Human herpesvirus 6 open reading frame U14 protein and cellular p53 interact with each other and are contained in the virion

A mass spectroscopic analysis of proteins from human herpesvirus 6 (HHV-6)-infected cells showed that the HHV-6 U14 protein coimmunoprecipitated with the tumor suppressor p53. The binding of U14 to p53 was verified by coimmunoprecipitation experiments in both Molt-3 cells infected with HHV-6 and 293 cells cotransfected with U14 and p53 expression vectors. Indirect immunofluorescence assays (IFAs) showed that by 18 h postinfection (hpi) U14 localized to the dot-like structures observed in both the nucleus and cytoplasm where p53 was partly accumulated. Despite Northern blotting evidence that U14 follows late kinetics, the U14 protein was detected immediately after infection (at 3 hpi) by IFA. In addition, by Western blotting, U14 was detected at 0 hpi or in the presence of cycloheximide which completely abolished the expression of IE1 protein. In addition to U14, p53 was detected at 0 hpi although it was not detected in mock-infected cells. Furthermore, both U14 and p53 were clearly detected in the viral particles by Western blotting and immunoelectron microscopy, supporting the idea that U14 and p53 are incorporated into virions. Our study provides the first evidence of the incorporation of cellular p53 into viral particles and suggests that p53 may play an important role in viral infection.     

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.     

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.     

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.     

Small RNA deep sequencing identifies microRNAs and other small noncoding RNAs from human herpesvirus 6B

Roseolovirus, or human herpesvirus 6 (HHV-6), is a ubiquitous human pathogen infecting over 95% of the population by the age of 2 years. As with other herpesviruses, reactivation of HHV-6 can present with severe complications in immunocompromised individuals. Recent studies have highlighted the importance of herpesvirus-derived microRNAs (miRNAs) in modulating both cellular and viral gene expression. An initial report which computed the likelihood of various viruses to encode miRNAs did not predict HHV-6 miRNAs. To experimentally screen for small HHV-6-encoded RNAs, we conducted large-scale sequencing of Sup-T-1 cells lytically infected with a laboratory strain of HHV-6B. This revealed an abundant, 60- to 65-nucleotide RNA of unknown function derived from the lytic origin of replication (OriLyt) that gave rise to smaller RNA species of 18 or 19 nucleotides. In addition, we identified four pre-miRNAs whose mature forms accumulated in Argonaute 2. In contrast to the case for other betaherpesviruses, HHV-6B miRNAs are expressed from direct repeat regions (DR(L) and DR(R)) located at either side of the genome. All miRNAs are conserved in the closely related HHV-6A variant, and one of them is a seed ortholog of the human miRNA miR-582-5p. Similar to alphaherpesvirus miRNAs, they are expressed in antisense orientation relative to immediate-early open reading frames (ORFs) and thus have the potential to regulate key viral genes.     

Recent topics related to human herpesvirus 6 cell tropism

Human herpesvirus 6 (HHV-6) is a T lymphotropic herpes virus that is categorized into two variants, A (HHV-6A) and B (HHV-6B), on the basis of distinct genetic, immunological and biological characteristics. HHV-6 uses human CD46 as a cellular receptor. Without viral replication, HHV-6A induces cell–cell fusion between cells expressing human CD46. Some HHV-6B strains can also induce CD46-mediated cell–cell fusion. A multiple glycoprotein complex composed of glycoprotein (g) H-gL complexed with gQ1 and gQ2 has been identified, and found to be a viral ligand for the human CD46 receptor. Moreover, a novel complex consisting of gH/gL/gO, which does not associate with CD46, has also been identified. The evidence suggests that an additional receptor for HHV-6B or both variants may play a role in determining the cell tropism of this virus. Finally, cholesterol in the HHV-6 envelope and plasma membrane of the host cells plays an important role in HHV-6 entry, although how this function relates to cell–envelope fusion remains to be elucidated.     

A sensitive quantification of HHV-6B by real-time PCR

Human herpesvirus (HHV)-6B is a pathogen causing latent infection in virtually all humans. Nevertheless, the interaction of HHV-6B with its host cells is poorly understood. Although HHV-6B is approximately 90% homologous to HHV-6A, it expresses certain B-specific genes. In order to quantify the amount of expressed viral mRNA we have developed a method using real-time PCR on a LightCycler instrument. Here we describe an assay for the detection of the HHV-6B B6 mRNA, but our approach can easily be extended to involve other mRNAs. This method is useful during the study of HHV-6B biology and offers reliable and reproducible, quantitative detection of viral mRNA below the attomol range. Published: December 9, 2002     

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.     

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.     

Human herpesvirus 6B origin: sequence diversity, requirement for two binding sites for origin-binding protein, and enhanced replication from origin multimers.

A previously identified human herpesvirus 6B (HHV-6B) origin of DNA replication contains two binding sites for the origin-binding protein (OBPH6B). We have investigated the functional significance of these sites by determining the replication efficiencies of mutated origin sequences, using a transient replication assay. The results indicate that both sites are required for DNA replication. In addition, we have tested the functional consequences of linear sequence amplifications in the origin. The data show that tandemized origin elements are more efficiently replicated than single-copy origins. Finally, we have determined the extent of interstrain origin sequence variation that exists among HHV-6 isolates by cloning, sequencing, and analyzing origins from a number of virus isolates, including examples of both HHV-6A and HHV-6B.     

A Simple Proteomics-Based Approach to Identification of Immunodominant Antigens from a Complex Pathogen: Application to the CD4 T Cell Response against Human Herpesvirus 6B

Most of humanity is chronically infected with human herpesvirus 6 (HHV-6), with viral replication controlled at least in part by a poorly characterized CD4 T cell response. Identification of viral epitopes recognized by CD4 T cells is complicated by the large size of the herpesvirus genome and a low frequency of circulating T cells responding to the virus. Here, we present an alternative to classical epitope mapping approaches used to identify major targets of the T cell response to a complex pathogen like HHV-6B. In the approach presented here, extracellular virus preparations or virus-infected cells are fractionated by SDS-PAGE, and eluted fractions are used as source of antigens to study cytokine responses in direct ex vivo T cell activation studies. Fractions inducing significant cytokine responses are analyzed by mass spectrometry to identify viral proteins, and a subset of peptides from these proteins corresponding to predicted HLA-DR binders is tested for IFN-γ production in seropositive donors with diverse HLA haplotypes. Ten HHV-6B viral proteins were identified as immunodominant antigens. The epitope-specific response to HHV-6B virus was complex and variable between individuals. We identified 107 peptides, each recognized by at least one donor, with each donor having a distinctive footprint. Fourteen peptides showed responses in the majority of donors. Responses to these epitopes were validated using in vitro expanded cells and naturally expressed viral proteins. Predicted peptide binding affinities for the eight HLA-DRB1 alleles investigated here correlated only modestly with the observed CD4 T cell responses. Overall, the response to the virus was dominated by peptides from the major capsid protein U57 and major antigenic protein U11, but responses to other proteins including glycoprotein H (U48) and tegument proteins U54 and U14 also were observed. These results provide a means to follow and potentially modulate the CD4 T-cell immune response to HHV-6B.     

Identification of the Human Herpesvirus 6A gQ1 Domain Essential for Its Functional Conformation

Human herpesvirus 6 is a T lymphotropic herpesvirus, long classified into variants A and B (HHV-6A and HHV-6B) based on differences in sequence and pathogenicity. Recently, however, HHV-6A and HHV-6B were reclassified as different species. Here, we isolated a neutralizing monoclonal antibody (Mab) named AgQ 1-1 that was specific for HHV-6A glycoprotein Q1 (AgQ1), and we showed that amino acid residues 494 to 497 of AgQ1 were critical for its recognition by this Mab. This region was also essential for AgQ1''s complex formation with gH, gL, and gQ2, which might be important for viral binding to the cellular receptor, CD46. In addition, amino acid residues 494 to 497 are essential for viral replication. Interestingly, this sequence corresponds to the domain on HHV-6B gQ1 that is critical for recognition by an HHV-6B-specific neutralizing Mab. Within this domain, only Q at position 496 of HHV-6A is distinct from the HHV-6B sequence; however, the mutant AgQ1(Q496E) was still clearly recognized by the Mab AgQ 1-1. Surprisingly, replacement of the adjacent amino acid, in mutant AgQ1(C495A), resulted in poor recognition by Mab AgQ 1-1, and AgQ1(C495A) could not form the gH/gL/gQ1/gQ2 complex. Furthermore, the binding ability of mutant AgQ1(L494A) with CD46 decreased, although it could form the gH/gL/gQ1/gQ2 complex and it showed clear reactivity to Mab AgQ 1-1. These data indicated that amino acid residues 494 to 497 of AgQ1 were critical for the recognition by Mab AgQ 1-1 and essential for AgQ1''s functional conformation.     

Characterization of the DNA polymerase gene of human herpesvirus 6.

The construction of a recombinant bacteriophage lambda library containing overlapping clones covering 155 kbp of the 161-kbp genome of the Ugandan U1102 isolate of human herpesvirus 6 (HHV-6) is described. The use of degenerate-primer polymerase chain reaction allowed the isolation of a DNA probe for the DNA polymerase gene of HHV-6, which was subsequently used to isolate and position the pol gene on the physical map of the viral genome. A 4.4-kbp EcoRI DNA restriction fragment containing the pol gene was isolated and sequenced. The open reading frames flanking the pol gene code for the HHV-6 glycoprotein B gene and the human cytomegalovirus UL53 homolog. This arrangement is different from that seen in the alpha and gamma herpesvirus families, lending further support to the notion that HHV-6 is a member of the beta herpesvirus group.     

Human herpesvirus 6 infection arrests cord blood mononuclear cells in G(2) phase of the cell cycle

We here report that after infection with human herpesvirus 6A, human cord blood mononuclear cells accumulate in G₂/M phase of the cell cycle. Experiments with foscarnet or ultraviolet (UV)-irradiated virus stocks pointed at an (immediate-)early, newly formed viral protein to be responsible for the arrest. At the molecular level, p53, cyclin B₁, cyclin A and tyrosine¹⁵-phosphorylated cdk1 accumulated after HHV-6A infection, indicating an arrest in G₂. However, no change was observed in the levels of downstream effectors of p53 in establishing a G₂ arrest, i.e. p21 and 14-3-3σ. We thus conclude that the HHV-6A-induced G₂ arrest occurs independently of p53 accumulation.     

Human herpesvirus 8 encodes a homolog of interleukin-6.

Kaposi's sarcoma is a multifocal lesion that is reported to be greatly influenced by cytokines such as interleukin-6 (IL-6) and oncostatin M. DNA sequences of a novel human gammaherpesvirus, termed human herpesvirus 8 (HHV-8) or Kaposi sarcoma-associated herpesvirus, have been identified in all epidemiological forms of Kaposi's sarcoma with high frequency. The presence of HHV-8 DNA is also clearly associated with certain B-cell lymphomas (body cavity-based lymphomas) and multicentric Castleman's disease. Sequence analysis of a 17-kb fragment revealed that adjacent to a block of conserved herpesvirus genes (major DNA-binding protein, glycoprotein B, and DNA polymerase), the genome of HHV-8 encodes structural homolog of IL-6. This cytokine is involved not only in the pathogenesis of Kaposi's sarcoma but also in certain B-cell lymphomas and multicentric Castleman's disease. The viral counterpart of IL-6 (vIL-6) has conserved important features such as cysteine residues involved in disulfide bridging or an amino-terminal signal peptide. Most notably, the region known to be involved in receptor binding is highly conserved in vIL-6. This conservation of essential features and the remarkable overlap between diseases associated with HHV-8 and diseases associated with IL-6 disregulation clearly suggest that vIL-6 is involved in HHV-8 pathogenesis.