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.     

Histone modification pattern of the T-cellular Herpesvirus saimiri genome in latency

Herpesvirus saimiri (HVS) subgroup C strains are able to growth transform human T lymphocytes in vitro. The stably persisting and nonintegrating HVS episome represents an optimal prerequisite for the investigation of the epigenetic state of latent herpesvirus genomes in vitro. Quantitative chromatin immunoprecipitation experiments using seven different histone acetylation- or methylation-specific antibodies revealed repressive marks at four lytic gene promoters and a variable pattern at the weakly transcribed LANA/orf73 promoter. The constitutive stpC/tip promoter regulating the viral oncoproteins and, more interestingly, the noncoding repetitive H-DNA elements flanking the coding region, showed a permissive chromatin structure. This study provides an appropriate model for the analysis of epigenetic herpesvirus genome modifications and their dynamics in T cells.     

Analysis of viral cis elements conferring Kaposi's sarcoma-associated herpesvirus episome partitioning and maintenance

Maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) episomes in latently infected cells is dependent on the latency-associated nuclear antigen (LANA). LANA binds to the viral terminal repeats (TR), leading to recruitment of cellular origin recognition complex proteins. Additionally, LANA tethers episomes to chromosomes via interactions with histones H2A and H2B (A. J. Barbera et al., Science 311:856-861, 2006). Despite these molecular details, less is known about how episomes are established after de novo infection. To address this, we measured short-term retention rates of green fluorescent protein-expressing replicons in proliferating lymphoid cells. In the absence of antibiotic selection, LANA significantly reduced the loss rate of TR-containing replicons. Additionally, we found that LANA can support long-term stability of KSHV replicons for more than 2 months under nonselective conditions. Analysis of cis elements within TR that confer episome replication and partitioning revealed that these activities can occur independently, and furthermore, both events contribute to episome stability. We found that replication-deficient plasmids containing LANA binding sites (LBS1/2) exhibited measurable retention rates in the presence of LANA. To confirm these observations, we uncoupled KSHV replication and partitioning by constructing hybrid origins containing the Epstein-Barr virus (EBV) dyad symmetry for plasmid replication and KSHV LBS1/2. We demonstrate that multiple LBS1/2 function in a manner analogous to that of the EBV family of repeats by forming an array of LANA binding sites for partitioning of KSHV genomes. Our data suggest that the efficiency with which KSHV establishes latency is dependent on multiple LANA activities, which stabilize viral genomes early after de novo infection.     

ORF73 of herpesvirus Saimiri strain C488 tethers the viral genome to metaphase chromosomes and binds to cis-acting DNA sequences in the terminal repeats

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), a human oncogenic gamma-2-herpesvirus, transforms human endothelial cells and establishes latent infection at a low efficiency in vitro. During latent infection, only a limited number of genes are expressed, and the circularized viral genome is maintained as a multicopy episome. Latency-associated nuclear antigen (LANA), exclusively expressed during latency, has been shown to have a multifunctional role in KS pathogenesis. LANA tethers the viral episome to the host chromosome, thus ensuring efficient persistence of the viral genome during successive rounds of cell division. Besides episome maintenance, LANA modulates the expression of genes of various cellular and viral pathways, including those of retinoblastoma protein and p53. Herpesvirus saimiri (HVS), another gamma-2-herpesvirus, primarily infects New World primates. Orf73, encoding the nuclear antigen of HVS, is the positional homolog of the LANA gene, and the ORF73 protein has some sequence homology to KSHV LANA. However, the function of ORF73 of HVS has not been thoroughly investigated. In this report, we show that HVS ORF73 may be important for episome persistence and colocalizes with the HVS genomic DNA on metaphase chromosomes. Furthermore, HVS terminal repeats (TRs) contain a cis-acting sequence similar to that in KSHV TRs, suggesting that the LANA binding sequence is conserved between these two viruses. This cis-acting element is sufficient to bind HVS ORF73 from strains C488 and A11, and plasmids containing the HVS C488 TR element are maintained and replicate in HVS C488 ORF73-expressing cells.     

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 1 mediates episome persistence through cis-acting terminal repeat (TR) sequence and specifically binds TR DNA

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) (also known as human herpesvirus 8) latently infects KS tumors, primary effusion lymphomas (PELs), and PEL cell lines. In latently infected cells, KSHV DNA is maintained as circularized, extrachromosomal episomes. To persist in proliferating cells, KSHV episomes must replicate and efficiently segregate to progeny nuclei. In uninfected B-lymphoblastoid cells, KSHV latency-associated nuclear antigen (LANA1) is necessary and sufficient for persistence of artificial episomes containing specific KSHV DNA. In previous work, the cis-acting sequence required for episome persistence contained KSHV terminal-repeat (TR) DNA and unique KSHV sequence. We now show that cis-acting KSHV TR DNA is necessary and sufficient for LANA1-mediated episome persistence. Furthermore, LANA1 binds TR DNA in mobility shift assays and a 20-nucleotide LANA1 binding sequence has been identified. Since LANA1 colocalizes with KSHV episomes along metaphase chromosomes, these results are consistent with a model in which LANA1 may bridge TR DNA to chromosomes during mitosis to efficiently segregate KSHV episomes to progeny nuclei.     

Long-term-infected telomerase-immortalized endothelial cells: a model for Kaposi's sarcoma-associated herpesvirus latency in vitro and in vivo

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. Most KS tumor cells are latently infected with KSHV and are of endothelial origin. While PEL-derived cell lines maintain KSHV indefinitely, all KS tumor-derived cells to date have lost viral genomes upon ex vivo cultivation. To study KSHV latency and tumorigenesis in endothelial cells, we generated telomerase-immortalized human umbilical vein endothelial (TIVE) cells. TIVE cells express all KSHV latent genes 48 h postinfection, and productive lytic replication could be induced by RTA/Orf50. Similar to prior models, infected cultures gradually lost viral episomes. However, we also obtained, for the first time, two endothelial cell lines in which KSHV episomes were maintained indefinitely in the absence of selection. Long-term KSHV maintenance correlated with loss of reactivation in response to RTA/Orf50 and complete oncogenic transformation. Long-term-infected TIVE cells (LTC) grew in soft agar and proliferated under reduced-serum conditions. LTC, but not parental TIVE cells, formed tumors in nude mice. These tumors expressed high levels of the latency-associated nuclear antigen (LANA) and expressed lymphatic endothelial specific antigens as found in KS (LYVE-1). Furthermore, host genes, like those encoding interleukin 6, vascular endothelial growth factor, and basic fibroblast growth factor, known to be highly expressed in KS lesions were also induced in LTC-derived tumors. KSHV-infected LTCs represent the first xenograft model for KS and should be of use to study KS pathogenesis and for the validation of anti-KS drug candidates.     

Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures

Angiogenic Kaposi's sarcoma (KS) skin lesions found in both AIDS and non-AIDS patients are universally associated with infection by the presumed causative agent, known as KS-associated herpesvirus (KSHV) or human herpesvirus 8. KSHV genomes expressing latent state virus-encoded mRNAs and the LANA1 (latent nuclear antigen 1) protein are consistently present in spindle-like tumor cells that are thought to be of endothelial cell origin. Although the KSHV lytic cycle can be induced in rare latently infected primary effusion lymphoma (PEL) cell lines, the ability to transmit or assay infectious KSHV has so far eluded investigators. Here, we demonstrate that infection with supernatant virions derived from three different tetradecanoyl phorbol acetate-induced PEL cell lines can induce cultured primary human dermal microvascular endothelial cells (DMVEC) to form colonies of proliferating latently infected spindle-shaped cells, all of which express the KSHV-encoded LANA1 protein. Although their initial infectivity varied widely (JSC1 > > BC3 > BCP1), virions from all three cell lines produced distinctive spindle cell colonies and plaques without affecting the contact-inhibited cobblestone-like phenotype of adjacent uninfected DMVEC. Each infected culture could also be expanded into a completely spindloid persistently infected culture displaying aggregated swirls of spindle cells resembling those in KS lesions. Formation of new colonies and plaques was inhibited in the presence of phosphonoacetic acid or gangciclovir, but these antiherpesvirus agents had little effect on the propagation of already latently infected spindloid cultures. In persistently infected secondary cultures, patches of up to 10% of the spindloid cells constitutively expressed several early viral lytic cycle proteins, and 1 to 2% of the cells also formed typical herpesvirus DNA replication compartments, displayed cytopathic rounding effects, and expressed late viral antigens. We conclude that de novo KSHV infection induces a spindle cell conversion phenotype in primary DMVEC cultures that is directly associated with latent state expression of the LANA1 protein. However, these cultures also spontaneously reactivate to produce an unusual combination of both latent and productive but slow lytic cycle infection. The formation of spindle cell colonies and plaques in DMVEC cultures provides for the first time a quantitative assay for directly measuring the infectivity of KSHV virion preparations.     

Kaposi's sarcoma-associated herpesvirus-encoded latency-associated nuclear antigen inhibits lytic replication by targeting Rta: a potential mechanism for virus-mediated control of latency

Like other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV, also designated human herpesvirus 8) can establish a latent infection in the infected host. During latency a small number of genes are expressed. One of those genes encodes latency-associated nuclear antigen (LANA), which is constitutively expressed in cells during latent as well as lytic infection. LANA has previously been shown to be important for the establishment of latent episome maintenance through tethering of the viral genome to the host chromosomes. Under specific conditions, KSHV can undergo lytic replication, with the production of viral progeny. The immediate-early Rta, encoded by open reading frame 50 of KSHV, has been shown to play a critical role in switching from viral latent replication to lytic replication. Overexpression of Rta from a heterologous promoter is sufficient for driving KSHV lytic replication and the production of viral progeny. In the present study, we show that LANA down-modulates Rta's promoter activity in transient reporter assays, thus repressing Rta-mediated transactivation. This results in a decrease in the production of KSHV progeny virions. We also found that LANA interacts physically with Rta both in vivo and in vitro. Taken together, our results demonstrate that LANA can inhibit viral lytic replication by inhibiting expression as well as antagonizing the function of Rta. This suggests that LANA may play a critical role in maintaining latency by controlling the switch between viral latency and lytic replication.     

Inhibition of polo-like kinase 1 (PLK1) facilitates reactivation of gamma-herpesviruses and their elimination

Both Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) establish the persistent, life-long infection primarily at the latent status, and associate with certain types of tumors, such as B cell lymphomas, especially in immuno-compromised individuals including people living with HIV (PLWH). Lytic reactivation of these viruses can be employed to kill tumor cells harboring latently infected viral episomes through the viral cytopathic effects and the subsequent antiviral immune responses. In this study, we identified that polo-like kinase 1 (PLK1) is induced by KSHV de novo infection as well as lytic switch from KSHV latency. We further demonstrated that PLK1 depletion or inhibition facilitates KSHV reactivation and promotes cell death of KSHV-infected lymphoma cells. Mechanistically, PLK1 regulates Myc that is critical to both maintenance of KSHV latency and support of cell survival, and preferentially affects the level of H3K27me3 inactive mark both globally and at certain loci of KSHV viral episomes. Furthremore, we recognized that PLK1 inhibition synergizes with STAT3 inhibition to efficiently induce KSHV reactivation. We also confirmed that PLK1 depletion or inhibition yields the similar effect on EBV lytic reactivation and cell death of EBV-infected lymphoma cells. Lastly, we noticed that PLK1 in B cells is elevated in the context of HIV infection and caused by HIV Nef protein to favor KSHV/EBV latency.     

The size and conformation of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) DNA in infected cells and virions.

The genome of a novel human herpesvirus has been detected in specimens of Kaposi's sarcoma (KS) and in several AIDS-related lymphoproliferative disorders. Here we examine the size and genomic conformation of the DNA of this virus (known as KS-associated herpesvirus or human herpesvirus 8) in latently and lytically infected cells and in virions. Pulsed-field gel electrophoresis of viral DNA shows that the viral genome is similar in size to those of other gammaherpesviruses (160 to 170 kb). As with Epstein-Barr virus, KS-associated herpesvirus DNA is stably maintained in latently infected B cells as episomal monomer circles and induction from latency is associated with the selective accumulation of linear genomic forms.     

Herpesvirus saimiri episomal persistence is maintained via interaction between open reading frame 73 and the cellular chromosome-associated protein MeCP2

Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus, which naturally infects the squirrel monkey Saimiri sciureus, causing an asymptomatic but persistent infection. The latent phase of gamma-2 herpesviruses is characterized by their ability to persist in a dividing cell population while expressing a limited subset of latency-associated genes. In HVS only three genes, open reading frame 71 (ORF71), ORF72, and ORF73, are expressed from a polycistronic mRNA. ORF73 has been shown to be the only gene essential for HVS episomal maintenance and can therefore be functionally compared to the human gammaherpesvirus latency-associated proteins, EBNA-1 and Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA). HVS ORF73 is the positional homologue of KSHV LANA and, although it shares limited sequence homology, has significant structural and functional similarities. Investigation of KSHV LANA has demonstrated that it is able to mediate KSHV episomal persistence by tethering the KSHV episome to host mitotic chromosomes via interactions with cellular chromosome-associated proteins. These include associations with core and linker histones, several bromodomain proteins, and the chromosome-associated proteins methyl CpG binding protein 2 (MeCP2) and DEK. Here we show that HVS ORF73 associates with MeCP2 via a 72-amino-acid domain within the ORF73 C terminus. Furthermore, we have assessed the functional significance of this interaction, using a variety of techniques including small hairpin RNA knockdown, and show that association between ORF73 and MeCP2 is essential for HVS chromosomal attachment and episomal persistence.