Distinct p53, p53:LANA, and LANA Complexes in Kaposi's Sarcoma-Associated Herpesvirus Lymphomas

Abstract

The role of p53 in primary effusion lymphoma (PEL) is complicated. The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) binds p53. Despite this interaction, we had found that p53 was functional in PEL, i.e., able to induce apoptosis in response to DNA damage (C. E. Petre, S. H. Sin, and D. P. Dittmer, J. Virol. 81:1912-1922, 2007), and that hdm2 was overexpressed. To further elucidate the relationship between LANA, p53, and hdm2, we purified LANA complexes from PEL by column chromatography. This confirmed that LANA bound p53. However, the LANA:p53 complexes were a minority compared to hdm2:p53 and p53:p53 complexes. The half-life of p53 was not extended, which is in contrast to the half-life of simian virus 40 T antigen-transformed cells. p53:p53, LANA:p53, and LANA:LANA complexes coexisted in PEL, and each protein was able to bind to its cognate DNA element. These data suggest that under normal conditions, p53 is inactive in PEL, thus allowing for exponential growth, but that this inactivation is driven by the relative stoichiometries of LANA, hdm2, and p53. If p53 is activated by DNA damage or nutlin-3a, the complex falls apart easily, and p53 exercises its role as guardian of the genome.Kaposi's sarcoma-associated herpesvirus (KSHV) is found in Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and tumors from patients with the plasmablastic variant of multicentric Castleman disease (MCD) (reviewed in reference 15). The latency-associated nuclear antigen (LANA) is encoded by open reading frame 73 (ORF 73) of the viral genome. It is expressed in every KSHV-infected cell during the latent as well as lytic phase of the viral life cycle. LANA is required for replication and maintenance of the viral DNA during latent infection (2, 13). Experimental abrogation of LANA expression through small interfering RNA (siRNA) or genomic knockout leads to loss of KSHV from latently infected cells, genetically demonstrating that LANA is necessary for maintenance of latency (28,103). LANA is a large protein composed of 1,162 amino acids (KSHV M type, reference sequence NC_003409). It has many known biochemical activities, and many more have yet to be determined. One way to think about LANA is as a nuclear scaffolding protein for viral DNA replication and transcription, analogous to the large T antigen of the polyomaviruses.We hypothesized that there exist multiple biochemically distinct LANA complexes in KSHV-infected PEL, as both LANA and its many potential interaction partners are present at physiological molar ratios to each other. Biochemical analysis of purified native complexes in PEL allowed us to establish that not all of LANA was part of a LANA:p53 complex and that not all of p53 was bound to LANA.The C terminus of the LANA protein has sequence-specific DNA binding activity. LANA can bind to a 20-bp repeated cis element in the terminal repeats (TRs) of the viral genome (3,26,27,45,50,78,85,101). We previously identified a similar cis element that was present as a single c...

Cytoplasmic isoforms of Kaposi sarcoma herpesvirus LANA recruit and antagonize the innate immune DNA sensor cGAS

“…Cells (1.5 × 10 8 ) from each stable cell line (BCBL-1 GFP, BCBL-1 GFP-LANA-FL, BCBL-1 GFP-LANA-NC) were washed once in PBS and resuspended in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl 2 , 5 mM DTT, and protease inhibitors) for 30 min on ice and homogenized with 15 strokes in a cell douncer (73). Nuclei were pelleted by centrifugation for 10 min at 1,200 rpm (Eppendorf centrifuge 5417R, rotor F45-30-11, Hamburg, Germany) and 4°C and resuspended in 2 mL TBST lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% TritonX-100).…”

Ribosomal Protein S6 Interacts with the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus

“…(iv) LANA binds to Rb (61) as well as to p53 (18,80). The LANA-p53 complex can be destroyed by the mdm-2/p53 interaction inhibitor nutlin (9,58,67), which leads to p53-dependent apoptosis in PEL. Because of its ability to decorate host chromosomes, LANA can induce chromosome instability phenotypes that are akin to p53 inactivation (54,70).…”

“…Column fractions containing LANA were collected and loaded onto Hiprep 16/10 heparin FF column (Amersham, Inc.), followed by a wash with 20 column volumes of 0.2 M KCl in buffer D and elution with 0.5 M KCl in buffer D. The fractions were then loaded onto a Mono-Q column (Sigma, Inc.). We collected two peaks at 0.4 M buffer D and 0.7 M KCl in buffer D. Immunoprecipitation with anti-LANA antibody (LN53; ABI, Inc.) was from a the second peak as described previously (9). Proteins were resolved by 8 to 16% gradient SDS-PAGE and stained with Coomassie blue (Bio-Rad, Inc.).…”

p53 Tumor Suppressor Protein Stability and Transcriptional Activity Are Targeted by Kaposi's Sarcoma-Associated Herpesvirus-Encoded Viral Interferon Regulatory Factor 3

“…vIRF-4 regulates p53 stability by interacting with Mdm2 as well as with HAUSP, resulting in decreased relative levels of p53 via 26S proteasome-mediated degradation (82,83). Also latency-associated nuclear antigen (LANA) forms complexes with p53 in the nuclei of latently infected cells (84) and induces p53 ubiquitination and degradation (85,86). Moreover, LANA markedly elevates the expression of Aurora A oncogenic kinase, which leads to phosphorylation of p53 at S215 and S315 and subsequent p53 ubiquitination and proteasome-mediated degradation (87).…”