HIV-1 Preintegration Complex Preferentially Integrates the Viral DNA into Nucleosomes Containing Trimethylated Histone 3-Lysine 36 Modification and Flanking Linker DNA.

HIV-1 DNA is preferentially integrated into chromosomal hot spots by the preintegration complex (PIC). To understand the mechanism, we measured the DNA integration activity of PICs-extracted from infected cells-and intasomes, biochemically assembled PIC substructures using a number of relevant target substrates. We observed that PIC-mediated integration into human chromatin is preferred compared to genomic DNA. Surprisingly, nucleosomes lacking histone modifications were not preferred integration compared to the analogous naked DNA. Nucleosomes containing the trimethylated histone 3 lysine 36 (H3K36me3), an epigenetic mark linked to active transcription, significantly stimulated integration, but the levels remained lower than the naked DNA. Notably, H3K36me3-modified nucleosomes with linker DNA optimally supported integration mediated by the PIC but not by the intasome. Interestingly, optimal intasome-mediated integration required the cellular cofactor LEDGF. Unexpectedly, LEDGF minimally affected PIC-mediated integration into naked DNA but blocked integration into nucleosomes. The block for the PIC-mediated integration was significantly relieved by H3K36me3 modification. Mapping the integration sites in the preferred substrates revealed that specific features of the nucleosome-bound DNA are preferred for integration, whereas integration into naked DNA was random. Finally, biochemical and genetic studies demonstrate that DNA condensation by the H1 protein dramatically reduces integration, providing further evidence that features inherent to the open chromatin are preferred for HIV-1 integration. Collectively, these results identify the optimal target substrate for HIV-1 integration, report a mechanistic link between H3K36me3 and integration preference, and importantly, reveal distinct mechanisms utilized by the PIC for integration compared to the intasomes. IMPORTANCE HIV-1 infection is dependent on integration of the viral DNA into the host chromosomes. The preintegration complex (PIC) containing the viral DNA, the virally encoded integrase (IN) enzyme, and other viral/host factors carries out HIV-1 integration. HIV-1 integration is not dependent on the target DNA sequence, and yet the viral DNA is selectively inserted into specific "hot spots" of human chromosomes. A growing body of literature indicates that structural features of the human chromatin are important for integration targeting. However, the mechanisms that guide the PIC and enable insertion of the PIC-associated viral DNA into specific hot spots of the human chromosomes are not fully understood. In this study, we describe a biochemical mechanism for the preference of the HIV-1 DNA integration into open chromatin. Furthermore, our study defines a direct role for the histone epigenetic mark H3K36me3 in HIV-1 integration preference and identify an optimal substrate for HIV-1 PIC-mediated viral DNA integration.

Evaluation of liver failure in a pediatric transplant recipient of a liver allograft with inherited chromosomally integrated HHV-6B.

BACKGROUND: Active infections of human herpesvirus 6B (HHV-6B) are frequent in immunocompromised recipients after transplantation. Nevertheless, they need to be distinguished from latent inherited chromosomally integrated genomes (iciHHV-6) present in about 1% of the population to avoid unnecessary administration of toxic antivirals. METHODS: A 5-year-old child presented with acute liver allograft rejection associated with HHV-6 DNA in plasma, which led to an unfavorable outcome. We investigated the possibility of HHV-6 infection derived from an iciHHV-6 present in the donor's liver using molecular and histopathology studies in various tissues, including quantification of HHV-6 DNA, genotyping, sequencing for antiviral resistance genes, relative quantification of viral transcripts, and detection of gB and gH viral proteins. RESULTS: The presence of iciHHV-6B was evidenced in the donor with signs of reactivation in the gallbladder and transplanted liver (detection of HHV-6B mRNA and late proteins). This localized expression could have played a role in liver rejection. Low viral loads in the recipient's plasma, with identical partial U39 sequences, were in favor of viral DNA released from the transplanted liver rather than a systemic infection. CONCLUSIONS: Determination of iciHHV-6 status before transplantation should be considered to guide clinical decisions, such as antiviral prophylaxis, viral load monitoring, and antiviral therapy.

The Epstein-Barr Virus Episome Maneuvers between Nuclear Chromatin Compartments during Reactivation.

The human genome is structurally organized in three-dimensional space to facilitate functional partitioning of transcription. We learned that the latent episome of the human Epstein-Barr virus (EBV) preferentially associates with gene-poor chromosomes and avoids gene-rich chromosomes. Kaposi's sarcoma-associated herpesvirus behaves similarly, but human papillomavirus does not. Contacts on the EBV side localize to OriP, the latent origin of replication. This genetic element and the EBNA1 protein that binds there are sufficient to reconstitute chromosome association preferences of the entire episome. Contacts on the human side localize to gene-poor and AT-rich regions of chromatin distant from transcription start sites. Upon reactivation from latency, however, the episome moves away from repressive heterochromatin and toward active euchromatin. Our work adds three-dimensional relocalization to the molecular events that occur during reactivation. Involvement of myriad interchromosomal associations also suggests a role for this type of long-range association in gene regulation.IMPORTANCE The human genome is structurally organized in three-dimensional space, and this structure functionally affects transcriptional activity. We set out to investigate whether a double-stranded DNA virus, Epstein-Barr virus (EBV), uses mechanisms similar to those of the human genome to regulate transcription. We found that the EBV genome associates with repressive compartments of the nucleus during latency and with active compartments during reactivation. This study advances our knowledge of the EBV life cycle, adding three-dimensional relocalization as a novel component to the molecular events that occur during reactivation. Furthermore, the data add to our understanding of nuclear compartments, showing that disperse interchromosomal interactions may be important for regulating transcription.

Outcomes of hematopoietic cell transplantation using donors or recipients with inherited chromosomally integrated HHV-6.

Human herpesvirus 6 (HHV-6) species have a unique ability to integrate into chromosomal telomeres. Mendelian inheritance via gametocyte integration results in HHV-6 in every nucleated cell. The epidemiology and clinical effect of inherited chromosomally integrated HHV-6 (iciHHV-6) in hematopoietic cell transplant (HCT) recipients is unclear. We identified 4319 HCT donor-recipient pairs (8638 subjects) who received an allogeneic HCT and had archived pre-HCT peripheral blood mononuclear cell samples. We screened these samples for iciHHV-6 and compared characteristics of HCT recipients and donors with iciHHV-6 with those of recipients and donors without iciHHV-6, respectively. We calculated Kaplan-Meier probability estimates and Cox proportional hazards models for post-HCT outcomes based on recipient and donor iciHHV-6 status. We identified 60 HCT recipients (1.4%) and 40 donors (0.9%) with iciHHV-6; both recipient and donor harbored iciHHV-6 in 13 HCTs. Thus, there were 87 HCTs (2%) in which the recipient, donor, or both harbored iciHHV-6. Acute graft-versus-host disease (GVHD) grades 2-4 was more frequent when recipients or donors had iciHHV-6 (adjusted hazard ratios, 1.7-1.9; P = .004-.001). Cytomegalovirus viremia (any and high-level) was more frequent among recipients with iciHHV-6 (adjusted HRs, 1.7-3.1; P = .001-.040). Inherited ciHHV-6 status did not significantly affect risk for chronic GVHD, hematopoietic cell engraftment, overall mortality, or nonrelapse mortality. Screening for iciHHV-6 could guide donor selection and post-HCT risk stratification and treatment. Further study is needed to replicate these findings and identify potential mechanisms.

Inherited Chromosomally Integrated Human Herpesvirus 6 Genomes Are Ancient, Intact, and Potentially Able To Reactivate from Telomeres.

The genomes of human herpesvirus 6A (HHV-6A) and HHV-6B have the capacity to integrate into telomeres, the essential capping structures of chromosomes that play roles in cancer and ageing. About 1% of people worldwide are carriers of chromosomally integrated HHV-6 (ciHHV-6), which is inherited as a genetic trait. Understanding the consequences of integration for the evolution of the viral genome, for the telomere, and for the risk of disease associated with carrier status is hampered by a lack of knowledge about ciHHV-6 genomes. Here, we report an analysis of 28 ciHHV-6 genomes and show that they are significantly divergent from the few modern nonintegrated HHV-6 strains for which complete sequences are currently available. In addition, ciHHV-6B genomes in Europeans are more closely related to each other than to ciHHV-6B genomes from China and Pakistan, suggesting regional variation of the trait. Remarkably, at least one group of European ciHHV-6B carriers has inherited the same ciHHV-6B genome, integrated in the same telomere allele, from a common ancestor estimated to have existed 24,500 ± 10,600 years ago. Despite the antiquity of some, and possibly most, germ line HHV-6 integrations, the majority of ciHHV-6B (95%) and ciHHV-6A (72%) genomes contain a full set of intact viral genes and therefore appear to have the capacity for viral gene expression and full reactivation.IMPORTANCE Inheritance of HHV-6A or HHV-6B integrated into a telomere occurs at a low frequency in most populations studied to date, but its characteristics are poorly understood. However, stratification of ciHHV-6 carriers in modern populations due to common ancestry is an important consideration for genome-wide association studies that aim to identify disease risks for these people. Here, we present full sequence analysis of 28 ciHHV-6 genomes and show that ciHHV-6B in many carriers with European ancestry most likely originated from ancient integration events in a small number of ancestors. We propose that ancient ancestral origins for ciHHV-6A and ciHHV-6B are also likely in other populations. Moreover, despite their antiquity, all of the ciHHV-6 genomes appear to retain the capacity to express viral genes, and most are predicted to be capable of full viral reactivation. These discoveries represent potentially important considerations in immunocompromised patients, in particular in organ transplantation and in stem cell therapy.

Chromosomal Integration by Human Herpesviruses 6A and 6B.

Upon infection and depending on the infected cell type, human herpesvirus 6A (HHV-6A) and 6B (HHV-6B) can replicate or enter a state of latency. HHV-6A and HHV-6B can integrate their genomes into host chromosomes as one way to establish latency. Viral integration takes place near the subtelomeric/telomeric junction of chromosomes. When HHV-6 infection and integration occur in gametes, the virus can be genetically transmitted. Inherited chromosomally integrated HHV-6 (iciHHV-6)-positive individuals carry one integrated HHV-6 copy per somatic cell. The prevalence of iciHHV-6+ individuals varies between 0.6% and 2%, depending on the geographical region sampled. In this chapter, the mechanisms leading to viral integration and reactivation from latency, as well as some of the biological and medical consequences associated with iciHHV-6, were discussed.

Possible chromosomal and germline integration of human herpesvirus 7.

Human herpesvirus 7 (HHV-7) is a betaherpesvirus, and is phylogenetically related to both HHV-6A and HHV-6B. The presence of telomeric repeat sequences at both ends of its genome should make it equally likely to integrate into the human telomere as HHV-6. However, numerous studies have failed to detect germline integration of HHV-7, suggesting an important difference between the HHV-6A/-6B and HHV-7 genomes. In search of possible germline integrated HHV-7, we developed a sensitive and quantitative real-time PCR assay and discovered that primers designed against some parts of the HHV-7 genome can frequently miss HHV-7 positive clinical samples even though they work efficiently in cell-culture-derived HHV-7 positive materials. Using a primer pair against the U90 ORF of HHV-7, we identified a possible case of germline integration of HHV-7 with one copy of viral genome per cell in both peripheral blood cells and hair follicles. Chromosomal integration of HHV-7 in these individuals was confirmed by fluorescence in situ hybridization analysis. Germline integration of HHV-7 was further confirmed by detection of ~2.6 copies of HHV-7 in the hair follicles of one of the parents. Our results shed light on the complex nature of the HHV-7 genome in human-derived materials in comparison to cell-culture-derived materials and show the need for stringent criteria in the selection of primers for epidemiological HHV-7 studies.

HIV infection reveals widespread expansion of novel centromeric human endogenous retroviruses.

Human endogenous retroviruses (HERVs) make up 8% of the human genome. The HERV-K (HML-2) family is the most recent group of these viruses to have inserted into the genome, and we have detected the activation of HERV-K (HML-2) proviruses in the blood of patients with HIV-1 infection. We report that HIV-1 infection activates expression of a novel HERV-K (HML-2) provirus, termed K111, present in multiple copies in the centromeres of chromosomes throughout the human genome yet not annotated in the most recent human genome assembly. Infection with HIV-1 or stimulation with the HIV-1 Tat protein leads to the activation of K111 proviruses. K111 is present as a single copy in the genome of the chimpanzee, yet K111 is not found in the genomes of other primates. Remarkably, K111 proviruses appear in the genomes of the extinct Neanderthal and Denisovan, while modern humans have at least 100 K111 proviruses spread across the centromeres of 15 chromosomes. Our studies suggest that the progenitor K111 integrated before the Homo-Pan divergence and expanded in copy number during the evolution of hominins, perhaps by recombination. The expansion of K111 provides sequence evidence suggesting that recombination between the centromeres of various chromosomes took place during the evolution of humans. K111 proviruses show significant sequence variations in each individual centromere, which may serve as markers in future efforts to annotate human centromere sequences. Further, this work is an example of the potential to discover previously unknown genomic sequences through the analysis of nucleic acids found in the blood of patients.

Prevalence of chromosomally integrated human herpesvirus 6 in patients with human herpesvirus 6-central nervous system dysfunction.

We identified 37 hematopoietic cell transplantation recipients with human herpesvirus 6 (HHV-6) central nervous system dysfunction and tested donor-recipient pairs for chromosomally integrated HHV-6 (ciHHV-6). One patient had ciHHV-6A with possible HHV-6A reactivation and encephalitis. There was no ciHHV-6 enrichment in this group, but larger studies are needed to determine if patients with ciHHV-6 are at increased risk for HHV-6-associated diseases or other complications.

Analyses of germline, chromosomally integrated human herpesvirus 6A and B genomes indicate emergent infection and new inflammatory mediators.

Human herpesvirus-6A (HHV-6A) is rarer than HHV-6B in many infant populations. However, they are similarly prevalent as germline, chromosomally integrated genomes (ciHHV-6A/B). This integrated form affects 0.1-1 % of the human population, where potentially virus gene expression could be in every cell, although virus relationships and health effects are not clear. In a Czech/German patient cohort ciHHV-6A was more common and diverse than ciHHV-6B. Quantitative PCR, nucleotide sequencing and telomeric integration site amplification characterized ciHHV-6 in 44 German myocarditis/cardiomyopathy and Czech malignancy/inflammatory disease (MI) patients plus donors. Comparisons were made to sequences from global virus reference strains, and blood DNA from childhood-infections from Zambia (HHV-6A mainly) and Japan (HHV-6B). The MI cohort were 86 % (18/21) ciHHV-6A, the cardiac cohort 65 % (13/20) ciHHV-6B, suggesting different disease links. Reactivation was supported by findings of 1) recombination between ciHHV-6A and HHV-6B genes in 20 % (4/21) of the MI cohort; 2) expression in a patient subset, of early/late transcripts from the inflammatory mediator genes chemokine receptor U51 and chemokine U83, both identical to ciHHV-6A DNA sequences; and 3) superinfection shown by deep sequencing identifying minor virus-variants only in ciHHV-6A, which expressed transcripts, indicating virus infection reactivates latent ciHHV-6A. Half the MI cohort had more than two copies per cell, median 5.2, indicative of reactivation. Remarkably, the integrated genomes encoded the secreted-active form of virus chemokines, rare in virus from childhood-infections. This shows integrated virus genomes can contribute new human genes with links to inflammatory pathology and supports ciHHV-6A reactivation as a source for emergent infection.

Human cytomegalovirus major immediate early 1 protein targets host chromosomes by docking to the acidic pocket on the nucleosome surface.

The 72-kDa immediate early 1 (IE1) protein encoded by human cytomegalovirus (hCMV) is a nuclearly localized promiscuous regulator of viral and cellular transcription. IE1 has long been known to associate with host mitotic chromatin, yet the mechanisms underlying this interaction have not been specified. In this study, we identify the cellular chromosome receptor for IE1. We demonstrate that the viral protein targets human nucleosomes by directly binding to core histones in a nucleic acid-independent manner. IE1 exhibits two separable histone-interacting regions with differential binding specificities for H2A-H2B and H3-H4. The H2A-H2B binding region was mapped to an evolutionarily conserved 10-amino-acid motif within the chromatin-tethering domain (CTD) of IE1. Results from experimental approaches combined with molecular modeling indicate that the IE1 CTD adopts a ß-hairpin structure, docking with the acidic pocket formed by H2A-H2B on the nucleosome surface. IE1 binds to the acidic pocket in a way similar to that of the latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus. Consequently, the IE1 and LANA CTDs compete for binding to nucleosome cores and chromatin. Our work elucidates in detail how a key viral regulator is anchored to human chromosomes and identifies the nucleosomal acidic pocket as a joint target of proteins from distantly related viruses. Based on the striking similarities between the IE1 and LANA CTDs and the fact that nucleosome targeting by IE1 is dispensable for productive replication even in "clinical" strains of hCMV, we speculate that the two viral proteins may serve analogous functions during latency of their respective viruses.

Efficient replication of Epstein-Barr virus-derived plasmids requires tethering by EBNA1 to host chromosomes.

The EBNA1 protein of Epstein-Barr virus enables plasmids carrying oriP both to duplicate and to segregate efficiently in proliferating cells. EBNA1 recruits the origin recognition complex (ORC) to establish a replication origin at one element of oriP, DS (dyad symmetry); at another element, FR (family of repeats), EBNA1 binds to an array of sites from which it tethers plasmids to host chromosomes for mitotic stability. We report experiments leading to the conclusion that tethering by EBNA1 to host chromosomes is also needed within interphase nuclei in order for plasmids to be replicated efficiently from oriP. The DNA-binding domain of EBNA1, which lacks chromosome-binding ability, was found to support weak, DS-specific replication in HEK293 cells after transient transfection, being 17% as active as wild-type EBNA1. The low efficiency of replication was not due to the failure of the DNA-binding domain to retain plasmids within nuclei, because plasmids were recovered in similar amounts and entirely from the nuclear fraction of these transiently transfected cells. A derivative of EBNA1 with its chromosome-tethering domains replaced by a 22-amino-acid nucleosome-binding domain was fully active in supporting oriP functions. The implication is that EBNA1's DNA-binding domain is able to recruit ORC to DS, but either this step or subsequent replication is only efficient if the plasmid is tethered to a host chromosome. Finally, with some cell lines, DS can hardly support even transient plasmid replication without FR. A loss of plasmids lacking FR from nuclei cannot account for this requirement, suggesting that the stronger tethering to chromosomes by FR is needed for plasmid replication within the nuclei of such cells.

Identification of chromosomally integrated human herpesvirus 6 by droplet digital PCR.

BACKGROUND: Human herpesvirus 6 (HHV-6) latently infects a majority of adults. In about 1% of the population HHV-6 exists in a chromosomally integrated form (ciHHV-6) that resides in every somatic and germ cell and can be transmitted through the germ line. Patients with ciHHV-6 have been misdiagnosed and unnecessarily treated for active HHV-6 infection, sometimes with important side effects, based on results from quantitative molecular HHV-6 tests. METHODS: A droplet digital PCR (ddPCR) assay was developed to identify ciHHV-6 in cellular patient samples by precisely determining the ratio of HHV-6 to cellular DNA. We validated the assay on confirmed ciHHV-6 patient samples and a cell line derived from a ciHHV-6 patient, and we analyzed hematopoietic stem cell transplant patients suspected of having ciHHV-6. We additionally evaluated whether the assay could be applied to stored plasma samples from a study of clinical correlates of HHV-6. RESULTS: The ddPCR assay accurately identified ciHHV-6 in cellular samples (buffy coat, peripheral blood mononuclear cells), giving a ratio very close to 1 HHV-6/cell [mean (SD), 1.02 (0.03)] in fluorescence in situ hybridization-confirmed sample). In stored plasma samples, the assay performance was set by design to have 100% sensitivity, which resulted in 82% specificity for ciHHV-6. CONCLUSIONS: The possibility of ciHHV-6 is often overlooked in patients with detectable HHV-6 viral loads by quantitative PCR. Our ddPCR test provides rapid and accurate laboratory identification of ciHHV-6 from easily obtained cellular samples. In addition, the assay provides excellent sensitivity and specificity using stored plasma samples, facilitating retrospective analysis of the clinical significance of ciHHV-6.

An immunocompetent child with chromosomally integrated human herpesvirus 6B accidentally identified during the care of Mycoplasma pneumoniae infection.

Human herpesvirus 6 (HHV-6) is the only virus known to integrate into human chromosomes and be transmitted from parents to offspring. Less than 1% of the population carries integrated HHV-6 in their genomes. Here, we report the case of a 9-year-old Japanese girl with an extraordinarily high copy number of HHV-6B in her genome. The integrated virus genome was detected by real-time polymerase chain reaction (PCR) in cerebrospinal fluid and serum during the treatment of meningoencephalitis and pneumonia caused by Mycoplasma pneumoniae infection. Furthermore, the HHV-6B genome was detected in hair follicle, plasma, and whole blood in the patient and her mother, but not in the patient's father. Fluorescence in situ hybridization revealed that the viral genome was integrated into chromosome 22. Therefore, these results emphasize the importance of screening for chromosomally integrated HHV-6 prior to starting unnecessary antiviral therapies, particularly for patients harboring HHV-6 with a high copy number.

Sequence analysis of transplacentally acquired human herpesvirus 6 DNA is consistent with transmission of a chromosomally integrated reactivated virus.

The majority of human herpesvirus 6 (HHV-6) congenital infections (86%) originate from germ line transmission of chromosomally integrated HHV-6 (ciHHV-6). To determine whether transplacentally acquired HHV-6 could derive from the transmission of reactivated maternal ciHHV-6, we identified mother-infant pairs in which infants had proven transplacentally acquired HHV-6 and mothers had documented ciHHV-6, and we sequenced and compared the HHV-6 gB gene sequences for each pair. Our data indicate that the gB gene sequence found in each cord blood specimen was identical to that of the corresponding mother but divergent from that of other known HHV-6 isolates. These results are consistent with transplacentally acquired HHV-6 originating from the transmission of reactivated ciHHV-6.

Identification of host-chromosome binding sites and candidate gene targets for Kaposi's sarcoma-associated herpesvirus LANA.

LANA is essential for tethering the Kaposi's sarcoma-associated herpesvirus (KSHV) genome to metaphase chromosomes and for modulating host-cell gene expression, but the binding sites in the host-chromosome remain unknown. Here, we use LANA-specific chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to identify LANA binding sites in the viral and host-cell genomes of a latently infected pleural effusion lymphoma cell line BCBL1. LANA bound with high occupancy to the KSHV genome terminal repeats (TR) and to a few minor binding sites in the KSHV genome, including the LANA promoter region. We identified 256 putative LANA binding site peaks with P < 0.01 and overlap in two independent ChIP-Seq experiments. We validated several of the high-occupancy binding sites by conventional ChIP assays and quantitative PCR. Candidate cellular LANA binding motifs were identified and assayed for binding to purified recombinant LANA protein in vitro but bound with low affinity compared to the viral TR binding site. More than half of the LANA binding sites (170/256) could be mapped to within 2.5 kb of a cellular gene transcript. Pathways and Gene Ontogeny (GO) analysis revealed that LANA binds to genes within the p53 and tumor necrosis factor (TNF) regulatory network. Further analysis revealed partial overlap of LANA and STAT1 binding sites in several gamma interferon (IFN-γ)-regulated genes. We show that ectopic expression of LANA can downmodulate IFN-γ-mediated activation of a subset of genes, including the TAP1 peptide transporter and proteasome subunit beta type 9 (PSMB9), both of which are required for class I antigen presentation. Our data provide a potential mechanism through which LANA may regulate several host cell pathways by direct binding to gene regulatory elements.

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 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.

The chromatin-tethering domain of human cytomegalovirus immediate-early (IE) 1 mediates associations of IE1, PML and STAT2 with mitotic chromosomes, but is not essential for viral replication.

Human cytomegalovirus (HCMV) immediate-early (IE) 1 protein associates with chromosomes in mitotic cells using its carboxyl-terminal 16 aa region. However, the role of this IE1 activity in viral growth has not been evaluated in the context of mutant virus infection. We produced a recombinant HCMV encoding mutant IE1 with the carboxyl-terminal chromosome-tethering domain (CTD) deleted. This IE1(ΔCTD) virus grew like the wild-type virus in fibroblasts, indicating that the CTD is not essential for viral replication in permissive cells. Unlike wild-type virus infections, PML and STAT2, which interact with IE1, did not accumulate at mitotic chromosomes in IE1(ΔCTD) virus-infected fibroblasts, demonstrating that their associations with chromosomes are IE1 CTD-dependent. IE1 SUMOylation did not affect IE1 association with chromosomes. Our results provide genetic evidence that the CTD is required for the associations of IE1, PML and STAT2 with mitotic chromosomes, but that these IE1-related activities are not essential for viral replication in fibroblasts.

Chromosomally integrated human herpesvirus-6 in transplant recipients.

Human herpesvirus-6 (HHV-6) is unique among human herpesviruses because of its ability to integrate into chromosomes. This entity, termed chromosomally integrated HHV-6 (CIHHV-6), is often mistaken for active infection and treated unnecessarily. The clinical significance of CIHHV-6 in transplant recipients is not defined. Herein, the clinical characteristics of 7 liver transplant patients with CIHHV-6 from our recent study, together with 14 other published cases of CIHHV-6 were reviewed. Of the 21 cases, CIHHV-6B was reported most commonly among solid organ transplant recipients, while CIHHV-6A was mostly seen in allogeneic hematopoietic stem cell recipients. None of the 21 patients developed clinical symptoms related to HHV-6 after transplantation. However, antiviral therapy was administered to 5 asymptomatic patients mistaken to have HHV-6 infection because of their very high HHV-6 DNA levels, 3 who developed symptomatic cytomegalovirus disease, and 1 with graft-versus-host disease that was mistaken for HHV-6 infection. In patients who received antiviral therapy, there was no apparent decline in HHV-6 DNA load, although change in viral kinetics is difficult to discern in the setting of high baseline HHV-6 DNA load. Clinicians should be aware of this entity of CIHHV-6 so that antiviral therapy can be considered in the proper clinical context.