is important for latent persistence of the virus.

Verma Subhash C SC Bajaj Bharat G BG Cai Qiliang Q Si Huaxin H Seelhammer Todd T Robertson Erle S ES

Journal of virology 20060823 22

Latency-associated nuclear antigen (LANA) of KSHV is expressed in all forms of Kaposi's sarcoma-associated herpesvirus (KSHV)-mediated tumors and is important for TR-mediated replication and persistence of the virus. LANA does not exhibit any enzymatic activity by itself but is critical for replication and maintenance of the viral genome. To identify LANA binding proteins, we used a LANA binding sequence 1 DNA affinity column and determined the identities of a number of proteins associated with ...[more]

PMID: 16928741

Similar Datasets

Project description:The regulation of latency is central to herpesvirus biology. Recent transcriptome-wide surveys have uncovered evidence for promiscuous transcription across the entirety of the Kaposi's sarcoma-associated herpesvirus (KSHV) genome and postulated the existence of multiple viral long noncoding RNAs (lncRNAs). Next-generation sequencing studies are highly dependent on the specific experimental approach and particular algorithms of analysis and therefore benefit from independent confirmation of the results. The antisense-to-latency transcript (ALT) lncRNA was discovered by genome-tiling microarray (Chandriani et al., J Virol 86:7934-7942, 2010, https://doi.org/10.1128/JVI.00645-10). To characterize ALT in detail, we physically isolated this lncRNA by a strand-specific hybrid capture assay and then employed transcriptome sequencing and novel reverse transcription-PCR (RT-PCR) assays to distinguish all RNA species in the KSHV latency region. These methods confirm that ALT initiates at positions 120739/121012 and encodes a single splice site, which is shared with the 3'-coterminal K14-vGPCR/ORF74 mRNA, terminating at 130873 (GenBank accession number GQ994935), resulting in an ?10,000-nucleotide transcript. No shorter ALT isoforms were identified. This study also identified a novel intron within the LANA 5' untranslated region using a splice acceptor at 127888. In summary, ALT joins PAN/nut1/T1.1 as a bona fide lncRNA of KSHV with potentially important roles in viral gene regulation and pathogenesis. IMPORTANCE:Increasing data support the importance of noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and lncRNAs, which have been shown to exert critical regulatory functions without coding for recognizable proteins. Defining the sequences of these ncRNAs is essential for future studies aiming to functionally characterize a specific ncRNA. Most lncRNA studies are highly dependent on high-throughput sequencing and bioinformatic analyses, few studies follow up on the initial predictions, and analyses are at times discordant. The manuscript characterizes one key viral lncRNA, ALT, by physically isolating ALT and by a sequencing-independent assay. It provides for a simple assay to monitor lncRNA expression in experimental and clinical samples. ALT is expressed antisense to the major viral latency transcripts encoding LANA as well as the viral miRNAs and thus has the potential to regulate this key part of the viral life cycle.

Dataset Information

Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus recruits uracil DNA glycosylase 2 at the terminal repeats and is important for latent persistence of the virus.

Publications

Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus recruits uracil DNA glycosylase 2 at the terminal repeats and is important for latent persistence of the virus.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure