Human herpesvirus-6 and human herpesvirus-7 (HHV-6, HHV-7)

human herpesvirus 6 (HHV-6) is the major causative agent of exanthem subitum which is one of popular diseases in infant, and establishes latent infections in adults of more than 90%. Recently, the encephalitis caused by reactivated- HHV-6 has been shown in patients after transplantation. In addition, the relationship HHV-6 and drug-induced hypersensitivity syndrome has also been reported. human herpesvirus 7 (HHV-7) was isolated from the stimulated-peripheral blood lymphocytes of a healthy individual, and also causes exanthema subitum. Both viruses are related viruses which belong to betaherpesvirus subfamily, and replicate and produce progeny viruses in T cells.     

Transactivation of human immunodeficiency virus promoter by human herpesvirus 6.

Patients with acquired immunodeficiency syndrome (AIDS) are often infected with a number of other heterologous viruses in addition to the initial human immunodeficiency virus (HIV) infection, and these agents could act as potential reactivating agents of latent HIV. A new antigenically distinct herpesvirus, designated human herpesvirus 6 (HHV-6), has recently been isolated from patients with AIDS and has been shown to infect a number of different human cells, specifically human T cells, B cells, and glial cells. Since these are some of the same cells that harbor the AIDS virus, it is quite important to determine any interaction between this new herpesvirus and HIV. In this report, we demonstrate that HHV-6 can trans-activate the HIV promoter in human T-cell lines as measured by the expression of the bacterial gene chloramphenicol acetyltransferase. This indicates that stimulation of HIV gene expression by HHV-6 could play a role in HIV pathogenesis.     

Tamplicon-7, a Novel T-Lymphotropic Vector Derived from Human Herpesvirus 7

We describe the derivation of a novel T-cell-defective virus vector employing the human herpesvirus 7 (HHV-7). The new vector, designated Tamplicon-7, replicates in CD4(+) T cells. The system is composed of a helper virus and defective virus genomes derived by the replication of the input Tamplicon vector. There are two cis-acting functions required for the replication and packaging of the defective virus genomes in the presence of the helper virus: the viral DNA replication origin and the composite cleavage and packaging signal, which directs the cleavage and packaging of defective virus genomes. Viral DNA replication is compatible with the rolling circle mechanism, producing large head-to-tail concatemers of the Tamplicon vector. Thus, in the presence of the helper virus, the replicated vectors are packaged and secreted into the medium. Furthermore, we have shown that the vector can be employed to express a foreign gene, encoding the green fluorescent protein, in the T cells infected with the HHV-7 helper virus. We predict that the Tamplicon-7 vector might be potentially useful for gene therapy of diseases affecting the human CD4(+) T cells, including autoimmune diseases, T-cell lymphomas, and AIDS.     

Transposase-mediated construction of an integrated adeno-associated virus type 5 helper plasmid

Adeno-associated viruses (AAVs) are replication-defective parvoviruses that require helper virusfunctionsfor efficient productive replication. The AAVs are currently premier candidates as vectors for human gene therapy applications. In particular; much recent interest has been expressed concerning recombinant AAV serotype 5 (rAAV-5) vectors, as they appear to utilize cellular receptors distinctfrom those of the prototypical AAV serotype (AAV-2) and have been reported to have transduction properties in vivo that differ significantly from those of the prototype. One of the most popular current methodsfor the production of rAAVs involves co-transfection of human 293 cells with three plasmids: (i) an adenovirus (Ad)-derived helper plasmid containing Ad genes required for AAV replication, (ii) an AAV-derived plasmid encoding complementing AAV genes (ie., the viral rep and cap genes), and (iii) a target plasmid containing a transgene of interestflanked by AAV inverted terminal repeats (ITRs) that confer packaging and replication capabilities upon the ITR-flanked heterologous DNA. Here we describe novel plasmid reagents designed for convenient and efficient production of rAAV-S. An integrated helper plasmid containing all Ad genes requiredfor the efficient production of recombinant AAV as well as the complementing AAV genes on the same plasmid backbone, was constructed via transposase-mediated insertion into an Ad helper plasmid of a transposable element containing the AAV-5 rep and cap genes linked to a selectable marker This simple strategy can be used in the rapid and efficient construction of integrated helper plasmids derived from any reported AAV serotype for which a molecular clone exists.     

Human CD4+ T cell response to human herpesvirus 6

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.     

Pathogenic effects of human herpesvirus 6 in human lymphoid tissue ex vivo

Human herpesvirus 6 (HHV-6) is a potentially immunosuppressive agent that has been suggested to act as a cofactor in the progression of human immunodeficiency virus disease. However, the lack of suitable experimental models has hampered the elucidation of the mechanisms of HHV-6-mediated immune suppression. Here, we used ex vivo lymphoid tissue to investigate the cellular tropism and pathogenic mechanisms of HHV-6. Viral strains belonging to both HHV-6 subgroups (A and B) were able to productively infect human tonsil tissue fragments in the absence of exogenous stimulation. The majority of viral antigen-expressing cells were CD4(+) T lymphocytes expressing a nonnaive phenotype, while CD8(+) T cells were efficiently infected only with HHV-6A. Accordingly, HHV-6A infection resulted in the depletion of both CD4(+) and CD8(+) T cells, whereas in HHV-6B-infected tissue CD4(+) T cells were predominantly depleted. The expression of different cellular antigens was dramatically altered in HHV-6-infected tissues: whereas CD4 was upregulated, both CD46, which serves as a cellular receptor for HHV-6, and CD3 were downmodulated. However, CD3 downmodulation was restricted to infected cells, while the loss of CD46 expression was generalized. Moreover, HHV-6 infection markedly enhanced the production of the CC chemokine RANTES, whereas other cytokines and chemokines were only marginally affected. These results provide the first evidence, in a physiologically relevant study model, that HHV-6 can severely affect the physiology of secondary lymphoid organs through direct infection of T lymphocytes and modulation of key membrane receptors and chemokines.     

Development of Virus-Specific CD4+ and CD8+ Regulatory T Cells Induced by Human Herpesvirus 6 Infection

Human herpesvirus 6 (HHV-6) is an important immunosuppressive and immunomodulatory virus. The mechanisms by which HHV-6 establishes latency and immunosuppression in its host are not well understood. Here we characterized HHV-6-specific T cells in peripheral blood mononuclear cells (PBMCs) from HHV-6-infected donors. Our results showed that HHV-6 infection could induce both CD4⁺ and CD8⁺ HHV-6-specific regulatory T (Treg) cells. These HHV-6-specific Treg cells had potent suppressive activity and expressed high levels of Treg-associated molecules CD25, FoxP3, and GITR. Both CD4⁺ and CD8⁺ Treg cells secreted gamma interferon (IFN-γ) and interleukin-10 (IL-10) but little or no IL-2, IL-4, or transforming growth factor β (TGF-β). Furthermore, HHV-6-specifc Treg cells not only could suppress naive and HHV-6-specific CD4⁺ effector T cell immune responses but also could impair dendritic cell (DC) maturation and functions. In addition, the suppressive effects mediated by HHV-6-specific Treg cells were mainly through a cell-to-cell contact-dependent mechanism but not through the identified cytokines. These results suggest that HHV-6 may utilize the induction of Treg cells as a strategy to escape antivirus immune responses and maintain the latency and immunosuppression in infected hosts.     

Cre levels limit packaging signal excision efficiency in the Cre/loxP helper-dependent adenoviral vector system

Helper-dependent (HD) adenovirus vectors devoid of all viral coding sequences have a large cloning capacity and provide long-term transgene expression in vivo with negligible toxicity, making them attractive vectors for gene therapy. Currently, the most efficient means of producing HD vectors involves coinfecting 293 cells expressing Cre with the HD vector and a helper virus bearing a packaging signal flanked by loxP sites. Cre-mediated packaging signal excision renders the helper virus genome unpackageable but still able to replicate and provide helper functions for HD vector propagation. Typically, helper virus contamination is < or =1% pre- and < or =0.1% postpurification by CsCl banding. While these contamination levels are low, further reduction is desirable. However, this objective has not been realized since the Cre/loxP system was first developed. This lack of progress is due, at least in part, to our lack of understanding of the origins of the contaminating helper virus, thus rendering its reduction or elimination difficult to achieve. This study was designed to investigate the possible sources of contaminating helper virus persisting during HD vector amplification. The results revealed that Cre is limiting in helper virus-infected Cre-expressing 293 cells, thereby permitting helper viruses to escape packaging signal excision and propagate. The results of this study should provide a foundation for developing rational strategies to further reduce or possibly eliminate the contaminating helper virus.     

Autonomous parvovirus vectors: preventing the generation of wild-type or replication-competent virus

The preferential expression of autonomous parvoviruses in tumour cells and their oncolytic activity has attracted attention to the potential use of these viruses as vectors for cancer gene therapy. Moreover, they are non-pathogenic in adult animals and they seem to be associated with low or no immunogenicity. Other interesting features are their episomal replication and high stability. Vectors derived from the autonomous parvoviruses MVM(p) or H1 express proteins that can directly or indirectly interfere with tumour development. They retain cis- and trans-acting sequences required for viral DNA amplification; the transgene replaces part of the capsid coding genes. Their development has been hampered by low titres and contamination with replication-competent virus (RCV) that is generated through homologous recombination with helper plasmids. Several approaches have been used to avoid recombination between vectors and helpers. In most instances, reducing the homology up- or downstream of the transgene in either the vector or the helper did not significantly affect RCV production. However, completely eliminating homology downstream of the transgene, splitting VP genes on different helpers or pseudotyping vectors resulted in the production of RCV-free stocks. Although VP-containing particles could sometimes be identified in these stocks by in situ hybridisation, they did not amplify and are therefore not true RCV. The integration of capsid-coding sequences into packaging cells also reduced contamination by RCV and allowed for the amplification of vectors through serial infections. Great progress has been made recently towards the generation of truly RCV-free stocks of vectors derived from autonomous parvoviruses H1 and MVMp. Combining these new vectors with a new packaging cell line should greatly facilitate their development.     

Efficient propagation of human herpesvirus 6B in a T-cell line derived from a patient with adult T-cell leukemia/lymphoma.

The efficient propagation of the OK strain of the B variant of human herpesvirus 6 (HHV-6B) was demonstrated in a line of T cells, TaY, established from the peripheral blood lymphocytes of a patient with adult T-cell leukemia/lymphoma (ATL). Growth of TaY cells depends on the presence of IL-2 and the cells harbor HTLV-I genomes. A severe cytopathic effect (CPE) was observed in many HHV-6B(OK)-infected TaY cells one week after infection. The release of virus from HHV-6B(OK)-infected TaY cells [TaY(OK)] was first detected after three days and increased rapidly for up to seven days after infection, as demonstrated by PCR. The titer of HHV-6B(OK) in the supernatant was comparable to the value of 10(3.5) TCID50/ml obtained with PHA-activated cord blood lymphocytes (CBL) that had been infected with HHV-6B(OK). The replication of the virus was shown to depend to a considerable extent on cell viability. Electron microscopy revealed many herpesvirus-type capsid- and enveloped-viruses in the nuclei and cytoplasm of degenerated cells in TaY(OK) cultures. The U1102 strain of HHV-6A and the Z29 strain of HHV-6B also infected TaY cells productively, as detected by PCR and an immunofluorescence test. These results suggest that the activation of CD4+ T lymphocytes with mitogens such as PHA or IL-2 and the expression of some cellular gene or the HTLV-I gene might be essential for efficient propagation of HHV-6B. TaY cells should play an important role in future investigations of cell-virus interactions and genetic variations or cell tropism of HHV-6 isolates since no cell line that shows propagation of both HHV-6A and HHV-6B has been reported to date.     

Flow cytometric evaluation of antiviral agents against human herpesvirus 6

Antiviral activities of acyclovir (9-[(2-hydroxyethoxy) methyl] guanine, ACV), penciclovir (9-[4-hydroxy-3-(hydroxymethyl) butyl] guanine, PCV), ganciclovir ([9-(1,3-dihydroxy-2-propoxy) methyl] guanine, GCV), and foscarnet (phosphonoformic acid, PFA) were determined against Human Herpesvirus 6 (HHV-6) by flow cytometric technique. The technique is based on the detection of gp116 antigen expression in virus infected cells. Susceptibility was defined in terms of drug concentration which reduced the number of cells expressing HHV-6 gp116 antigen with a mean fluorescent intensity (MFI) by 50% as compared to virus infected untreated cells. GCV was found to be most effective against HHV-6 followed by PFA, PCV and ACV. For HHV-6A, the mean 50% inhibitory concentrations (IC50) of GCV and PFA were found to be 3.4 microM and 34.7 microM respectively, whereas the IC50 of ACV and PCV were found to be 53.7 microM and 37.9 microM respectively. For HHV-6B, the IC50 of GCV and PFA were found to be 5.7 microM and 71.4 microM respectively, whereas the IC50 of ACV and PCV were found to be 119.0 microM and 77.8 microM respectively. Flow cytometry is a valuable technique for the evaluation of antiviral compounds against viruses including HHV-6.     

Herpes simplex virus type 1/adeno-associated virus hybrid vectors mediate site-specific integration at the adeno-associated virus preintegration site,AAVS1, on human chromosome 19

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors have a large transgene capacity and can efficiently infect many different cell types. One disadvantage of HSV-1 vectors is their instability of transgene expression. By contrast, vectors based on adeno-associated virus (AAV) can either persist in an episomal form or integrate into the host cell genome, thereby supporting long-term gene expression. AAV expresses four rep genes, rep68, -78, -40, and -52. Of those, rep68 or rep78 are sufficient to mediate site-specific integration of the AAV DNA into the host cell genome. The major disadvantage of AAV vectors is the small transgene capacity ( approximately 4.6 kb). In this study, we constructed HSV/AAV hybrid vectors that contained, in addition to the standard HSV-1 amplicon elements, AAV rep68, rep78, both rep68 and -78, or all four rep genes and a reporter gene that was flanked by the AAV inverted terminal repeats (ITRs). Southern blots of Hirt DNA from cells transfected with the hybrid vectors and HSV-1 helper DNA demonstrated that both the AAV elements and the HSV-1 elements were functional in the context of the hybrid vector. All hybrid vectors could be packaged into HSV-1 virions, although those containing rep sequences had lower titers than vectors that did not. Site-specific integration at AAVS1 on human chromosome 19 was directly demonstrated by PCR and sequence analysis of ITR-AAVS1 junctions in hybrid vector-transduced 293 cells. Cell clones that stably expressed the transgene for at least 12 months could easily be isolated without chemical selection. In the majority of these clones, the transgene cassette was integrated at AAVS1, and no sequences outside the ITR cassette, rep in particular, were present as determined by PCR, ITR rescue/replication assays, and Southern analysis. Some of the clones contained random integrations of the transgene cassette alone or together with sequences outside the ITR cassette. These data indicate that the long-term transgene expression observed following transduction with HSV/AAV hybrid vectors is, at least in part, supported by chromosomal integration of the transgene cassette, both randomly and site specifically.     

Altered cytokine production after human herpes virus type 6 infection.

Several strategies allow viruses to elude the surveillance of the immune system and to establish persistent infection in the host. One of such mechanisms is the immunosuppression caused by the direct infection and functional impairment of immune cells. Human Herpes virus type 6 (HHV-6) is a typical immunosuppressive agent, as suggested by its tropism for both CD4+ and CD8+ T cells, B cells, monocytes/macrophages, megakaryocytes and NK cells. In this study the production of IL-10 and IL-12 by peripheral blood mononuclear cells (PBMC) was evaluated during HHV-6 infection "in vitro". Our results demonstrate that HHV-6 up-regulates IL-10 production by PBMC. Furthermore, our data suggest that rhIFN gamma addition counteracts the effect of HHV-6 in promoting IL-10 release. To gain more insight into the role of IFN gamma, anti-IFN gamma monoclonal antibodies were added to PBMC stimulated with LPS. Neutralization of endogenous IFN gamma upregulated IL-10 release. Furthermore, HHV-6 infection inhibited IFN gamma release induced by LPS in PBMC. No basal production of IL-12 was found in PBMC. Moreover, HHV-6 infection did not induce IL-12 release by PBMC. On the contrary, IL-12 was detected in the supernatants of PBMC treated with LPS with or without rhIFN gamma. In these experimental conditions the further addition of HHV-6 markedly impaired IL-12 production. Moreover, the neutralization of IL-10 resulted in a significant up-regulation of IL-12. Finally our data suggest that the immunodysregulation induced by HHV-6 could be accounted for by a shift from a Th-1 to a Th-2 type cytokine profile.     

Nucleotide sequence analysis of a 38.5-kilobase-pair region of the genome of human herpesvirus 6 encoding human cytomegalovirus immediate-early gene homologs and transactivating functions.

Human herpesvirus 6 (HHV-6) is prevalent in the human population, with primary infection occurring early in life. Its predominant CD4+ T-lymphocyte tropism, its ability to activate human immunodeficiency virus type 1 (HIV-1) gene expression in vitro, and its upregulation of CD4 expression has led to speculation that HHV-6 may act as a positive cofactor in the progression of HIV infection to AIDS in individuals infected with both viruses. Previous sequencing studies of restricted regions of the 161.5-kbp genome of HHV-6 have demonstrated unequivocally that it is a member of the betaherpesvirus subgroup and have indicated that the HHV-6 genome is generally collinear with the unique long (UL) component of human cytomegalovirus (HCMV). In the work described in this report we have extended these sequencing studies by determining the primary structure of 38.5-kbp of the HHV-6 genome (genomic position 21.0 to 59.5 kbp). Within the sequenced region lie 31 open reading frames, 20 of which are homologous to positional counterparts in HCMV. Of particular significance is the identification of homologs of the HCMV UL36-38 and US22-type genes, which have been shown to encode transactivating proteins. We show that DNA sequences encoding these HHV-6 homologs were able to transactivate HIV-1 long terminal repeat-directed chloramphenicol acetyltransferase expression in cotransfection assays, thus demonstrating functional as well as structural conservation of these betaherpesvirus-specific gene products. Our data therefore confirm the close relationship between HHV-6 and HCMV and identify putative immediate-early regulatory genes of HHV-6 likely to play key roles in lytic replication and possibly also in the interactions between HHV-6 and HIV in dually infected cells.     

Cation-independent mannose 6-phosphate receptor blocks apoptosis induced by herpes simplex virus 1 mutants lacking glycoprotein D and is likely the target of antiapoptotic activity of the glycoprotein

Herpes simplex virus 1 mutants lacking the gene encoding glycoprotein D (gD) and the gD normally present in the envelope of the virus (gD(-/-) stocks) or mutants lacking the gD gene but containing trans-induced gD in their envelopes (gD(-/+)) cause apoptosis in human SK-N-SH cells. The gD(-/-) virions are taken up by endocytosis and are degraded, whereas gD(-/+) viruses replicate but produce gD(-/-) virus. Apoptosis is blocked by delivery of the gD gene in trans. Studies designed to test several hypotheses concerning the role of gD in apoptosis revealed the following. (i) gD(-/-) and gD(-/+) stocks induce fragmentation of cellular DNA in SK-N-SH, HEp-2, HeLa, and Vero cell lines. (ii) Chloroquine blocks apoptosis induced by gD(-/-) stocks but not by gD(-/+) stocks. The drug also rescues gD(-/-) from degradation. (iii) Cells transduced with cation-independent mannose 6-phosphate receptor (CI-MPR) block apoptosis induced by either gD(-/-) or gD(-/+) virus. (iv) Expression of sequences antisense to the cloned CI-MPR gene induced apoptosis by themselves. Wild-type virus but not gD(-/-) or gD(-/+) stocks of mutant virus blocked apoptosis induced by the expression of CI-MPR antisense sequences. These results exclude the possibility that to block apoptosis, gD must interact with its HveA receptor, a member of the tumor necrosis factor alpha receptor family. Instead, the data suggest that gD blocks the influx of lysosomal enzymes into the endosomal compartment by binding to CI-MPR. This conclusion is consistent with published reports that phosphorylated gD interacts with CI-MPR.