Reactivation of Chromosomally Integrated Human Herpesvirus-6 by Telomeric Circle Formation

More than 95% of the human population is infected with human herpesvirus-6 (HHV-6) during early childhood and maintains latent HHV-6 genomes either in an extra-chromosomal form or as a chromosomally integrated HHV-6 (ciHHV-6). In addition, approximately 1% of humans are born with an inheritable form of ciHHV-6 integrated into the telomeres of chromosomes. Immunosuppression and stress conditions can reactivate latent HHV-6 replication, which is associated with clinical complications and even death. We have previously shown that Chlamydia trachomatis infection reactivates ciHHV-6 and induces the formation of extra-chromosomal viral DNA in ciHHV-6 cells. Here, we propose a model and provide experimental evidence for the mechanism of ciHHV-6 reactivation. Infection with Chlamydia induced a transient shortening of telomeric ends, which subsequently led to increased telomeric circle (t-circle) formation and incomplete reconstitution of circular viral genomes containing single viral direct repeat (DR). Correspondingly, short t-circles containing parts of the HHV-6 DR were detected in cells from individuals with genetically inherited ciHHV-6. Furthermore, telomere shortening induced in the absence of Chlamydia infection also caused circularization of ciHHV-6, supporting a t-circle based mechanism for ciHHV-6 reactivation.     

Human Herpesvirus 6A Infection and Immunopathogenesis in Humanized Rag2?/?γc?/? Mice

Although serious human diseases have been correlated with human herpesvirus 6A (HHV-6A) and HHV-6B, the lack of animal models has prevented studies which would more definitively link these viral infections to disease. HHV-6A and HHV-6B have recently been classified as two distinct viruses, and in this study we focused specifically on developing an in vivo model for HHV-6A. Here we show that Rag2^−/−γc^−/− mice humanized with cord blood-derived human hematopoietic stem cells produce human T cells that express the major HHV-6A receptor, CD46. Both cell-associated and cell-free viral transmission of HHV-6A into the peritoneal cavity resulted in detectable viral DNA in at least one of the samples (blood, bone marrow, etc.) analyzed from nearly all engrafted mice. Organs and cells positive for HHV-6A DNA were the plasma and cellular blood fractions, bone marrow, lymph node, and thymic samples; control mice had undetectable viral DNA. We also noted viral pathogenic effects on certain T cell populations. Specific thymocyte populations, including CD3⁻ CD4⁺ CD8⁻ and CD3⁺ CD4⁻ cells, were significantly modified in humanized mice infected by cell-associated transmission. In addition, we detected significantly increased proportions of CD4⁺ CD8⁺ cells in the blood of animals infected by cell-free transmission. These findings provide additional evidence that HHV-6A may play a role in human immunodeficiencies. These results indicate that humanized mice can be used to study HHV-6A in vivo infection and replication as well as aspects of viral pathogenesis.     

Functional Identification and Analysis of cis-Acting Sequences Which Mediate Genome Cleavage and Packaging in Human Herpesvirus 6

Sequences present at the genomic termini of herpesviruses become linked during lytic-phase replication and provide the substrate for cleavage and packaging of unit length viral genomes. We have previously shown that homologs of the consensus herpesvirus cleavage-packaging signals, pac1 and pac2, are located at the left and right genomic termini of human herpesvirus 6 (HHV-6), respectively. Immediately adjacent to these elements are two distinct arrays of human telomeric repeat sequences (TRS). We now show that the unique sequence element formed at the junction of HHV-6B genome concatemers (pac2-pac1) is necessary and sufficient for virally mediated cleavage of plasmid DNAs containing the HHV-6B lytic-phase origin of DNA replication (oriLyt). The concatemeric junction sequence also allowed for the packaging of these plasmid molecules into intracellular nucleocapsids as well as mature, infectious viral particles. In addition, this element significantly enhanced the replication efficiency of oriLyt-containing plasmids in virally infected cells. Experiments revealed that the concatemeric junction sequence possesses an unusual, S1 nuclease-sensitive conformation (anisomorphic DNA), which might play a role in this apparent enhancement of DNA replication—although additional studies will be required to test this hypothesis. Finally, we also analyzed whether the presence of flanking viral TRS had any effect on the functional activity of the minimal concatemeric junction (pac2-pac1). These experiments revealed that the TRS motifs, either alone or in combination, had no effect on the efficiency of virally mediated DNA replication or DNA cleavage. Taken together, these data show that the cleavage and packaging of HHV-6 DNA are mediated by cis-acting consensus sequences similar to those found in other herpesviruses, and that these sequences also influence the efficiency of HHV-6 DNA replication. Since the adjacent TRS do not influence either viral cleavage and packaging or viral DNA replication, their function remains uncertain.     

Inhibition of p53-Dependent,but Not p53-Independent,Cell Death by U19 Protein from Human Herpesvirus 6B

Infection with human herpesvirus (HHV)-6B alters cell cycle progression and stabilizes tumor suppressor protein p53. In this study, we have analyzed the activity of p53 after stimulation with p53-dependent and -independent DNA damaging agents during HHV-6B infection. Microarray analysis, Western blotting and confocal microscopy demonstrated that HHV-6B-infected cells were resistant to p53-dependent arrest and cell death after γ irradiation in both permissive and non-permissive cell lines. In contrast, HHV-6B-infected cells died normally through p53-independet DNA damage induced by UV radiation. Moreover, we identified a viral protein involved in inhibition of p53 during HHV-6B-infection. The protein product from the U19 ORF was able to inhibit p53-dependent signaling following γ irradiation in a manner similar to that observed during infection. Similar to HHV-6B infection, overexpression of U19 failed to rescue the cells from p53-independent death induced by UV radiation. Hence, infection with HHV-6B specifically blocks DNA damage-induced cell death associated with p53 without inhibiting the p53-independent cell death response. This block in p53 function can in part be ascribed to the activities of the viral U19 protein.     

Human herpesvirus 8 infects and replicates in primary cultures of activated B lymphocytes through DC-SIGN

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. Although latent HHV-8 DNA can be detected in B cells from persons with these cancers, there is little information on the replication of HHV-8 in B cells. Indeed, B cells are relatively resistant to HHV-8 infection in vitro. We have recently shown that DC-SIGN, a C-type lectin first identified on dendritic cells (DC), is an entry receptor for HHV-8 on DC and macrophages. We have also demonstrated previously that B lymphocytes from peripheral blood and tonsils express DC-SIGN and that this expression increases after B-cell activation. Here we show that activated blood and tonsillar B cells can be productively infected with HHV-8, as measured by an increase in viral DNA, the expression of viral lytic and latency proteins, and the production of infectious virus. The infection of B cells with HHV-8 was blocked by the pretreatment of the cells with antibody specific for DC-SIGN or with mannan but not antibody specific for xCT, a cystine/glutamate exchange transporter that has been implicated in HHV-8 fusion to cells. The infection of B cells with HHV-8 resulted in increased expression of DC-SIGN and a decrease in the expression of CD20 and major histocompatibility complex class I. HHV-8 could also infect and replicate in B-cell lines transduced to express full-length DC-SIGN but not in B-cell lines transduced to express DC-SIGN lacking the transmembrane domain, demonstrating that the entry of HHV-8 into B cells is related to DC-SIGN-mediated endocytosis. The role of endocytosis in viral entry into activated B cells was confirmed by blocking HHV-8 infection with endocytic pathway inhibitors. Thus, the expression of DC-SIGN is essential for productive HHV-8 infection of and replication in B cells.     

Imbalanced Oxidative Stress Causes Chlamydial Persistence during Non-Productive Human Herpes Virus Co-Infection

Both human herpes viruses and Chlamydia are highly prevalent in the human population and are detected together in different human disorders. Here, we demonstrate that co-infection with human herpes virus 6 (HHV6) interferes with the developmental cycle of C. trachomatis and induces persistence. Induction of chlamydial persistence by HHV6 is independent of productive virus infection, but requires the interaction and uptake of the virus by the host cell. On the other hand, viral uptake is strongly promoted under co-infection conditions. Host cell glutathione reductase activity was suppressed by HHV6 causing NADPH accumulation, decreased formation of reduced glutathione and increased oxidative stress. Prevention of oxidative stress restored infectivity of Chlamydia after HHV6-induced persistence. We show that co-infection with Herpes simplex virus 1 or human Cytomegalovirus also induces chlamydial persistence by a similar mechanism suggesting that Chlamydia -human herpes virus co-infections are evolutionary shaped interactions with a thus far unrecognized broad significance.     

GP96 Interacts with HHV-6 during Viral Entry and Directs It for Cellular Degradation

CD46 and CD134 mediate attachment of Human Herpesvirus 6A (HHV-6A) and HHV-6B to host cell, respectively. But many cell types interfere with viral infection through rapid degradation of viral DNA. Hence, not all cells expressing these receptors are permissive to HHV-6 DNA replication and production of infective virions suggesting the involvement of additional factors that influence HHV-6 propagation. Here, we used a proteomics approach to identify other host cell proteins necessary for HHV-6 binding and entry. We found host cell chaperone protein GP96 to interact with HHV-6A and HHV-6B and to interfere with virus propagation within the host cell. In human peripheral blood mononuclear cells (PBMCs), GP96 is transported to the cell surface upon infection with HHV-6 and interacts with HHV-6A and -6B through its C-terminal end. Suppression of GP96 expression decreased initial viral binding but increased viral DNA replication. Transient expression of human GP96 allowed HHV-6 entry into CHO-K1 cells even in the absence of CD46. Thus, our results suggest an important role for GP96 during HHV-6 infection, which possibly supports the cellular degradation of the virus.     

Optimization of chemical induction conditions for human herpesvirus 8 (HHV-8) reactivation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) from latently-infected BC-3 cells

Human herpesvirus 8 (HHV-8) persists as episomal DNA in latently-infected cells and can establish two alternative life cycles, latent or lytic. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is a known inducer of HHV-8 in several human primary effusion lymphoma cell lines and has been widely used for HHV-8 reactivation; however, induction conditions have differed, resulting in varying levels of virus expression. We have used HHV-8 latently-infected BC-3 cells as a model to determine critical parameters for optimizing virus reactivation by TPA. We found that cell growth properties and drug treatment conditions were important for maximum reactivation of HHV-8. Addition of TPA to cells in the early log phase of a sigmoidal growth curve, which was tightly associated with high percentage of the cells in early S phase and with lower histone deacetylase activity in the cells, provided the optimum cell conditions for latent virus to switch to lytic replication. Furthermore, increasing TPA concentration (up to 320 ng per ml) at 48 h exposure time resulted in increased virus production. The results demonstrate the use of a step-wise strategy with chemical induction that may facilitate broad detection of latent DNA viruses and novel virus discovery.     

Direct Repeat 6 from Human Herpesvirus-6B Encodes a Nuclear Protein that Forms a Complex with the Viral DNA Processivity Factor p41

The SalI-L fragment from human herpesvirus 6A (HHV-6A) encodes a protein DR7 that has been reported to produce fibrosarcomas when injected into nude mice, to transform NIH3T3 cells, and to interact with and inhibit the function of p53. The homologous gene in HHV-6B is dr6. Since p53 is deregulated in both HHV-6A and -6B, we characterized the expression of dr6 mRNA and the localization of the translated protein during HHV-6B infection of HCT116 cells. Expression of mRNA from dr6 was inhibited by cycloheximide and partly by phosphonoacetic acid, a known characteristic of herpesvirus early/late genes. DR6 could be detected as a nuclear protein at 24 hpi and accumulated to high levels at 48 and 72 hpi. DR6 located in dots resembling viral replication compartments. Furthermore, a novel interaction between DR6 and the viral DNA processivity factor, p41, could be detected by confocal microscopy and by co-immunoprecipitation analysis. In contrast, DR6 and p53 were found at distinct subcellular locations. Together, our data imply a novel function of DR6 during HHV-6B replication.     

In Vitro and In Vivo Human Herpesvirus 8 Infection of Placenta

Herpesvirus infection of placenta may be harmful in pregnancy leading to disorders in fetal growth, premature delivery, miscarriage, or major congenital abnormalities. Although a correlation between human herpesvirus 8 (HHV-8) infection and abortion or low birth weight in children has been suggested, and rare cases of in utero or perinatal HHV-8 transmission have been documented, no direct evidence of HHV-8 infection of placenta has yet been reported. The aim of this study was to evaluate the in vitro and in vivo susceptibility of placental cells to HHV-8 infection. Short-term infection assays were performed on placental chorionic villi isolated from term placentae. Qualitative and quantitative HHV-8 detection were performed by PCR and real-time PCR, and HHV-8 proteins were analyzed by immunohistochemistry. Term placenta samples from HHV-8-seropositive women were analyzed for the presence of HHV-8 DNA and antigens. In vitro infected histocultures showed increasing amounts of HHV-8 DNA in tissues and supernatants; cyto- and syncitiotrophoblasts, as well as endothelial cells, expressed latent and lytic viral antigens. Increased apoptotic phenomena were visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end-labeling method in infected histocultures. Ex vivo, HHV-8 DNA and a latent viral antigen were detected in placenta samples from HHV-8-seropositive women. These findings demonstrate that HHV-8, like other human herpesviruses, may infect placental cells in vitro and in vivo, thus providing evidence that this phenomenon might influence vertical transmission and pregnancy outcome in HHV-8-infected women.     

Human herpesvirus 6A U27 plays an essential role for the virus propagation

Human herpesvirus 6A (HHV-6A) is a member of the genus Roseolovirus and the subfamily Betaherpesvirinae. It is similar to and human cytomegalovirus (HCMV). HHV-6A encodes a 41 kDa nuclear phosphoprotein, U27, which acts as a processivity factor in the replication of the viral DNA. HHV-6A U27 has 43% amino acid sequence homology with HCMV UL44, which is important for DNA replication. A previous study on HHV-6A U27 revealed that it greatly increases the in vitro DNA synthesis activity of HHV-6A DNA polymerase. However, the role of U27 during the HHV-6A virus replication process remains unclear. In this study, we constructed a U27-deficient HHV-6A mutant (HHV-6ABACU27mut) with a frameshift insertion at the U27 gene using an HHV-6A bacterial artificial chromosome (BAC) system. Viral reconstitution from the mutant BAC DNA was not detected, in contrast to the wild type and the revertant from the U27 mutant. This suggests that U27 plays a critical role in the life cycle of HHV-6A.     

The human vascular endothelial cell line HUV-EC-C harbors the integrated HHV-6B genome which remains stable in long term culture

Human herpes virus 6 (HHV-6) is a common human pathogen that is most often detected in hematopoietic cells. Although human cells harboring chromosomally integrated HHV-6 can be generated in vitro, the availability of such cell lines originating from in vivo tissues is limited. In this study, chromosomally integrated HHV-6B has been identified in a human vascular endothelial cell line, HUV-EC-C (IFO50271), derived from normal umbilical cord tissue. Sequence analysis revealed that the viral genome was similar to the HHV-6B HST strain. FISH analysis using a HHV-6 DNA probe showed one signal in each cell, detected at the distal end of the long arm of chromosome 9. This was consistent with a digital PCR assay, validating one copy of the viral DNA. Because exposure of HUV-EC-C to chemicals did not cause viral reactivation, long term cell culture of HUV-EC-C was carried out to assess the stability of viral integration. The growth rate was altered depending on passage numbers, and morphology also changed during culture. SNP microarray profiles showed some differences between low and high passages, implying that the HUV-EC-C genome had changed during culture. However, no detectable change was observed in chromosome 9, where HHV-6B integration and the viral copy number remained unchanged. Our results suggest that integrated HHV-6B is stable in HUV-EC-C despite genome instability.     

Amplification and excision of integrated polyoma DNA sequences require a functional origin of replication

Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination.     

Activation of the Epstein-Barr virus replicative cycle by human herpesvirus 6.

One common attribute of herpesviruses is the ability to establish latent, life-long infections. The role of virus-virus interaction in viral reactivation between or among herpesviruses has not been studied. Preliminary experiments in our laboratory had indicated that infection of Epstein-Barr virus (EBV) genome-positive human lymphoid cell lines with human herpesvirus 6 (HHV-6) results in EBV reactivation in these cells. To further our knowledge of this complex phenomenon, we investigated the effect of HHV-6 infection on expression of the viral lytic cycle proteins of EBV. Our results indicate that HHV-6 upregulates, by up to 10-fold, expression of the immediate-early Zebra antigen and the diffuse and restricted (85 kDa) early antigens (EA-D and EA-R, respectively) in both EBV producer and nonproducer cell lines (i.e., P3HR1, Akata, and Raji). Maximal EA-D induction was observed at 72 h post-HHV-6 infection. Furthermore, expression of late EBV gene products, namely, the viral capsid antigen (125 kDa) and viral membrane glycoprotein gp350, was also increased in EBV producer cells (P3HR1 and Akata) following infection by HHV-6. By using dual-color membrane immunofluorescence, it was found that most of the cells expressing viral membrane glycoprotein gp350 were also positive for HHV-6 antigens, suggesting a direct effect of HHV-6 replication on induction of the EBV replicative cycle. No expression of late EBV antigens was observed in Raji cells following infection by HHV-6, implying a lack of functional complementation between the deleted form of EBV found in Raji cells and the superinfecting HHV-6. The susceptibility of the cell lines to infection by HHV-6 correlated with increased expression of various EBV proteins in that B95-8 cells, which are not susceptible to HHV-6 infection, did not show an increase in expression of EBV antigens following treatment with HHV-6. Moreover, UV light-irradiated or heat-inactivated HHV-6 had no upregulating effect on the Zebra antigen or EA-D in Raji cells, indicating that infectious virus is required for the observed effects of HHV-6 on these EBV products. These results show that HHV-6, another lymphotropic human herpesvirus, can activate EBV replication and may thus contribute to the pathogenesis of EBV-associated diseases.     

Herpesviruses and Chromosomal Integration

Herpesviruses are members of a diverse family of viruses that colonize all vertebrates from fish to mammals. Although more than one hundred herpesviruses exist, all are nearly identical architecturally, with a genome consisting of a linear double-stranded DNA molecule (100 to 225 kbp) protected by an icosahedral capsid made up of 162 hollow-centered capsomeres, a tegument surrounding the nucleocapsid, and a viral envelope derived from host membranes. Upon infection, the linear viral DNA is delivered to the nucleus, where it circularizes to form the viral episome. Depending on several factors, the viral cycle can proceed either to a productive infection or to a state of latency. In either case, the viral genetic information is maintained as extrachromosomal circular DNA. Interestingly, however, certain oncogenic herpesviruses such as Marek''s disease virus and Epstein-Barr virus can be found integrated at low frequencies in the host''s chromosomes. These findings have mostly been viewed as anecdotal and considered exceptions rather than properties of herpesviruses. In recent years, the consistent and rather frequent detection (in approximately 1% of the human population) of human herpesvirus 6 (HHV-6) viral DNA integrated into human chromosomes has spurred renewed interest in our understanding of how these viruses infect, replicate, and propagate themselves. In this review, we provide a historical perspective on chromosomal integration by herpesviruses and present the current state of knowledge on integration by HHV-6 with the possible clinical implications associated with viral integration.Integration of viral genomes into the host''s chromosomes is mandatory for the successful completion of the life cycles of several viruses, including retroviruses and adeno-associated viruses (AAV). In contrast, herpesviruses maintain their genomes as extrachromosomal circular episomes in the nuclei of infected cells without the need for integration. However, there have been several reports of chromosomally integrated herpesvirus (CIHHV) DNA over the years, suggesting that herpesviruses can indeed integrate into the host''s chromosomes under certain conditions. In addition, for a virus such as human herpesvirus 6 (HHV-6), found integrated into the germ lines of approximately 1% of the world''s population, integration may represent more than a sporadic or anecdotal event.Considering that replication of nonintegrated herpesvirus DNA occurs through the well-accepted rolling-circle mechanism, yielding long DNA concatemers that are subsequently cleaved into single-genome equivalents during nucleic acid encapsidation, how replication of linear CIHHV DNA can occur (if it does) remains unknown. In this document, we review cases and reports of integrated nonhuman and human herpesviruses and discuss the outcomes of such events on the life cycles of the viruses and the potential medical consequences of integration.Chromosomal insertions of alphaherpesvirus DNA segments, including those from herpes simplex viruses and equine herpesvirus types 1 and 3, have been reported on numerous occasions in the past (10, 11, 71, 77, 81, 87, 106). In most instances, these events were detected following infection with defective interfering particles or UV-irradiated viral preparations or transfection of sheared or subgenomic viral DNA fragments. The integrated viral genome therefore consists mostly of subgenomic fragments, and there is no possibility for the production of infectious viral particles to occur. Many of the cells carrying integrated viral DNA displayed a transformed phenotype, fueling hypotheses on the oncogenic nature of these viruses. Although the integration of foreign (viral) DNA into chromosomes can cause several anomalies, the intent of this review is to focus on viruses for which integration of full-length viral DNA is documented and to raise, at least theoretically, the possibility that viral replication can occur following integration. Viruses that meet these criteria include Marek''s disease virus (MDV), Epstein-Barr virus (EBV), and HHV-6.     

Gamma delta+ T cells involvement in viral immune control of chronic human herpesvirus 8 infection

Little is known about what effector populations are associated with the control of human herpesvirus 8 (HHV-8) infection in vivo. We compared T lymphocyte subsets among HIV-HHV-8+ and HIV-HHV-8- infected human individuals. alphabeta+ T cells from HHV-8-infected individuals displayed a significantly higher percentage of differentiated effector cells among both CD4+ and CD8+ T cell subsets. HHV-8 infection was associated with significant expansion of gammadelta+ Vdelta1 T cells expressing a differentiated effector cell phenotype in peripheral blood. In vitro stimulation of PBMC from HHV-8-infected individuals with either infectious viral particles or different HHV-8 viral proteins resulted in gammadelta Vdelta1 T cell activation. In addition, gammadelta Vdelta1 T cells displayed a strong reactivity against HHV-8-infected cell lines and prevented the release of infectious viral particles following the induction of lyric replication. These data indicate that gammadelta T cells play a role in both innate and adaptive T cell responses against HHV-8 in immunocompetent individuals.     

Dual Role of Novel Ingenol Derivatives from Euphorbia tirucalli in HIV Replication: Inhibition of De Novo Infection and Activation of Viral LTR

HIV infection is not cleared by antiretroviral drugs due to the presence of latently infected cells that are not eliminated with current therapies and persist in the blood and organs of infected patients. New compounds to activate these latent reservoirs have been evaluated so that, along with HAART, they can be used to activate latent virus and eliminate the latently infected cells resulting in eradication of viral infection. Here we describe three novel diterpenes isolated from the sap of Euphorbia tirucalli, a tropical shrub. These molecules, identified as ingenols, were modified at carbon 3 and termed ingenol synthetic derivatives (ISD). They activated the HIV-LTR in reporter cell lines and human PBMCs with latent virus in concentrations as low as 10 nM. ISDs were also able to inhibit the replication of HIV-1 subtype B and C in MT-4 cells and human PBMCs at concentrations of EC₅₀ 0.02 and 0.09 µM respectively, which are comparable to the EC₅₀ of some antiretroviral currently used in AIDS treatment. Control of viral replication may be caused by downregulation of surface CD4, CCR5 and CXCR4 observed after ISD treatment in vitro. These compounds appear to be less cytotoxic than other diterpenes such as PMA and prostratin, with effective dose versus toxic dose TI>400. Although the mechanisms of action of the three ISDs are primarily attributed to the PKC pathway, downregulation of surface receptors and stimulation of the viral LTR might be differentially modulated by different PKC isoforms.     

Requirements for excision and amplification of integrated viral DNA molecules in polyoma virus-transformed cells

The integration of polyoma virus DNA into the genome of transformed rat cells generally takes place in a tandem head-to-tail arrangement. A functional viral large tumor antigen (T-Ag) renders this structure unstable, as manifested by free DNA production and excision or amplification of the integrated viral DNA. All of these phenomena involve the mobilization of precise genomic “units,” suggesting that they result from intramolecular homologous recombination events occurring in the repeated viral DNA sequences within the integrated structures. We studied polyoma ts-a-transformed rat cell lines, which produced large T-Ag but contained less than a single copy of integrated viral DNA. In all of these lines, reversion to a normal phenotype (indicative of excision) was extremely low and independent of the presence of a functional large T-Ag. The revertants were either phenotypic or had undergone variable rearrangements of the integrated sequences that seemed to involve flanking host DNA. In two of these cell lines (ts-a 4A and ts-a 3B), we could not detect any evidence of amplification even after 2 months of propagation under conditions permissive for large T-Ag. An amplification event was detected in a small subpopulation of the ts-a R5-1 line after 2 months of growth at 33°C. This involved a DNA fragment of 5.1 kilobases, consisting of the left portion of the viral insertion and about 2.5 kilobases of adjacent host DNA sequences. None of these lines spontaneously produced free viral DNA, but after fusion with 3T3 mouse fibroblasts, R5-1 and 4A produced a low level of heterogeneous free DNA molecules, which contained both viral and flanking host DNA. In contrast, the ts-a 9 cell line, whose viral insertion consists of a partial tandem of ~1.2 viral genomes, underwent a high rate of excision or amplification when propagated at temperatures permissive for large T-Ag function. These results indicate that the high rate of excision and amplification of integrated viral genomes observed in polyoma-transformed rat cells requires the presence of regions of homology (i.e., repeats) in the integrated viral sequences. Therefore, these events occur via homologous intramolecular recombination, which is promoted directly or indirectly by the large viral T-Ag.