Kaposi's Sarcoma-associated Herpesvirus Latency-associated Nuclear Antigen Research Articles

The latency-associated nuclear antigen of Kaposi sarcoma-associated herpesvirus induces B cell hyperplasia and lymphoma

Kaposi sarcoma-associated herpesvirus (KSHV) is a human lymphotropic herpesvirus. It is implicated in B cell neoplasias such as primary effusion lymphoma and multicentric Castleman disease in AIDS patients. The KSHV latency-associated nuclear antigen (LANA) is consistently expressed in all KSHV-associated tumor cells and was shown to bind the tumor suppressor proteins p53 and pRb. To test LANA's contribution to lymphomagenesis in vivo we generated transgenic mice expressing LANA under the control of its own promoter, which is B cell specific. All of the transgenic mice developed splenic follicular hyperplasia due to an expansion of IgM+ IgD+ B cells and showed increased germinal center formation. We also observed lymphomas, implying that LANA can activate B cells and provide the first step toward lymphomagenesis.

ORC, MCM, and Histone Hyperacetylation at the Kaposi's Sarcoma-Associated Herpesvirus Latent Replication Origin

The viral genome of Kaposi's sarcoma-associated herpesvirus (KSHV) persists as an extrachromosomal plasmid in latently infected cells. The KSHV latency-associated nuclear antigen (LANA) stimulates plasmid maintenance and DNA replication by binding to an approximately 150-bp region within the viral terminal repeats (TR). We have used chromatin immunoprecipitation assays to demonstrate that LANA binds specifically to the replication origin sequence within the KSHV TR in latently infected cells. The latent replication origin within the TR was also bound by LANA-associated proteins CBP, double-bromodomain-containing protein 2 (BRD2), and the origin recognition complex 2 protein (ORC2) and was enriched in hyperacetylated histones H3 and H4 relative to other regions of the latent genome. Cell cycle analysis indicated that the minichromosome maintenance complex protein, MCM3, bound TR in late-G(1)/S-arrested cells, which coincided with the loss of histone H3 K4 methylation. Micrococcal nuclease studies revealed that TRs are embedded in a highly ordered nucleosome array that becomes disorganized in late G(1)/S phase. ORC binding to TR was LANA dependent when reconstituted in transfected plasmids. DNA affinity purification confirmed that LANA, CBP, BRD2, and ORC2 bound TR specifically and identified the histone acetyltransferase HBO1 (histone acetyltransferase binding to ORC1) as a potential TR binding protein. Disruption of ORC2, MCM5, and HBO1 expression by small interfering RNA reduced LANA-dependent DNA replication of TR-containing plasmids. These findings are the first demonstration that cellular replication and origin licensing factors are required for KSHV latent cycle replication. These results also suggest that the KSHV latent origin of replication is a unique chromatin environment containing histone H3 hyperacetylation within heterochromatic tandem repeats.

Accumulation of heterochromatin components on the terminal repeat sequence of Kaposi's sarcoma-associated herpesvirus mediated by the latency-associated nuclear antigen.

In the latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), its 160-kb circularized episomal DNA is replicated and maintained in the host nucleus. KSHV latency-associated nuclear antigen (LANA) is a key factor for maintaining viral latency. LANA binds to the terminal repeat (TR) DNA of the viral genome, leading to its localization to specific dot structures in the nucleus. In such an infected cell, the expression of the viral genes is restricted by a mechanism that is still unclear. Here, we found that LANA interacts with SUV39H1 histone methyltransferase, a key component of heterochromatin formation, as determined by use of a DNA pull-down assay with a biotinylated DNA fragment that contained a LANA-specific binding sequence and a maltose-binding protein pull-down assay. The diffuse localization of LANA on the chromosomes of uninfected cells changed to a punctate one with the introduction of a bacterial artificial chromosome containing most of the TR region, and SUV39H1 clearly colocalized with the LANA-associated dots. Thus, the LANA foci in KSHV-infected cells seemed to include SUV39H1 as well as heterochromatin protein 1. Furthermore, a chromatin immunoprecipitation assay revealed that the TR and the open reading frame (ORF) K1 and ORF50/RTA genes, but not the ORF73/LANA gene, lay within the heterochromatin during KSHV latency. Taken together, these observations indicate that LANA recruits heterochromatin components to the viral genome, which may lead to the establishment of viral latency and govern the transcription program.

Seroprevalence of Kaposi's sarcoma-associated herpesvirus in various populations in Cuba.

Little is known about the prevalence and distribution of Kaposi's sarcoma-associated herpesvirus (KSHV) infection in the Caribbean. The aim of this study was to determine rates of KSHV seropositivity in various populations in Cuba. During the years 1998 to 2002 we screened serum samples from 410 subjects in Cuba. Serologic screening for KSHV antibodies was a two-step process using (1) indirect immunofluorescence assay (IFA) specifically reactive to the KSHV latency-associated nuclear antigen (LANA) encoded by open reading frame 73 (ORF73), and (2) confirmatory immunoblot using recombinant KSHV ORF65.2, a lytically expressed, 20-kilodalton protein as the target antigen. Five different populations were studied: (1) 45 AIDS patients with Kaposi's sarcoma (AIDS-KS), (2) 154 HIV-1-infected patients without clinical evidence of KS, (3) 171 HIV-negative blood donors, (4) 27 consecutive kidney transplant recipients, who were HIV-negative, and (5) 13 contacts (sexual contacts or relatives) of the AIDS-KS-affected patients. Among the 45 AIDS-KS subjects, 35 of them (77.8%) were KSHV-seropositive. Thirty-two of the 154 HIV-positive patients without KS (20.8% of them) were KSHV-seropositive, and 6 of the 13 contacts of KS-affected patients (46.2% of them) were infected with KSHV. In contrast to other researchers, we did not find in the populations that we studied in Cuba that KSHV seropositivity was associated with male homosexual or bisexual activity. We found high KSHV seropositivity rates among women reporting sexual contact with bisexual men and among men who had acquired an HIV infection in Africa. There were low rates of KSHV infection among the blood donors (1.2%) and the renal transplant recipients (0.0%). The low rates of KSHV infection that we found among the non-HIV-infected populations in Cuba are similar to patterns found in populations in Europe and in the United States. Together with similar results from Brazil, Jamaica, and the United States of America, our results suggest that KSHV infection is uncommon in some populations in the Western Hemisphere and that KSHV is largely confined to patients with AIDS-associated KS.

The gammaherpesvirus 68 latency-associated nuclear antigen homolog is critical for the establishment of splenic latency.

Open reading frame 73 (ORF 73) is conserved among the gamma-2-herpesviruses (rhadinoviruses) and, in Kaposi's sarcoma-associated herpesvirus (KSHV) and herpesvirus saimiri (HVS), has been shown to encode a latency-associated nuclear antigen (LANA). The KSHV and HVS LANAs have also been shown to be required for maintenance of the viral genome as an episome during latency. LANA binds both the viral latency-associated origin of replication and the host cell chromosome, thereby ensuring efficient partitioning of viral genomes to daughter cells during mitosis of a latently infected cell. In gammaherpesvirus 68 (gammaHV68), the role of the LANA homolog in viral infection has not been analyzed. Here we report the construction of a gammaHV68 mutant containing a translation termination codon in the LANA ORF (73.STOP). The 73.STOP mutant virus replicated normally in vitro, in both proliferating and quiescent murine fibroblasts. In addition, there was no difference between wild-type (WT) and 73.STOP virus in the kinetics of induction of lethality in mice lacking B and T cells (Rag 1(-/-)) infected with 1000 PFU of virus. However, compared to WT virus, the 73.STOP mutant exhibited delayed kinetics of replication in the lungs of immunocompetent C57BL/6 mice. In addition, the 73.STOP mutant exhibited a severe defect in the establishment of latency in the spleen of C57BL/6 mice. Increasing the inoculum of 73.STOP virus partially overcame the acute replication defected observed in the lungs at day 4 postinfection but did not ameliorate the severe defect in the establishment of splenic latency. Thus, consistent with its proposed role in replication of the latent viral episome, LANA appears to be a critical determinant in the establishment of gammaHV68 latency in the spleen post-intranasal infection.

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen prolongs the life span of primary human umbilical vein endothelial cells.

Tumor spindle cells in all clinical types of Kaposi's sarcoma (KS) are infected with Kaposi's sarcoma-associated herpesvirus (KSHV). Although KSHV contains more than 80 genes, only a few are expressed in tumor spindle cells, including latency-associated nuclear antigen (LANA) and k-cyclin (kCYC). To assess the oncogenic potential of LANA and kCYC, primary human umbilical vein endothelial cells (HUVEC) and murine NIH 3T3 cells were stably transduced by using recombinant retroviruses expressing these genes or the known viral oncogene simian virus 40 large T antigen (LTAg). Interestingly, LANA-transduced HUVEC proliferated faster and demonstrated a greatly prolonged life span (mean +/- standard deviation, 38.3 +/- 11.0 passages) than untransduced cells and vector-transduced cells (<20 passages). By contrast, kCYC-transduced HUVEC did not proliferate faster or live longer than control cells. LANA- and kCYC-transduced HUVEC, but not LTAg-transduced HUVEC, retained the ability to form normal vessel-like structures in an in vitro model of angiogenesis. In cellular assays of transformation, LANA- and kCYC-transduced NIH 3T3 cells demonstrated minimal or no anchorage-independent growth in soft agar and no tumorigenicity when injected into nude mice, unlike LTAg-transduced NIH 3T3 cells. Lastly, gene expression profiling revealed down-regulation, or silencing, of a number of genes within LANA-transduced HUVEC. Taken together, these results suggest that KSHV LANA is capable of inducing prolonged life span, but not transformation, in primary human cells. These findings may explain why LANA-expressing spindle cells proliferate within KS tumors, yet most often do not demonstrate biologic characteristics of transformation or true malignant conversion.

The Kaposi sarcoma–associated herpesvirus (KSHV) induces cellular interleukin 6 expression: role of the KSHV latency-associated nuclear antigen and the AP1 response element

AbstractCellular interleukin 6 (IL-6) is an important growth factor for Kaposi sarcoma– associated herpesvirus (KSHV)–associated neoplasms, which include human immunodeficiency virus (HIV)–related and -unrelated cases of Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). Increased IL-6 levels are found in tissues affected with these diseases, and KSHV exists in a latent state in the majority of virally infected cells. In addition, acute infection with KSHV up-regulates IL-6 expression in endothelial cells. Thus, the hypothesis was considered that a latent KSHV gene product up-regulates IL-6 expression. To evaluate this hypothesis, the KSHV latency-associated nuclear antigen (LANA) was expressed in human embryonal kidney 293 cells and a bone marrow stromal cell line. LANA up-regulates IL-6 expression by inducing transcription from the IL-6 promoter, and the AP1 response element within the IL-6 promoter is necessary for and mediates IL-6 up-regulation by LANA. Thus, LANA may play a key pathophysiologic role in KSHV-associated neoplasms by functioning to up-regulate expression of IL-6.

The KSHV latency-associated nuclear antigen: a multifunctional protein.

Kaposi's sarcoma(KS)-associated herpes virus (KSHV) or human herpesvirus 8 (HHV-8) is highly associated with KS, primary effusion lymphoma (PEL), and multicentric Castleman's disease, an aggressive lymphoproliferative disorder (1-3). Most tumor cells are latently infected with KSHV in which a small subset of viral genes are expressed (4-6). Of these latently expressed genes, the latency-associated nuclear antigen (LANA1, LNA, or LNA1) is the only protein consistently shown to be highly expressed by in situ hybridization and immunohistochemistry (7-10). In the past few years multiple functions have been demonstrated for LANA1. Here we review LANA1's roles in KSHV infection. Topics discussed include LANA1's roles in episome persistence, regulation of transcription and interaction with cellular proteins.