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1.  Analysis of a Neutralizing Antibody for Human Herpesvirus 6B Reveals a Role for Glycoprotein Q1 in Viral Entry ▿  
Journal of Virology  2011;85(24):12962-12971.
Human herpesvirus 6 (HHV-6) is a T cell-tropic betaherpesvirus. HHV-6 can be classified into two variants, HHV-6A and HHV-6B, based on differences in their genetic, antigenic, and growth characteristics and cell tropisms. The function of HHV-6B should be analyzed more in its life cycle, as more than 90% of people have the antibodies for HHV-6B but not HHV-6A. It has been shown that the cellular receptor for HHV-6A is human CD46 and that the viral ligand for CD46 is the envelope glycoprotein complex gH/gL/gQ1/gQ2; however, the receptor-ligand pair used by HHV-6B is still unknown. In this study, to identify the glycoprotein(s) important for HHV-6B entry, we generated monoclonal antibodies (MAbs) that inhibit infection by HHV-6B. Most of these MAbs were found to recognize gQ1, indicating that HHV-6B gQ1 is critical for virus entry. Interestingly, the recognition of gQ1 by the neutralizing MAb was enhanced by coexpression with gQ2. Moreover, gQ1 deletion or point mutants that are not recognized by the MAb could nonetheless associate with gQ2, indicating that although the MAb recognized the conformational epitope of gQ1 exposed by the gQ2 interaction, this epitope was not related to the gQ2 binding domain. Our study shows that HHV-6B gQ1 is likely a ligand for the HHV-6B receptor, and the recognition site for this MAb will be a promising target for antiviral agents.
PMCID: PMC3233151  PMID: 21957287
2.  Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Infection of Human Fibroblast Cells Occurs through Endocytosis 
Journal of Virology  2003;77(14):7978-7990.
Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV-8) DNA and transcripts have been detected in the B cells, macrophages, keratinocytes, and endothelial and epithelial cells of KS patients. In vitro, HHV-8 infects human B, endothelial, epithelial, and fibroblast cells, as well as animal cells, and the infection is characterized by (i) absence of lytic replication by the input virus and (ii) latent infection. For its initial binding to target cells, HHV-8 uses ubiquitous heparan sulfate molecules via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8 also interacts with the α3β1 integrin via its glycoprotein gB, and virus binding studies suggest that α3β1 is one of the HHV-8 entry receptors (S. M. Akula, N. P. Pramod, F. Z. Wang, and B. Chandran, Cell 108:407-419, 2002). In this study, morphological and biochemical techniques were used to examine the entry of HHV-8 into human foreskin fibroblasts (HFF). HHV-8 was detected in coated vesicles and in large, smooth-surfaced endocytic vesicles. Fusion of viral envelope with the vesicle wall was also observed. In immune electron microscopy, anti-HHV-8 gB antibodies colocalized with virus-containing endocytic vesicles. In fluorescence microscopic analyses, transferrin was colocalized with HHV-8. HHV-8 infection was significantly inhibited by preincubation of cells with chlorpromazine HCl, which blocks endocytosis via clathrin-coated pits, but not by nystatin and cholera toxin B, which blocks endocytosis via caveolae and induces the dissociation of lipid rafts, respectively. Infection was also inhibited by blocking the acidification of endosomes by NH4Cl and bafilomycin A. Inhibition of HHV-8 open reading frame 73 gene expression by chlorpromazine HCl, bafilomycin A, and NH4Cl demonstrated that the virions in the vesicles could proceed to cause an infection. Taken together, these findings suggest that for its infectious entry into HFF, HHV-8 uses clathrin-mediated endocytosis and a low-pH intracellular environment.
PMCID: PMC161913  PMID: 12829837
3.  Immunomodulation and immunosuppression by human herpesvirus 6A and 6B 
Future virology  2013;8(3):273-287.
Like other members of the Herpesviridae family, human herpesvirus (HHV)-6A and HHV-6B have developed a wide variety of strategies to modulate or suppress host immune responses and, thereby, facilitate their own spread and persistence in vivo. Long considered two variants of the same virus, HHV-6A and HHV-6B have recently been reclassified as distinct viral species, although the established nomenclature has been maintained. In this review, we summarize the distinctive profiles of interaction of these two viruses with the human immune system. Both HHV-6A and HHV-6B display a tropism for CD4+ T lymphocytes, but they can also infect, in a productive or nonproductive fashion, other cells of the immune system. However, there are important differences regarding the ability of each virus to infect cytotoxic effector cells, as HHV-6A has been shown to productively infect several of these cells, whereas HHV-6B infects them inefficiently at best. In addition to direct cytopathic effects, both HHV-6A and HHV-6B can interfere with immunologic functions to varying degrees via cytokine modulation, including blockade of IL-12 production by professional antigen-presenting cells, modulation of cell-surface molecules essential for T-cell activation, and expression of viral chemokines and chemokine receptors. Some of these effects are related to signaling through and downregulation of the viral receptor, CD46, a key molecule linking innate and adaptive immune responses. Increasing attention has recently been focused on the importance of viral interactions with dendritic cells, which may serve both as targets of virus-mediated immunosuppression and as vehicles for viral transfer to CD4+ T cells. Our deepening knowledge of the mechanisms developed by HHV-6A and HHV-6B to evade immunologic control may lead to new strategies for the prevention and treatment of the diseases associated with these viruses. Moreover, elucidation of these viral mechanisms may uncover new avenues to therapeutically manipulate or modulate the immune system in immunologically mediated human diseases.
PMCID: PMC3806647  PMID: 24163703
antigen-presenting cells; CD46; chemokines; cytokines; HHV-6A; HHV-6B; immunomodulation; immunosuppression; receptors; T cells
4.  Human Herpesvirus 6 Variant A but Not Variant B Induces Fusion from Without in a Variety of Human Cells through a Human Herpesvirus 6 Entry Receptor, CD46 
Journal of Virology  2002;76(13):6750-6761.
Human herpesvirus 6 (HHV-6) is a lymphotropic betaherpesvirus that productively infects T cells and monocytes. HHV-6 isolates can be differentiated into two groups, variants A and B (HHV-6A and HHV-6B). Here, we show a functional difference between HHV-6A and -6B in that HHV-6A induced syncytium formation of diverse human cells but HHV-6B did not. The syncytium formation induced by HHV-6A was observed 2 h after infection; moreover, it was found in the presence of cycloheximide, indicating that HHV-6A induced fusion from without (FFWO) in the target cells. Furthermore, the fusion event was dependent on the expression of the HHV-6 entry receptor, CD46, on the target cell membrane. In addition, we determined that short consensus repeat 2 (SCR2), -3, and -4 of the CD46 ectodomain were essential for the formation of the virus-induced syncytia. Monoclonal antibodies against glycoproteins B and H of HHV-6A inhibited the fusion event, indicating that the syncytium formation induced by HHV-6A required glycoproteins H and B. These findings suggest that FFWO, which HHV-6A induced in a variety of cell lines, may play an important role in the pathogenesis of HHV-6A, not only in lymphocytes but also in various tissues, because CD46 is expressed ubiquitously in human tissues.
PMCID: PMC136280  PMID: 12050388
5.  Association of Human Herpesvirus-6B with Mesial Temporal Lobe Epilepsy 
PLoS Medicine  2007;4(5):e180.
Human herpesvirus-6 (HHV-6) is a β-herpesvirus with 90% seroprevalence that infects and establishes latency in the central nervous system. Two HHV-6 variants are known: HHV-6A and HHV-6B. Active infection or reactivation of HHV-6 in the brain is associated with neurological disorders, including epilepsy, encephalitis, and multiple sclerosis. In a preliminary study, we found HHV-6B DNA in resected brain tissue from patients with mesial temporal lobe epilepsy (MTLE) and have localized viral antigen to glial fibrillary acidic protein (GFAP)–positive glia in the same brain sections. We sought, first, to determine the extent of HHV-6 infection in brain material resected from MTLE and non-MTLE patients; and second, to establish in vitro primary astrocyte cultures from freshly resected brain material and determine expression of glutamate transporters.
Methods and Findings
HHV-6B infection in astrocytes and brain specimens was investigated in resected brain material from MTLE and non-MTLE patients using PCR and immunofluorescence. HHV-6B viral DNA was detected by TaqMan PCR in brain resections from 11 of 16 (69%) additional patients with MTLE and from zero of seven (0%) additional patients without MTLE. All brain regions that tested positive by HHV-6B variant-specific TaqMan PCR were positive for viral DNA by nested PCR. Primary astrocytes were isolated and cultured from seven epilepsy brain resections and astrocyte purity was defined by GFAP reactivity. HHV-6 gp116/54/64 antigen was detected in primary cultured GFAP-positive astrocytes from resected tissue that was HHV-6 DNA positive—the first demonstration of an ex vivo HHV-6–infected astrocyte culture isolated from HHV-6–positive brain material. Previous work has shown that MTLE is related to glutamate transporter dysfunction. We infected astrocyte cultures in vitro with HHV-6 and found a marked decrease in glutamate transporter EAAT-2 expression.
Overall, we have now detected HHV-6B in 15 of 24 patients with mesial temporal sclerosis/MTLE, in contrast to zero of 14 with other syndromes. Our results suggest a potential etiology and pathogenic mechanism for MTLE.
Steve Jacobson and colleagues report finding human herpesvirus-6B DNA in brain resections from 11 of 16 patients with mesial temporal lobe epilepsy, strengthening the evidence for a role for this virus in this condition.
Editors' Summary
Epilepsy is a common brain disorder caused by a sudden, excessive electrical discharge in a cluster of neurons—the cells that transmit electrical messages between the body and the brain. Its symptoms depend on which part of the brain is affected by this electrical firestorm and how far the disturbance spreads. When only part of the brain is affected (a partial seizure or fit), patients may see or smell strange things, recall forgotten memories, or have part of their body jerk uncontrollably. When the electrical disturbance spreads across the whole brain (a generalized seizure), there may be loss of consciousness and/or the whole body may become rigid or jerk. Epilepsy is usually controlled with anti-epileptic drugs or, in very severe focal cases, surgery to the area of the brain where the seizure starts. Although head injuries or brain tumors can trigger epilepsy, the cause of most cases of epilepsy is unknown.
Why Was This Study Done?
Knowing what causes epilepsy might lead to better treatments for it. One possibility is that infections trigger epilepsy. The researchers in this study asked whether infections with human herpesvirus 6B (HHV-6B) are associated with a common type of epilepsy called mesial temporal lobe epilepsy (MTLE). Patients with MTLE often have extensive scarring in the hippocampus, a brain region responsible for memory that lies deep within a bigger region called the temporal lobe. Hippocampal scarring and MTLE are associated with a history of fever-induced fits, and HHV-6B infection can cause such fits in young children. Most people become infected with HHV-6B (or the closely related HHV-6A) early in life. The virus then remains latent for years within the brain and elsewhere. Given these facts and a previous investigation that showed that brain tissue from several patients with MTLE contained HHV-6B, the researchers reasoned that it was worth investigating HHV-6B as a cause of MTLE.
What Did the Researchers Do and Find?
The researchers first looked for HHV-6B DNA in brain tissue surgically removed from patients with MTLE or another type of epilepsy. Tissue from 11 of 16 patients with MTLE (but from 0 of 7 control patients) contained HHV-6B DNA. When the researchers grew astrocytes (a type of brain cell) from some of these samples, only those from HHV-6B DNA-positive samples from patients with MTLE expressed an HHV-6-specific protein. Next, the researchers investigated in detail a patient with MTLE who had four sequential operations to control his epilepsy. This patient's hippocampus, which was removed in his first operation, contained a higher level of HHV-6B DNA than the tissues removed in later operations. After the fourth operation (which removed half of his brain and cured his epilepsy), astrocytes grown from the temporal lobe and the frontal/parietal lobe (a brain region next to the temporal lobe) but not the frontal and occipital lobes contained HHV-6B DNA and expressed a viral protein. The researchers also measured the production by these various astrocytes of a substance that moves glutamate (an amino acid that also acts as a neurotransmitter) across cell membranes—MTLE has been associated with a glutamate transporter deficiency. Consistent with this, astrocytes from the patient's temporal lobe made no glutamate transporter mRNA (mRNA is an essential precursor for protein to be produced). Finally, infection of astrocytes isolated from a patient without MTLE with HHV-6B greatly reduced expression of glutamate transporter in these astrocytes.
What Do These Findings Mean?
These findings, together with those from the previous study, reveal that nearly two-thirds of patients with MTLE (but no patients with other forms of epilepsy) have an active HHV-6B infection in the brain region where their epilepsy originates. Overall, they provide strong support for the idea that HHV-6B infections might cause MTLE, particularly given the results obtained from the patient whose condition only improved after multiple brain operations had removed all the virally infected material. Furthermore, the demonstration that HHV-6B infection reduces glutamate transporter expression in astrocytes suggests that HHV-6B infection might cause astrocyte dysfunction. This dysfunction could lead to injury of the sensitive neurons in the hippocampus and trigger MTLE. Additional patients now need to be studied both to confirm the association between HHV-6B infection and MTLE and to discover exactly how this virus triggers epilepsy.
Additional Information.
Please access these Web sites via the online version of this summary at
MedlinePlus encyclopedia page on epilepsy (in English and Spanish)
World Health Organization fact sheet on epilepsy (in English, French, Spanish, Russian, Arabic, and Chinese)
US National Institute for Neurological Disorders and Stroke epilepsy information page (in English and Spanish)
UK National Health Service Direct information for patients on epilepsy (in several languages)
Neuroscience for kids, an educational Web site prepared by Eric Chudler (University of Washington, Seattle, Washington, United States), who also has a site that includes information on epilepsy and a list of links to epilepsy organizations (mainly in English but some sections in other languages as well)
A short scientific article on human herpes virus 6 in the journal Emerging Infectious Diseases
PMCID: PMC1880851  PMID: 17535102
6.  Complementation of the Function of Glycoprotein H of Human Herpesvirus 6 Variant A by Glycoprotein H of Variant B in the Virus Life Cycle 
Journal of Virology  2012;86(16):8492-8498.
Human herpesvirus 6 (HHV-6) is a T-cell-tropic betaherpesvirus. HHV-6 can be classified into two variants, HHV-6 variant A (HHV-6A) and HHV-6B, based on genetic, antigenic, and cell tropisms, although the homology of their entire genomic sequences is nearly 90%. The HHV-6A glycoprotein complex gH/gL/gQ1/gQ2 is a viral ligand that binds to the cellular receptor human CD46. Because gH has 94.3% amino acid identity between the variants, here we examined whether gH from one variant could complement its loss in the other. Recently, we successfully reconstituted HHV-6A from its cloned genome in a bacterial artificial chromosome (BAC) (rHHV-6ABAC). Using this system, we constructed HHV-6ABAC DNA containing the HHV-6B gH (BgH) gene instead of the HHV-6A gH (AgH) gene in Escherichia coli. Recombinant HHV-6ABAC expressing BgH (rHHV-6ABAC-BgH) was successfully reconstituted. In addition, a monoclonal antibody that blocks HHV-6B but not HHV-6A infection neutralized rHHV-6ABAC-BgH but not rHHV-6ABAC. These results indicate that HHV-6B gH can complement the function of HHV-6A gH in the viral infectious cycle.
PMCID: PMC3421736  PMID: 22647694
7.  The Telomeric Repeats of Human Herpesvirus 6A (HHV-6A) Are Required for Efficient Virus Integration 
PLoS Pathogens  2016;12(5):e1005666.
Human herpesvirus 6A (HHV-6A) and 6B (HHV-6B) are ubiquitous betaherpesviruses that infects humans within the first years of life and establishes latency in various cell types. Both viruses can integrate their genomes into telomeres of host chromosomes in latently infected cells. The molecular mechanism of viral integration remains elusive. Intriguingly, HHV-6A, HHV-6B and several other herpesviruses harbor arrays of telomeric repeats (TMR) identical to human telomere sequences at the ends of their genomes. The HHV-6A and HHV-6B genomes harbor two TMR arrays, the perfect TMR (pTMR) and the imperfect TMR (impTMR). To determine if the TMR are involved in virus integration, we deleted both pTMR and impTMR in the HHV-6A genome. Upon reconstitution, the TMR mutant virus replicated comparable to wild type (wt) virus, indicating that the TMR are not essential for HHV-6A replication. To assess the integration properties of the recombinant viruses, we established an in vitro integration system that allows assessment of integration efficiency and genome maintenance in latently infected cells. Integration of HHV-6A was severely impaired in the absence of the TMR and the virus genome was lost rapidly, suggesting that integration is crucial for the maintenance of the virus genome. Individual deletion of the pTMR and impTMR revealed that the pTMR play the major role in HHV-6A integration, whereas the impTMR only make a minor contribution, allowing us to establish a model for HHV-6A integration. Taken together, our data shows that the HHV-6A TMR are dispensable for virus replication, but are crucial for integration and maintenance of the virus genome in latently infected cells.
Author Summary
Herpesviruses are ubiquitous pathogens that persist in the host for life. Two human herpesviruses (HHV-6A and HHV-6B) can integrate their genetic material into the telomeres of host chromosomes. Integration also occurs in germ cells, resulting in individuals that harbor the virus in every single cells of their body and transmit it to their offspring, a condition that affects about 1% of the human population. We set to elucidate the integration mechanism that allows these viruses to maintain their genome in infected cells. Intriguingly, HHV-6A, HHV-6B and several other herpesviruses harbor telomere sequences at the end of their genome. Removal of these sequences in the genome of HHV-6A revealed that the viral telomeres are crucial for the integration of this human herpesvirus. In addition, we demonstrate that the telomere sequences at the right and left end of the virus genome play different roles in the integration process. Taken together, our data sheds light on the integration mechanism that allows HHV-6A to integrate into somatic cells and to enter into the germ line.
PMCID: PMC4887096  PMID: 27244446
8.  Virologic and Immunologic Evidence Supporting an Association between HHV-6 and Hashimoto's Thyroiditis 
PLoS Pathogens  2012;8(10):e1002951.
Hashimoto's thyroiditis (HT) is the most common of all thyroid diseases and is characterized by abundant lymphocyte infiltrate and thyroid impairment, caused by various cell- and antibody-mediated immune processes. Viral infections have been suggested as possible environmental triggers, but conclusive data are not available. We analyzed the presence and transcriptional state of human herpesvirus 6 (HHV-6) in thyroid fine needle aspirates (FNA) and peripheral blood mononuclear cells (PBMCs) from 34 HT patients and 28 controls, showing that HHV-6 DNA prevalence (82% vs. 10%, p≤0.001) and viral load were significantly increased in FNA from HT patients, and thyrocytes from HT FNA displayed a 100-fold higher HHV-6 DNA load compared to infiltrating lymphocytes. In addition, while HHV-6 was strictly latent in positive samples from controls, a low grade acute infection was detected in HT samples. HHV-6 variant characterization was carried out in 10 HT FNA samples, determining that all specimens harbored HHV-6 Variant A.
The tropism of HHV-6 for thyroid cells was verified by infection of Nthy-ori3-1, a thyroid follicular epithelial cell line, showing that thyrocytes are permissive to HHV-6 replication, which induces de novo expression of HLA class II antigens. Furthermore, HHV-6-infected Nthy-ori3-1 cells become targets for NK-mediated killing, NK cells from HT patients show a significantly more efficient killing of HHV-6 infected thyroid cells than healthy controls, and HT patients have increased T-cell responses to HHV-6 U94 protein, associated to viral latency. These observations suggest a potential role for HHV-6 (possibly variant A) in the development or triggering of HT.
Author Summary
Hashimoto's thyroiditis (HT) is a very common autoimmune disease of the thyroid. In addition to genetic background, several viruses, including herpesviruses, have been suggested to play a role as possible environmental triggers of disease, but conclusive data are still lacking. The anecdotal presence of human herpesvirus 6 (HHV-6) in HT specimens prompted us to study a possible association between HHV-6 and HT. Our analysis of fine needle thyroid aspirates and blood from HT patients and controls shows that HHV-6 prevalence and load are highly increased in HT patients. Furthermore, HT-derived thyrocytes harbor active virus, whereas HHV-6 is strictly latent in the few virus-positive controls. We also report that HHV-6 infects thyroid cells, inducing de novo expression of HLA-II surface antigens. Consequently, thyrocytes might behave as antigen presenting cells. Interestingly, immune cells from HT patients kill HHV-6-infected thyrocytes more efficiently than controls. Also, HT patients, but not controls, have specific T-cell responses to HHV-6 U94 protein. It is difficult to prove etiologic links between viral infections and diseases, especially in the case of a ubiquitous agent such as HHV -6. Nevertheless, our findings indicate that HHV-6 might contribute to HT development, and argue for a pathogenic association between HHV-6 and HT.
PMCID: PMC3464215  PMID: 23055929
9.  Human Herpesvirus 8 Glycoprotein B (gB), gH, and gL Can Mediate Cell Fusion 
Journal of Virology  2002;76(9):4390-4400.
Herpesvirus entry into cells and herpesvirus-induced cell fusion are related processes in that virus penetration proceeds by fusion of the viral envelope and cell membrane. To characterize the human herpesvirus 8 (HHV-8) glycoproteins that can mediate cell fusion, a luciferase reporter gene activation assay was used. Chinese hamster ovary (CHO) cells expressing the HHV-8 glycoproteins of interest along with a luciferase reporter gene under the control of the T7 promoter were cocultivated with human cells transfected with T7 RNA polymerase. Because HHV-8 glycoprotein B (gB) expressed in CHO cells localizes to the perinuclear region, a truncated form of gB (designated gBMUT) that lacks putative endocytosis signals was constructed by deletion of the distal 58 amino acids of the cytoplasmic tail. HHV-8 gBMUT was expressed efficiently on the surface of CHO cells. HHV-8 gB, gH, and gL could mediate the fusion of CHO cells with two different human cell types, embryonic kidney cells and B lymphocytes. Substituting gBMUT for gB significantly enhanced the fusion of CHO cells with human embryonic kidney cells but not B lymphocytes. Thus, two human cell types known to be susceptible to HHV-8 entry were also suitable targets for cell fusion induced by HHV-8 gB, gH, and gL. For human embryonic kidney cells and B cells at least, optimal fusion was noted with the expression of all three HHV-8 glycoproteins.
PMCID: PMC155071  PMID: 11932406
10.  CXC-Chemokine Receptor 4 Is Not a Coreceptor for Human Herpesvirus 7 Entry into CD4+ T Cells 
Journal of Virology  2000;74(4):2011-2016.
Human herpesvirus 7 (HHV-7) is a T-lymphotropic virus which utilizes the CD4 receptor as its main receptor to enter the target cells. Hence, HHV-7 can interfere with human immunodeficiency virus type 1 (HIV-1) infection in CD4+ T cells. It was recently suggested that the CXC chemokine receptor 4 (CXCR4), which was found to be a crucial coreceptor for T-tropic HIV-1 strains, may also play a role in the HHV-7 infection process. However, the results presented here demonstrate that CXCR4 is not involved in HHV-7 infection. The natural ligand of CXCR4, SDF-1α, was not able to inhibit HHV-7 infection in SupT1 cells or in CD8+ T-cell-depleted peripheral blood mononuclear cells. Also, AMD3100, a specific CXCR4 antagonist with potent antiviral activity against T-tropic HIV strains (50% inhibitory concentration [IC50], 1 to 10 ng/ml), completely failed to inhibit HHV-7 infection (IC50, >250 μg/ml). Thus, two different agents known to specifically interact with CXCR4 were not able to inhibit HHV-7 infection. Other T-lymphoid cell lines, expressing both CD4 and CXCR4 (e.g., HUT-78 and MT-4) could not be infected by HHV-7. In addition, the CD4-transfected cell lines HOS.CD4 and U87.CD4 and the CD4/CXCR4 double-transfected cell lines HOS.CD4.CXCR4 and U87.CD4.CXCR4 were not infectable with HHV-7. Also, we found no down-regulation of surface-bound or intracellular CXCR4 in HHV-7-infected CD4+ T cells. As compared to uninfected SupT1 cells, stromal cell-derived factor 1α (SDF-1α)/CXCR4-mediated intracellular calcium flux was unchanged in SupT1 cells that were acutely or persistently infected with HHV-7. All these data argue against CXCR4 as a receptor involved in the HHV-7 infection process.
PMCID: PMC111679  PMID: 10644374
11.  Kaposi's Sarcoma-Associated Herpesvirus Induces the Phosphatidylinositol 3-Kinase-PKC-ζ-MEK-ERK Signaling Pathway in Target Cells Early during Infection: Implications for Infectivity 
Journal of Virology  2003;77(2):1524-1539.
Human herpesvirus 8 (HHV-8) is implicated in the pathogenesis of Kaposi's sarcoma. HHV-8 envelope glycoprotein B (gB) possesses the RGD motif known to interact with integrin molecules, and HHV-8 infectivity was inhibited by RGD peptides, by antibodies against α3 and β1 integrins, and by soluble α3β1 integrin (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Anti-gB antibodies immunoprecipitated the virus α3 and β1 complexes, and virus-binding studies suggest a role for α3β1 in HHV-8 entry. HHV-8 infection induced the integrin-mediated activation of focal adhesion kinase (FAK), implicating a role for integrin and the associated signaling pathways in HHV-8 entry into the target cells. Immediately after infection, target cells exhibited morphological changes and cytoskeletal rearrangements, suggesting the induction of signal pathways. As early as 5 min postinfection, HHV-8 activated the MEK-ERK1/2 pathway. The focal adhesion components phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C-ζ (PKC-ζ) were recruited as upstream mediators of the HHV-8-induced ERK pathway. Anti-HHV-8 gB-neutralizing antibodies and soluble α3β1 integrin inhibited the virus-induced signaling pathways. Early kinetics of the cellular signaling pathway and its activation by UV-inactivated HHV-8 suggest a role for virus binding and/or entry but not viral gene expression in this induction. Studies with human α3 integrin-transfected Chinese hamster ovary cells and FAK-negative mouse DU3 cells suggest that the α3β1 integrin and FAK play roles in the HHV-8 mediated signal induction. Inhibitors specific for PI 3-kinase, PKC-ζ, MEK, and ERK significantly reduced the virus infectivity without affecting virus binding to the target cells. Examination of viral DNA entry suggests a role for PI 3-kinase in HHV-8 entry into the target cells and a role for PKC-ζ, MEK, and ERK at a post-viral entry stage of infection. These findings implicate a critical role for integrin-associated mitogenic signaling in HHV-8's infection of target cells and suggest that, by orchestrating the signal cascade, HHV-8 may create an appropriate intracellular environment to facilitate the infection.
PMCID: PMC140802  PMID: 12502866
12.  Detailed Study of the Interaction between Human Herpesvirus 6B Glycoprotein Complex and Its Cellular Receptor, Human CD134 
Journal of Virology  2014;88(18):10875-10882.
Recently, we identified a novel receptor, CD134, which interacts with the human herpesvirus 6B (HHV-6B) glycoprotein (g)H/gL/gQ1/gQ2 complex and plays a key role in the entry of HHV-6B into target cells. However, details of the interaction between the HHV-6B gH/gL/gQ1/gQ2 complex and CD134 were unknown. In this study, we identified a cysteine-rich domain (CRD), CDR2, of CD134 that is critical for binding to the HHV-6B glycoprotein complex and HHV-6B infection. Furthermore, we found that the expression of HHV-6B gQ1 and gQ2 subunits was sufficient for CD134 binding, which is different from the binding of human herpesvirus 6A (HHV-6A) to its receptor, CD46. Finally, we identified a region in gQ1 critical for HHV-6B gQ1 function. These results contribute much to our understanding of the interaction between this ligand and receptor.
IMPORTANCE We identified the domain in HHV-6B entry receptor CD134 and the components in the HHV-6B gH/gL/gQ1/gQ2 complex required for ligand-receptor binding during HHV-6B infection. Furthermore, we identified domains in gQ1 proteins of HHV-6A and -6B and a key amino acid residue in HHV-6B gQ1 required for its function. These data should be the basis for further investigation of ligand-receptor interaction in the study of HHV-6A and -6B.
PMCID: PMC4178835  PMID: 25008928
13.  Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Envelope Glycoprotein gB Induces the Integrin-Dependent Focal Adhesion Kinase-Src-Phosphatidylinositol 3-Kinase-Rho GTPase Signal Pathways and Cytoskeletal Rearrangements 
Journal of Virology  2004;78(8):4207-4223.
Human herpesvirus 8 (HHV-8; Kaposi's sarcoma-associated herpesvirus) envelope glycoprotein gB possesses an RGD motif, interacts with α3β1 integrin, and uses it as one of the entry receptors. HHV-8 induces the integrin-dependent focal adhesion kinase (FAK), a critical step in the outside-in signaling pathways necessary for the subsequent phosphorylation of other cellular kinases, cytoskeletal rearrangements, and other functions. As an initial step toward deciphering the role of HHV-8 gB-integrin interaction in infection, signal pathways induced by gB were examined. A truncated form of gB without the transmembrane and carboxyl domains (gBΔTM), a gBΔTM mutant form (gBΔTM-RGA) with an RGD-to-RGA mutation, and inhibitors of cellular kinases were used. HHV-8 gBΔTM, but not gBΔTM-RGA, induced FAK phosphorylation in target cells, which was in part dependent on the presence of α3β1 integrin. FAK was critical for the subsequent phosphorylation of Src by gBΔTM, and Src induction was essential for the phosphorylation of phosphatidylinositol 3-kinase (PI-3K). HHV-8 gBΔTM-induced PI-3K was essential for the induction of RhoA and Cdc42 Rho GTPases that was accompanied by the cytoskeletal rearrangements. These gB-induced morphological changes were inhibited by the PI-3K inhibitors. Ezrin, one of the essential elements required to cross-link the actin cytoskeleton with the plasma membrane and to induce the morphological changes, was induced by the Rho GTPases. Inhibition of cellular tyrosine kinases by the brief treatment of cells with 4′,5,7-trihydroxyisoflavone (genistein) blocked the entry of HHV-8 into target cells. These findings suggest that, independently of other viral glycoproteins and via its RGD motif, HHV-8 gB induces integrin-dependent pre-existing FAK-Src-PI-3K-Rho GTPase kinases. Since these signal pathways play vital roles in host cell endocytosis and movement of particulate materials in the cytoplasm, the early stages of HHV-8 gB interaction with host cells may provide a very conducive environment for the successful infection of target cells.
PMCID: PMC374261  PMID: 15047836
14.  Activation of the Epstein-Barr virus replicative cycle by human herpesvirus 6. 
Journal of Virology  1993;67(11):6768-6777.
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.
PMCID: PMC238118  PMID: 8411380
15.  Reactivation of Chromosomally Integrated Human Herpesvirus-6 by Telomeric Circle Formation 
PLoS Genetics  2013;9(12):e1004033.
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.
Author Summary
Human herpesviruses (HHVs) can reside in a lifelong non-infectious state displaying limited activity in their host and protected from immune responses. One possible way by which HHV-6 achieves this state is by integrating into the telomeric ends of human chromosomes, which are highly repetitive sequences that protect the ends of chromosomes from damage. Various stress conditions can reactivate latent HHV-6 thus increasing the severity of multiple human disorders. Recently, we have identified Chlamydia infection as a natural cause of latent HHV-6 reactivation. Here, we have sought to elucidate the molecular mechanism of HHV-6 reactivation. HHV-6 efficiently utilizes the well-organized telomere maintenance machinery of the host cell to exit from its inactive state and initiate replication to form new viral DNA. We provide experimental evidence that the shortening of telomeres, as a consequence of interference with telomere maintenance, triggers the release of the integrated virus from the chromosome. Our data provide a mechanistic basis to understand HHV-6 reactivation scenarios, which in light of the high prevalence of HHV-6 infection and the possibility of chromosomal integration of other common viruses like HHV-7 have important medical consequences for several million people worldwide.
PMCID: PMC3868596  PMID: 24367281
16.  Human CD4+ T Cell Response to Human Herpesvirus 6 
Journal of Virology  2012;86(9):4776-4792.
Following primary infection, human herpesvirus 6 (HHV-6) establishes a persistent infection for life. HHV-6 reactivation has been associated with transplant rejection, delayed engraftment, encephalitis, muscular dystrophy, and drug-induced hypersensitivity syndrome. The poor understanding of the targets and outcome of the cellular immune response to HHV-6 makes it difficult to outline the role of HHV-6 in human disease. To fill in this gap, we characterized CD4 T cell responses to HHV-6 using peripheral blood mononuclear cell (PBMC) and T cell lines generated from healthy donors. CD4+ T cells responding to HHV-6 in peripheral blood were observed at frequencies below 0.1% of total T cells but could be expanded easily in vitro. Analysis of cytokines in supernatants of PBMC and T cell cultures challenged with HHV-6 preparations indicated that gamma interferon (IFN-γ) and interleukin-10 (IL-10) were appropriate markers of the HHV-6 cellular response. Eleven CD4+ T cell epitopes, all but one derived from abundant virion components, were identified. The response was highly cross-reactive between HHV-6A and HHV-6B variants. Seven of the CD4+ T cell epitopes do not share significant homologies with other known human pathogens, including the closely related human viruses human herpesvirus 7 (HHV-7) and human cytomegalovirus (HCMV). Major histocompatibility complex (MHC) tetramers generated with these epitopes were able to detect HHV-6-specific T cell populations. These findings provide a window into the immune response to HHV-6 and provide a basis for tracking HHV-6 cellular immune responses.
PMCID: PMC3347333  PMID: 22357271
17.  Variant-Specific Tropism of Human Herpesvirus 6 in Human Astrocytes 
Journal of Virology  2005;79(15):9439-9448.
Though first described as a lymphotropic virus, human herpesvirus 6 (HHV-6) is highly neuropathogenic. Two viral variants are known: HHV-6A and HHV-6B. Both variants can infect glial cells and have been differentially associated with central nervous system diseases, suggesting an HHV-6 variant-specific tropism for glial cell subtypes. We have performed infections with both viral variants in human progenitor-derived astrocytes (HPDA) and monitored infected cell cultures for cytopathic effect (CPE), intra- and extracellular viral DNA load, the presence of viral particles by electronic microscopy, mRNA transcription, and viral protein expression. HHV-6A established a productive infection with CPE, visible intracellular virions, and high virus DNA loads. HHV-6B-infected HPDA showed no morphological changes, intracellular viral particles, and decreasing intra- and extracellular viral DNA over time. After long-term passage, HHV-6B-infected HPDA had stable but low levels of intracellular viral DNA load with no detectable viral mRNA. Our results demonstrate that HHV-6A and HHV-6B have differential tropisms and patterns of infection for HPDA in vitro, where HHV-6A results in a productive lytic infection. In contrast, HHV-6B was associated with a nonproductive infection. These findings suggest that HHV-6 variants might be responsible for specific infection patterns in glial cells in vivo. Astrocytes may be an important reservoir for this virus in which differential tropism of HHV-6A and HHV-6B may be associated with different disease outcomes.
PMCID: PMC1181567  PMID: 16014907
18.  Human Herpesvirus 8 Infects and Replicates in Primary Cultures of Activated B Lymphocytes through DC-SIGN▿  
Journal of Virology  2008;82(10):4793-4806.
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.
PMCID: PMC2346758  PMID: 18337571
19.  Identification of the Human Herpesvirus 6A gQ1 Domain Essential for Its Functional Conformation 
Journal of Virology  2013;87(12):7054-7063.
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.
PMCID: PMC3676137  PMID: 23596294
20.  Novel Marmoset (Callithrix jacchus) Model of Human Herpesvirus 6A and 6B Infections: Immunologic, Virologic and Radiologic Characterization 
PLoS Pathogens  2013;9(1):e1003138.
Human Herpesvirus 6 (HHV-6) is a ubiquitous virus with an estimated seroprevalence of 95% in the adult population. HHV-6 is associated with several neurologic disorders, including multiple sclerosis, an inflammatory demyelinating disease affecting the CNS. Animal models of HHV-6 infection would help clarify its role in human disease but have been slow to develop because rodents lack CD46, the receptor for cellular entry. Therefore, we investigated the effects of HHV-6 infections in a non-human primate, the common marmoset Callithrix jacchus. We inoculated a total of 12 marmosets with HHV-6A and HHV-6B intravenously and HHV-6A intranasally. Animals were monitored for 25 weeks post-inoculation clinically, immunologically and by MRI. Marmosets inoculated with HHV-6A intravenously exhibited neurologic symptoms and generated virus-specific antibody responses, while those inoculated intravenously with HHV-6B were asymptomatic and generated comparatively lower antibody responses. Viral DNA was detected at a low frequency in paraffin-embedded CNS tissue of a subset of marmosets inoculated with HHV-6A and HHV-6B intravenously. When different routes of HHV-6A inoculation were compared, intravenous inoculation resulted in virus-specific antibody responses and infrequent detection of viral DNA in the periphery, while intranasal inoculation resulted in negligible virus-specific antibody responses and frequent detection of viral DNA in the periphery. Moreover, marmosets inoculated with HHV-6A intravenously exhibited neurologic symptoms, while marmosets inoculated with HHV-6A intranasally were asymptomatic. We demonstrate that a marmoset model of HHV-6 infection can serve to further define the contribution of this ubiquitous virus to human neurologic disorders.
Author Summary
The human herpesviruses HHV-6A and HHV-6B are widely distributed in the human population, but also specifically associated with several central nervous system (CNS) diseases. We investigated HHV-6A and HHV-6B infections in the common marmoset, a non-human primate naturally susceptible to infection, unlike rodents. We inoculated marmosets with HHV-6A and HHV-6B intravenously, and with HHV-6A intranasally, to represent a more physiologic route of infection. Following intravenous HHV-6A inoculation, marmosets exhibited clinical symptoms with evidence of spinal cord pathology. Animals inoculated intravenously with HHV-6B were asymptomatic and without detectable CNS pathology. Both groups developed robust anti-viral antibody responses, and we detected viral DNA infrequently in the periphery. By contrast, marmosets inoculated intranasally with HHV-6A were asymptomatic, failed to generate anti-viral antibodies, and we frequently detected viral DNA in the periphery. Interestingly, HHV-6 DNA was detected in brain and spinal cord sections of several intravenously inoculated animals, demonstrating that HHV-6 can gain access to and persist in the CNS. These observations help to define the contributions of ubiquitous herpesviruses to neurologic disease development in a non-human primate. As little is known about the acquisition and host response to HHV-6A, this model may clarify how this virus may trigger or potentiate disease.
PMCID: PMC3561285  PMID: 23382677
21.  A Human Herpesvirus 6A-Encoded MicroRNA: Role in Viral Lytic Replication 
Journal of Virology  2014;89(5):2615-2627.
Human herpesvirus 6A (HHV-6A), a member of the betaherpesvirus family, is associated with several human diseases. Like all herpesviruses, HHV-6A establishes a lifelong, latent infection in its host. Reactivation of HHV-6A is frequent within the immunosuppressed and immunocompromised populations and results in lytic viral replication within multiple organs, often leading to severe disease. MicroRNAs (miRNAs) are key regulators of multiple cellular processes that regulate the translation of specific transcripts. miRNAs carried by herpesviruses play important roles in modulating the host cell, thereby facilitating a suitable environment for productive viral infection and/or latency. Currently, there are approximately 150 known human herpesvirus-encoded miRNAs, although an miRNA(s) encoded by HHV-6A has yet to be reported. We hypothesized that HHV-6A, like other members of the human herpesvirus family, encodes miRNAs, which function to promote viral infection. We utilized deep sequencing of small RNA species isolated from cells harboring HHV-6A to identify five novel small noncoding RNA species that originate from the viral genome, one of which has the characteristics of a viral miRNA. These RNAs are expressed during productive infection by either bacterial artificial chromosome (BAC)-derived virus in Jjhan cells or wild-type HHV-6A strain U1102 virus in HSB2 cells and are associated with the RNA induced silencing complex (RISC) machinery. Growth analyses of mutant viruses that lack each individual miRNA revealed that a viral miRNA candidate (miR-U86) targets the HHV-6A IE gene U86, thereby regulating lytic replication. The identification and biological characterization of this HHV-6A-specific miRNA is the first step to defining how the virus regulates its life cycle.
IMPORTANCE A majority of the human population is infected with human herpesvirus 6A (HHV-6A), a betaherpesvirus family member. Infections usually occur in young children, and upon resolution, the virus remains in a latent state within the host. Importantly, during times of weakened immune responses, the virus can reactivate and is correlated with significant disease states. Viruses encode many different types of factors that both undermine the host antiviral response and regulate viral replication, including small RNA species called microRNAs (miRNAs). Here we report that HHV-6A encodes at least one miRNA, which we named miR-U86. We have characterized the requirement of this viral miRNA and its impact on the viral life cycle and found that it functions to regulate a viral protein important for efficient viral replication. Our data suggest that viral miRNAs are important for HHV-6A and that they may serve as an important therapeutic target to inhibit the virus.
PMCID: PMC4325741  PMID: 25520507
22.  African Great Apes Are Naturally Infected with Roseoloviruses Closely Related to Human Herpesvirus 7 
Journal of Virology  2014;88(22):13212-13220.
Primates are naturally infected with herpesviruses. During the last 15 years, the search for homologues of human herpesviruses in nonhuman primates allowed the identification of numerous viruses belonging to the different herpesvirus subfamilies and genera. No simian homologue of human herpesvirus 7 (HHV7) has been reported to date. To investigate the putative existence of HHV7-like viruses in African great apes, we applied the consensus-degenerate hybrid oligonucleotide primers (CODEHOP) program-mediated PCR strategy to blood DNA samples from the four common chimpanzee subspecies (Pan troglodytes verus, P. t. ellioti, P. t. troglodytes, and P. t. schweinfurthii), pygmy chimpanzees (Pan paniscus), as well as lowland gorillas (Gorilla gorilla gorilla). This study led to the discovery of a novel roseolovirus close to HHV7 in each of these nonhuman primate species and subspecies. Generation of the partial glycoprotein B (1,111-bp) and full-length DNA polymerase (3,036/3,042-bp) gene sequences allowed the deciphering of their evolutionary relationships. Phylogenetic analyses revealed that HHV7 and its African great ape homologues formed well-supported monophyletic lineages whose topological resemblance to the host phylogeny is suggestive of virus-host codivergence. Notably, the evolutionary branching points that separate HHV7 from African great ape herpesvirus 7 are remarkably congruent with the dates of divergence of their hosts. Our study shows that African great apes are hosts of human herpesvirus homologues, including HHV7 homologues, and that the latter, like other DNA viruses that establish persistent infections, have cospeciated with their hosts.
IMPORTANCE Human herpesviruses are known to possess simian homologues. However, surprisingly, none has been identified to date for human herpesvirus 7 (HHV7). This study is the first to describe simian homologues of HHV7. The extensive search performed on almost all African great ape species and subspecies, i.e., common chimpanzees of the four subspecies, bonobos, and lowland gorillas, has allowed characterization of a specific virus in each. Genetic characterization of the partial glycoprotein B and full-length DNA polymerase gene sequences, followed by their phylogenetic analysis and estimation of divergence times, has shed light on the evolutionary relationships of these viruses. In this respect, we conclusively demonstrate the cospeciation between these new viruses and their hosts and report cases of cross-species transmission between two common chimpanzee subspecies in both directions.
PMCID: PMC4249110  PMID: 25187544
23.  No serological evidence for a role of HHV-6 infection in chronic fatigue syndrome 
Human herpesvirus 6A (HHV-6A) and human herpesvirus 6B (HHV-6B) are associated with a variety of conditions including rash, fever, and encephalitis and may play a role in several neurological diseases. Here luciferase immunoprecipitation systems (LIPS) was used to develop HHV-6 serologic diagnostic tests using antigens encoded by the U11 gene from HHV-6A (p100) and HHV-6B (p101). Analysis of the antibody responses against Renilla luciferase fusions with different HHV-6B p101 fragments identified an antigenic fragment (amino acids 389 to 858) that demonstrated ~86% seropositivity in serum samples from healthy US blood donors. Additional experiments detected a HHV-6A antigenic fragment (amino acids 751-870) that showed ~48% antibody seropositivity in samples from Mali, Africa, a known HHV-6A endemic region. In contrast to the high levels of HHV-6A immunoreactivity seen in the African samples, testing of US blood donors with the HHV-6A p100 antigenic fragment revealed little immunoreactivity. To potentially explore the role of HHV-6 infection in human disease, a blinded cohort of controls (n=59) and chronic fatigue syndrome (CFS) patients (n=72) from the US was examined for serum antibodies. While only a few of the controls and CFS patients showed high level immunoreactivity with HHV-6A, a majority of both the controls and CFS patients showed significant immunoreactivity with HHV-6B. However, no statistically significant differences in antibody levels or frequency of HHV-6A or HHV-6B infection were detected between the controls and CFS patients. These findings highlight the utility of LIPS for exploring the seroepidemiology of HHV-6A and HHV-6B infection, but suggest that these viruses are unlikely to play a role in the pathogenesis of CFS.
PMCID: PMC3493030  PMID: 23145212
Chronic Fatigue Syndrome (CFS); Human Herpes Virus-6 (HHV6); luciferase immunoprecipitation systems (LIPS)
24.  Human Herpesvirus 8 Envelope Glycoprotein B Mediates Cell Adhesion via Its RGD Sequence 
Journal of Virology  2003;77(5):3131-3147.
Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus, implicated in the pathogenesis of Kaposi's sarcoma, utilizes heparan sulfate-like molecules to bind the target cells via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8-gB possesses the Arg-Gly-Asp (RGD) motif, the minimal peptide region of many proteins known to interact with subsets of host cell surface integrins. HHV-8 utilizes α3β1 integrin as one of the receptors for its entry into the target cells via its gB interaction and induces the activation of focal adhesion kinase (FAK) (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Since FAK activation is the first step in the outside-in signaling necessary for integrin-mediated cytoskeletal rearrangements, cell adhesions, motility, and proliferation, the ability of HHV-8-gB to mediate the target cell adhesion was examined. A truncated form of gB without the transmembrane and carboxyl domains (gBΔTM) and a gBΔTM mutant (gBΔTM-RGA) with a single amino acid mutation (RGD to RGA) were expressed in a baculovirus system and purified. Radiolabeled HHV-8-gBΔTM, gBΔTM-RGA, and ΔTMgpK8.1A proteins bound to the human foreskin fibroblasts (HFFs), human dermal microvascular endothelial (HMVEC-d) cells, human B (BJAB) cells, and Chinese hamster ovary (CHO-K1) cells with equal efficiency, which was blocked by preincubation of proteins with soluble heparin. Maxisorp plate-bound gBΔTM protein induced the adhesion of HFFs and HMVEC-d and monkey kidney epithelial (CV-1) cells in a dose-dependent manner. In contrast, the gBΔTM-RGA and ΔTMgpK8.1A proteins did not mediate adhesion. Adhesion mediated by gBΔTM was blocked by the preincubation of target cells with RGD-containing peptides or by the preincubation of plate-bound gBΔTM protein with rabbit antibodies against gB peptide containing the RGD sequence. In contrast, adhesion was not blocked by the preincubation of plate-bound gBΔTM protein with heparin, suggesting that the adhesion is mediated by the RGD amino acids of gB, which is independent of the heparin-binding domain of gB. Integrin-ligand interaction is dependent on divalent cations. Adhesion induced by the gBΔTM was blocked by EDTA, thus suggesting the role of integrins in the observed adhesions. Focal adhesion components such as FAK and paxillin were activated by the binding of gBΔTM protein to the target cells but not by gBΔTM-RGA protein binding. Inhibition of FAK phosphorylation by genistein blocked gBΔTM-induced FAK activation and cell adhesion. These findings suggest that HHV-8-gB could mediate cell adhesion via its RGD motif interaction with the cell surface integrin molecules and indicate the induction of cellular signaling pathways, which may play roles in the infection of target cells and in Kaposi's sarcoma pathogenesis.
PMCID: PMC149745  PMID: 12584338
25.  Human herpesvirus 6. 
Clinical Microbiology Reviews  1997;10(3):521-567.
Human herpesvirus 6 variant A (HHV-6A) and human herpesvirus 6 variant B (HHV-6B) are two closely related yet distinct viruses. These visuses belong to the Roseolovirus genus of the betaherpesvirus subfamily; they are most closely related to human herpesvirus 7 and then to human cytomegalovirus. Over 95% of people older than 2 years of age are seropositive for either or both HHV-6 variants, and current serologic methods are incapable of discriminating infection with one variant from infection with the other. HHV-6A has not been etiologically linked to any human disease, but such an association will probably be found soon. HHV-6B is the etiologic agent of the common childhood illness exanthem subitum (roseola infantum or sixth disease) and related febrile illnesses. These viruses are frequently active and associated with illness in immunocompromised patients and may play a role in the etiology of Hodgkin's disease and other malignancies. HHV-6 is a commensal inhabitant of brains; various neurologic manifestations, including convulsions and encephalitis, can occur during primary HHV-6 infection or in immunocompromised patients. HHV-6 and distribution in the central nervous system are altered in patients with multiple sclerosis; the significance of this is under investigation.
PMCID: PMC172933  PMID: 9227865

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