Latency, Integration, and Reactivation of Human Herpesvirus-6.

Human herpesvirus-6A (HHV-6A) and human herpesvirus-6B (HHV-6B) are two closely related viruses that infect T-cells. Both HHV-6A and HHV-6B possess telomere-like repeats at the terminal regions of their genomes that facilitate latency by integration into the host telomeres, rather than by episome formation. In about 1% of the human population, human herpes virus-6 (HHV-6) integration into germline cells allows the viral genome to be passed down from one generation to the other; this condition is called inherited chromosomally integrated HHV-6 (iciHHV-6). This review will cover the history of HHV-6 and recent works that define the biological differences between HHV-6A and HHV-6B. Additionally, HHV-6 integration and inheritance, the capacity for reactivation and superinfection of iciHHV-6 individuals with a second strain of HHV-6, and the role of hypomethylation of human chromosomes during integration are discussed. Overall, the data suggest that integration of HHV-6 in telomeres represent a unique mechanism of viral latency and offers a novel tool to study not only HHV-6 pathogenesis, but also telomere biology. Paradoxically, the integrated viral genome is often defective especially as seen in iciHHV-6 harboring individuals. Finally, gaps in the field of HHV-6 research are presented and future studies are proposed.

Persistent human herpesvirus-6 infection in patients with an inherited form of the virus.

Human herpesvirus-6 (HHV-6)A and 6B are ubiquitous betaherpesviruses viruses with lymphotropic and neurotropic potential. As reported earlier, these viruses establish latency by integration into the telomeres of host chromosomes. Chromosomally integrated HHV-6 (CIHHV-6) can be transmitted vertically from parent to child. Some CIHHV-6 patients are suffering from neurological symptoms, while others remain asymptomatic. Four patients with CIHHV-6 and CNS dysfunction were treated with valganciclovir or foscarnet. HHV-6 replication was detected by reverse transcriptase polymerase chain reaction amplification of a late envelope glycoprotein. In this study we also compared the inherited and persistent HHV-6 viruses by DNA sequencing. The prevalence of CIHHV-6 in this cohort of adult patients from the USA suffering from a wide range of neurological symptoms including long-term fatigue were found significantly greater than the reported 0.8% in the general population. Long-term antiviral therapy inhibited HHV-6 replication as documented by loss of viral mRNA production. Sequence comparison of the mRNA and the inherited viral genome revealed that the transcript is produced by an exogenous virus. In conclusion, the data presented here document that some individuals with CIHHV-6 are infected persistently with exogenous HHV-6 strains that lead to a wide range of neurological symptoms; the proposed name for this condition is inherited herpesvirus 6 syndrome or IHS.

Mapping the telomere integrated genome of human herpesvirus 6A and 6B.

Human herpesvirus 6B (HHV-6B) is the causative agent of roseola infantum. HHV-6A and 6B can reactivate in immunosuppressed individuals and are linked with severe inflammatory response, organ rejection and central nervous system diseases. About 0.85% of the US and UK population carries an integrated HHV-6 genome in all nucleated cells through germline transmission. We have previously reported that the HHV-6A genome integrated in telomeres of patients suffering from neurological dysfunction and also in telomeres of tissue culture cells. We now report that HHV-6B also integrates in telomeres during latency. Detailed mapping of the integrated viral genomes demonstrates that a single HHV-6 genome integrates and telomere repeats join the left end of the integrated viral genome. When HEK-293 cells carrying integrated HHV-6A were exposed to the histone deacetylase inhibitor Trichostatin A, circularization and/or formation of concatamers were detected and this assay could be used to distinguish between lytic replication and latency.

Antiviral therapy of two patients with chromosomally-integrated human herpesvirus-6A presenting with cognitive dysfunction.

BACKGROUND: Human herpesvirus 6 (HHV-6) is a neurotropic virus implicated in central nervous system (CNS) dysfunction, multiple sclerosis, seizures and encephalitis. Inherited or "chromosomally integrated" HHV-6 (CIHHV-6) is a condition characterized by high DNA loads and germ line transmission of HHV-6 genomes, which are integrated into the telomere. OBJECTIVES: We previously reported that integrated HHV-6 can be reactivated by trichostatin A in vitro. Therefore, we hypothesized that a broad array of neurological symptoms of CIHHV-6 patients may respond to antiviral drug treatment. STUDY DESIGN: The patients have been treated with antiviral drugs and monitored for viral load, late mRNA, and clinical improvement. RESULTS: Antiviral therapy of two CIHHV patients resulted in successful clinical resolution. However, both patients relapsed on multiple occasions within 4-6 months of cessation of antiviral therapy. CONCLUSIONS: Successful antiviral drug treatment suggests that clinical symptoms of these patients were due to symptomatic reactivation of CIHHV-6. Alternatively, some CIHHV-6 patients may have a reduced resistance to community-acquired HHV-6 strains due to tolerance leading to persistent infections.

Chromosomally integrated human herpesvirus 6: questions and answers.

Chromosomally integrated human herpesvirus 6 (ciHHV-6) is a condition in which the complete HHV-6 genome is integrated into the host germ line genome and is vertically transmitted in a Mendelian manner. The condition is found in less than 1% of controls in the USA and UK, but has been found at a somewhat higher prevalence in transplant recipients and other patient populations in several small studies. HHV-6 levels in whole blood that exceed 5.5 log10 copies/ml are strongly suggestive of ciHHV-6. Monitoring DNA load in plasma and serum is unreliable, both for identifying and for monitoring subjects with ciHHV-6 due to cell lysis and release of cellular DNA. High HHV-6 DNA loads associated with ciHHV-6 can lead to erroneous diagnosis of active infection. Transplant recipients with ciHHV-6 may be at increased risk for bacterial infection and graft rejection. ciHHV-6 can be induced to a state of active viral replication in vitro. It is not known whether ciHHV-6 individuals are put at clinical risk by the use of drugs that have been associated with HHV-6 reactivation in vivo or in vitro. Nonetheless, we urge careful observation when use of such drugs is indicated in individuals known to have ciHHV-6. Little is known about whether individuals with ciHHV-6 develop immune tolerance for viral proteins. Further research is needed to determine the role of ciHHV-6 in disease.

The molecular biology of human herpesvirus-6 latency and telomere integration.

The genomes of herpesviruses establish latency as a circular episome. However, Human herpesvirus-6 (HHV-6) has been shown to specifically integrate into the telomeres of chromosomes during latency and vertically transmit through the germ-line. This review will focus on the telomere integration of HHV-6, the potential viral and cellular genes that mediate integration, and the clinical impact on the host.

The latent human herpesvirus-6A genome specifically integrates in telomeres of human chromosomes in vivo and in vitro.

Previous research has suggested that human herpesvirus-6 (HHV-6) may integrate into host cell chromosomes and be vertically transmitted in the germ line, but the evidence--primarily fluorescence in situ hybridization (FISH)--is indirect. We sought, first, to definitively test these two hypotheses. Peripheral blood mononuclear cells (PBMCs) were isolated from families in which several members, including at least one parent and child, had unusually high copy numbers of HHV-6 DNA per milliliter of blood. FISH confirmed that HHV-6 DNA colocalized with telomeric regions of one allele on chromosomes 17p13.3, 18q23, and 22q13.3, and that the integration site was identical among members of the same family. Integration of the HHV-6 genome into TTAGGG telomere repeats was confirmed by additional methods and sequencing of the integration site. Partial sequencing of the viral genome identified the same integrated HHV-6A strain within members of families, confirming vertical transmission of the viral genome. We next asked whether HHV-6A infection of naïve cell lines could lead to integration. Following infection of naïve Jjhan and HEK-293 cell lines by HHV-6, the virus integrated into telomeres. Reactivation of integrated HHV-6A virus from individuals' PBMCs as well as cell lines was successfully accomplished by compounds known to induce latent herpesvirus replication. Finally, no circular episomal forms were detected even by PCR. Taken together, the data suggest that HHV-6 is unique among human herpesviruses: it specifically and efficiently integrates into telomeres of chromosomes during latency rather than forming episomes, and the integrated viral genome is capable of producing virions.

Epigenetic regulation of Kaposi's sarcoma-associated herpesvirus replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma and B-lymphocyte disorders, primary effusion lymphoma (PEL) and Multicentric Castleman's Disease (MCD). KSHV usually exists in a latent form in which the viral genome is circularized into an extrachormosomal episome. However, induction of lytic replication by environmental stimuli or chemical agents is important for the spread of KSHV. The switch between latency and lytic replication is regulated by epigenetic factors. Hypomethylation of the promoter of replication and transcription activator (RTA), which is essential for the lytic switch, leads to KSHV reactivation. Histone acetylation induces KSHV replication by influencing protein-protein-associations and transcription factor binding. Histone modifications also determine chromatin structure and nucleosome positioning, which are important for KSHV DNA replication during latency. The association of KSHV proteins with chromatin remodeling complexes promotes the open chromatin structure needed for transcription factor binding and DNA replication. Additionally, post-translational modification of KSHV proteins is important for the regulation of RTA activity and KSHV replication. KSHV may also cause epigenetic modification of the host genome, contributing to promoter hypermethylation of tumor suppressor genes in KSHV-associated neoplasias.

Identification of mitochondrial genome concatemers in AIDS-associated lymphomas and lymphoid cell lines.

Since most oncogenic viruses persist as extrachromosomal covalently closed circular DNA (cccDNA) in tumor cells, we developed an assay to visualize and identify cccDNA in primary lymphomas. We identified concatemers of the mitochondrial genome in all samples analyzed, but not in normal lymphocytes. One AIDS-associated lymphoma (EL) was further studied in detail as its mitochondrial genome consisted of tandem head-to-tail duplications. Insertion of C-residues was noted near the origin of replication of EL mtDNA. EL cells responded weakly to Fas-apoptotic stimulus, displayed reduced mitochondrial activity and mass, and produced higher levels of reactive oxygen intermediates. Screening of several AIDS-associated lymphomas and established lymphoid cell lines also revealed the presence of mitochondrial genome concatemers consisting of interlinked monomer molecules. Taken together, our results suggest that formation of mtDNA concatemers is associated with oncogenic transformation in lymphoid cells.

Formation of Mitochondrial Genome Concatemers as an Alternative Mechanism Promoting Oncogenic Transformation of Lymphoid Cells.

We have previously shown that AIDS-associated lymphomas and lymphoma cell lines contain mitochondrial genome concatemers not present in normal T-lymphocytes. Since cellular homeostasis and energy production rely heavily on mitochondrial DNA (mtDNA) stability, mutations in the mtDNA have long been linked to the development of various types of cancers. In most of the cases, however, neoplastically transformed cells harbor non-mutated mtDNA. Herein, we propose an alternative mechanism that shows how the formation of mitochondrial genome concatemers may promote oncogenic transformation of normal lymphoid progenitor cells when no mtDNA mutations or chromosomal aberrations are present. We detected high reactive oxygen species (ROS) levels in the lymphoma samples tested despite no identification of putative mutations in the coding mtDNA. We propose that the formation of atypical mtDNA configurations (i.e. dimers and concatemers) interferes with normal mitochondrial function. Unstable mitochondria lead to abnormal assembly and dysfunction of the oxidative phosphorylation (OXPHOS) complexes, eventually leading to oxidative stress from elevated production of intracellular ROS. ROS have been reported to activate transcription factors associated with cellular proliferation and apoptosis inhibition. Therefore, we hypothesize that formation of mitochondrial genome concatemers can augment endogenous ROS levels capable of promoting oncogenic transformation of normal lymphoid progenitor cells.

Delta-9 tetrahydrocannabinol (THC) inhibits lytic replication of gamma oncogenic herpesviruses in vitro.

BACKGROUND: The major psychoactive cannabinoid compound of marijuana, delta-9 tetrahydrocannabinol (THC), has been shown to modulate immune responses and lymphocyte function. After primary infection the viral DNA genome of gamma herpesviruses persists in lymphoid cell nuclei in a latent episomal circular form. In response to extracellular signals, the latent virus can be activated, which leads to production of infectious virus progeny. Therefore, we evaluated the potential effects of THC on gamma herpesvirus replication. METHODS: Tissue cultures infected with various gamma herpesviruses were cultured in the presence of increasing concentrations of THC and the amount of viral DNA or infectious virus yield was compared to those of control cultures. The effect of THC on Kaposi's Sarcoma Associated Herpesvirus (KSHV) and Epstein-Barr virus (EBV) replication was measured by the Gardella method and replication of herpesvirus saimiri (HVS) of monkeys, murine gamma herpesvirus 68 (MHV 68), and herpes simplex type 1 (HSV-1) was measured by yield reduction assays. Inhibition of the immediate early ORF 50 gene promoter activity was measured by the dual luciferase method. RESULTS: Micromolar concentrations of THC inhibit KSHV and EBV reactivation in virus infected/immortalized B cells. THC also strongly inhibits lytic replication of MHV 68 and HVS in vitro. Importantly, concentrations of THC that inhibit virus replication of gamma herpesviruses have no effect on cell growth or HSV-1 replication, indicating selectivity. THC was shown to selectively inhibit the immediate early ORF 50 gene promoter of KSHV and MHV 68. CONCLUSIONS: THC specifically targets viral and/or cellular mechanisms required for replication and possibly shared by these gamma herpesviruses, and the endocannabinoid system is possibly involved in regulating gamma herpesvirus latency and lytic replication. The immediate early gene ORF 50 promoter activity was specifically inhibited by THC. These studies may also provide the foundation for the development of antiviral strategies utilizing non-psychoactive derivatives of THC.

Kaposi's sarcoma-associated herpesvirus-encoded viral interleukin-6 is secreted and modified differently than human interleukin-6: evidence for a unique autocrine signaling mechanism.

Viral interleukin-6 (vIL-6) is a homolog of cellular IL-6 that is encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) genome. vIL-6 binds to the IL-6 signal transducer gp130 without the cooperation of the IL-6 high affinity receptor to induce STAT3 DNA binding and cell proliferation. Although vIL-6 is believed to be important in the pathogenesis of KSHV-induced diseases, its secretion and post-translational modifications have not previously been characterized. Pulse-chase analysis revealed that the half-time of vIL-6 secretion is approximately 8-fold longer than that of human IL-6. Yet, the vIL-6 signal sequence targets human IL-6 secretion to nearly wild-type levels. Surprisingly, vIL-6 was not secreted from a cell line that does not express gp130 but expression of human gp130 in these cells enabled the secretion of vIL-6. Consistent with this observation, complete maturation of gp130 N-glycans is inhibited by vIL-6 coexpression, suggesting that the binding of the receptor to vIL-6 occurs intracellularly in early or pre-Golgi compartments. Furthermore, a vIL-6 mutant containing an endoplasmic reticulum retention signal is not secreted but does still induce receptor activation and signaling. Secreted vIL-6 is completely glycosylated at both possible N-glycosylaton sites and contains a large proportion of immature high-mannose glycans that is not typical of cytokines. These findings suggest that vIL-6 may induce gp130 signaling by an exclusively autocrine mechanism that relies on intracellular binding to its receptor. During KSHV infection, vIL-6 may only induce signaling in KSHV-infected cells to benefit the viral life cycle and promote oncogenic transformation.

The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus interacts preferentially with the terminal repeats of the genome in vivo and this complex is sufficient for episomal DNA replication.

The genome of Kaposi's sarcoma-associated herpesvirus (KSHV) persists in latently infected cells as a circular episome. The latency-associated nuclear antigen (LANA) has been shown to tether viral DNA fragments to chromosomes and is proposed to maintain the KSHV genome. In order to identify the in vivo-binding sites for LANA on the whole KSHV genome and to analyse the function of this protein-DNA interaction, different in vivo systems have been developed. Chromatin immunoprecipitation experiments using three different cell lines latently infected with KSHV demonstrated that LANA binds preferentially and directly to the terminal repeats (TRs) but not to other regions of the viral chromosome in vivo. In contrast, in vitro LANA-DNA binding was much less specific. To identify autonomously replicating sequences within the KSHV genome, BCBL-1 cells were transfected with cosmids representing the entire genome. Cosmid Z2, consisting of the right end of the unique region and TRs, persisted as an episome in short-term assays. Long term, stable episome replication was observed with constructs derived from Z2 containing TRs only. LANA expression constructs containing a variable number of TRs replicated stably as episomes in uninfected cells. A 424 bp subfragment of the 801 bp TR could mediate episome replication. These studies show that LANA is a trans-acting protein that binds preferentially to TRs in vivo and these two elements are sufficient for episome replication. These results also suggest that the LANA expression plasmids reported here could be utilized as episomal vectors in a manner similar to Epstein-Barr virus-based vectors.

The terminal repeats and latency-associated nuclear antigen of herpesvirus saimiri are essential for episomal persistence of the viral genome.

The simian herpesvirus saimiri (HVS) induces malignant T cell lymphomas and is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8). Both belong to the gamma-2 herpesvirus subgroup. The viral genome of HVS consists of a unique region (L-DNA) that contains all of the viral genes flanked by non-coding terminal repeats (H-DNA). Here we describe the cloning of a 113 kb restriction fragment containing the L-DNA of an oncogenic HVS strain in an F' replicon-based E. coli vector. Cloned DNA was infectious and the ends of the progeny viral genome consisted of amplified tandem alternating repeats of vector and a single H-DNA unit. T cells infected with these viruses contained the linear DNA typically found a few weeks after infection, but were unable to form episomal circular viral DNA, which is the latent form of the viral genome. Recombinant viruses with reconstructed H-DNA were generated and T cells infected with these rescued viruses contained high copy numbers of episomal DNA. Plasmids expressing the latency-associated nuclear antigen (LANA) and containing various numbers of H-DNA repeats stably replicated as episomes, but constructs containing three repeat units produced the highest copy numbers. These data show that intact and multiple terminal repeats are essential components for episomal replication in latently infected T cells. Moreover, LANA and terminal repeats are sufficient for stable plasmid persistence. Cloned HVS can also be utilized for mutagenesis of HVS and for the expression of foreign genes through efficient manipulation of plasmids in E. coli.

The herpesvirus saimiri tip484 and tip488 proteins both stimulate lck tyrosine protein kinase activity in vivo and in vitro.

Herpesvirus saimiri (HVS) of subgroup C efficiently induces leukemia in New World primates and transforms human lymphocytes. The viral tyrosine kinase interacting protein (Tip) binds to the tyrosine protein kinase Lck and is essential for transformation. Understanding how Tip modulates Lck activity is important for elucidating the mechanism of herpesvirus saimiri leukemogenesis. However, there are reports suggesting that whereas the Tip protein of HVS strain 484 stimulates the activity of Lck, the Tip protein of HVS strain 488 inhibits Lck. To determine whether these two divergent Tip proteins have opposite effects on Lck activity, we compared them in parallel. We found that both Tip proteins stimulated Lck kinase activity in vivo and in vitro and that both stimulated NF-AT- and STAT3-dependent transcription in T cells. Our data support the model that HVS infection increases the activity of Lck through the action of Tip.

Genetic requirements for the episomal maintenance of oncogenic herpesvirus genomes.

Herpesviruses are large double-stranded DNA viruses that are characterized by lifelong latency. Epstein-Barr virus (EBV), the recently discovered Kaposi's sarcoma associated herpesvirus (KSHV), also referred to as human herpesvirus-8 (HHV-8), and the simian Herpesvirus saimiri (HVS) are associated with malignant lymphoproliferative diseases. These viruses establish latent infection in lymphoid cells. During latency only a few viral genes are expressed and the viral genome persists as a multicopy circular episome. The episome contains repetitive sequences that serve as multiple cooperative binding sites for the viral DNA binding proteins Epstein-Barr virus nuclear antigen 1 (EBNA-1) of EBV and latency-associated nuclear antigen (LANA1) of KSHV and HVS, which are expressed during latency. The oligomerized proteins associate with the viral genome and tether it to host chromosomes, assuring continual lifelong persistence of the virus.