- Email: [email protected]
10: Analysis of HHV-6 envelope glycoproteins
Abstracts, 5th Int. Conf. on HHV-6 & 7, 1–3 May 2006, Barcelona, Spain / Journal of Clinical Virology 37 Suppl. 1 (2006) S97–118
S99
7 Differential tropism of human herpesvirus 6 (HHV-6) variants and induction of latency by HHV-6A in oligodendrocytes
9 Human herpesvirus 6 variants A and B (HHV-6A and HHV-6B) exhibit different tropism for human fibroblasts
J. Ahlqvist *, J. Fotheringham, N. Akhyani, K. Yao, A. Fogdell-Hahn, S. Jacobson. Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA E-mail address: [email protected]
D. Boutolleau1,2 *, P. Bonnafous1 , H. Agut1 , A. Gautheret-Dejean1,3 . de Virologie, Universit´e Pierre et Marie Curie-Paris 6, EA 2387, Groupe Hospitalier Piti´e-Salpˆetri`ere, Paris, France, 2 Laboratoire de Bact´ eriologie-Virologie, Facult´e de M´edecine Paris Sud, CHU de Bicˆetre, Le Kremlin-Bicˆetre, France, 3 Laboratoire de Microbiologie, Facult´e des Sciences Pharmaceutiques et Biologiques, Paris, France E-mail address: [email protected]
Background: Human herpesvirus 6A and 6B (HHV-6A and HHV-6B) are associated with a number of clinical disorders. HHV-6B causes the common childhood disease exhanthem subitum, while the pathologic characteristics for HHV-6A are less well defined it has been suggested that HHV-6A is more neurotropic. Objectives: To study tropisms and growth characteristics of HHV-6A and 6B in glial cells. Study Design: The human oligodendrocyte cell line MO3.13 was infected with HHV-6A and 6B. Results: HHV-6A infection induced significantly more cytopathic effect than infection with HHV-6B. HHV-6B induced an abortive infection associated with a decrease of the initial viral DNA load over time, early RNA expression, and no expression of viral antigen. In contrast, infection with HHV-6A DNA persisted in cells for at least 62 days. During the acute phase of infection with HHV-6A, intracellular and extra cellular viral load increased and cells expressed the viral protein IE-2 and gp116/54/64. No HHV-6A RNA or protein was expressed after 30 days post infection, suggesting that HHV-6A formed a latent infection. Conclusions: Our results suggest that HHV-6A and HHV-6B have different tropism in MO3.13 cells. Differences between HHV-6A and -6B infection in different neural cell types may be associated with different neurological diseases. 8 P53 exon 4 polymorphisms and the susceptibility to herpesvirus type 6 and type 1 infection in renal transplant recipients J.L. Leite *, J.A. Manfrinatto, M. Mazalli, L.S. Ward. FCM-UNICAMP, Campinas, Brazil E-mail address: [email protected] Background: Herpesviruses, like every infectious agent that requires the replication of its own genome in the host nucleus, have to overcome the barrier presented by p53. The ability of p53 inducing apoptosis is significantly reduced by 2 polymorphisms, at codons 47 and 72 of exon 4. Objectives and Study Design: In order to investigate the influence of this germline inheritance on the susceptibility to HHV6 and HHV1 infection, we examined 78 renal transplant recipients and 151 controls. Results: HHV6, but not HHV1, infection was more frequent among the renal transplant patients (35.89%) than in the control population (11.25%) (p < 0.001). There was no difference in the number of HHV6 or HHV1 infected patients between S47 and wild type codon 47 of p53 cases. Also, HHV1 infection rate was similar in all 72 p53 genotype cases. However, HHV6 positive cases were more frequent among renal transplant patients with 72 variants (17 out of the 28 HHV6 positive cases, 60.71%) than in patients presenting the wild-type Arg/Arg genotype (11 out of the 39 positive cases, 28.20%) (F; p = 0.001). In fact, the presence of a germline P72 genotype increased the risk for HHV-6 infection more than 5 times (OR = 5.479; 95% CI: 1.992– 15.069). Conclusions: Our data suggest that infection of cells presenting an impaired apoptotic system may favor survival and represent an evolutionary developed advantage for HHV6. Genotyping for 72 p53 polymorphism may be useful to screen for patients at higher risk for post-transplant infections hence identifying individuals that could benefit from preventive treatment.
1 Laboratoire
Background: HHV-6 is a ubiquitous betaherpesvirus for which two variants, A and B, have been described on the basis of different genetic, antigenic, and biological characteristics. Nevertheless, HHV-6A and HHV-6B are increasingly recognized as distinct pathogens, with specific epidemiologic and pathogenic properties. Indeed, HHV-6B accounts for the majority of HHV-6 primary infections, whereas the clinical expression of HHV-6A primary infection remains unknown, and HHV-6A seems to be preferentially associated with central nervous system (CNS) disorders. The differential tropism of HHV-6 variants may be one of the key factors to explain these different pathologic spectra. Objectives: To study HHV-6A and -6B tropisms for human embryonic lung fibroblasts (HELFs). Study Design: HELF cultures were infected with different strains of both variants and monitored for various virologic parameters during 8 days. Results: No cytopathic effect was observed in HHV-6-infected HELFs. Nevertheless, in HHV-6A-infected HELFs, the increase of intracellular viral load, the detection of both viral nuclear antigens and the late viral transcript of U100 gene were consistent with an efficient replication of HHV-6A. Moreover, the use of the monoclonal antibody J4.48 assessed that CD46 was used as a viral receptor. Conversely, HHV-6B was not able to replicate efficiently in HELFs, since viral load decreased continuously over the study period, and neither viral antigen nor late U100 gene transcript could be detected in infected cells. Conclusion: Only HHV-6A is able to establish an active infection in HELFs, as previously reported for different types of cells from CNS. This variant-specific tropism may help to better understand the variantspecific role of HHV-6 in pathology.
Molecular Biology 10 Analysis of HHV-6 envelope glycoproteins Y. Mori *. Laboratory of Virology and Vaccinology, Division of Biomedical Research, National Institute of Biomedical Innovation. Ibaraki, Osaka, Japan E-mail address: [email protected] Background: HHV-6 isolates can be classified into variants A and B (HHV-6A and HHV-B). The two variants have different cell tropisms, and human CD46 has been reported to be a cellular receptor for HHV-6. In the herpesvirus family, the envelope glycoproteins play critical roles in viral infection, including attachment, penetration, cellto-cell spread, and the envelopment and maturation of nascent viral particles. Objectives: The aim of this study was to analyze the roles of HHV-6 glycoproteins in virus entry. Study Design and Results: We found that HHV-6 gH/gL complex associates with glycoprotein Q1 and gQ2 (gQ1 and gQ2) on the viral envelope, and the gH/gL/gQ1/gQ2 complex is a viral ligand for its cellular receptor, CD46. Furthermore, we found that HHV-6 U47 gene which is a homolog of HCMV glycoprotein O (gO) gene, encoded another third component of the HHV-6 gH/gL-containing envelope complex. And the gH/gL/gO complex did not bind to human CD46, indicating that the complex is not a ligand for CD46.
S100 Abstracts, 5th Int. Conf. on HHV-6 & 7, 1–3 May 2006, Barcelona, Spain / Journal of Clinical Virology 37 Suppl. 1 (2006) S97–118 Conclusions: These results suggest that the viral envelope contains at least two kinds of gH/gL complexes, gH/gL/gQ1/gQ2 and gH/gL/gO, and that the gH/gL/gO complex may play different roles from gH/gL/ gQ1/gQ2 during viral infection. 11 Biological characterization of HHV-6 U94 D. Di Luca *. Department of Experimental and Diagnostic Medicine, University of Ferrara, Italy E-mail address: [email protected] Background: Human herpesvirus 6 (HHV-6) is the only human herpesvirus encoding U94/rep. HHV-6 U94 is homologous to REP68/78, a human parvovirus non-structural protein with pleiotropic effects. Parvoviral REP68/78 regulated viral gene expression, inhibits HIV LTR transcription and down regulates cellular oncogenes. Objectives: With the aim to characterize the biological activity of HHV-6 U94 we produced and purified the protein. Study Design: HHV-6 U94 was cloned in prokaryotic expression vectors, produced in bacteria, recovered from the medium and purified by chromatography. The purified protein was added to cell cultures infected with human beta-herpesviruses. Results: U94 was internalized and localized in the cell nucleus. U94 did not affect cell viability or cell replication. The replication of HHV-6, HHV-7 and HCMV was significantly inhibited by the presence of U94. The inhibitory effect was associated to ss-DNA binding activity. Conclusions: U94/REP plays a key role in the regulation of viral replication, and might be a promising candidate for the development of new specific antiviral agents. 12 Interactions of herpesviruses 6 and 7 with HIV-1 in human lymphoid tissue L. Margolis *. National Institute of Child Health and Human Development Bethesda, MD, USA E-mail address: [email protected] Background: HIV infection is often accompanied by infection with other pathogens that worsen the clinical course of HIV disease. However, several viruses have been shown to suppress HIV-1 replication in vivo and in vitro. Objectives: To study the effect of two herpesviruses, 6 and 7 (HHV-6 and HHV-7) on HIV-1 replication in human lymphoid tissue, where critical events of HIV disease occur. Study Design: Isolated blocks of human tonsillar tissue were coinfected with HIV-1 and HHV-6 or HHV-7 and viruses’ replication, cytokines production and cell surface markers expression were monitored. Results: Both herpesviruses selectively suppressed the replication of CCR5-tropic (R5) HIV-1 in coinfected tissue. However, the molecular mechanisms of HHV-7- and HHV-6-mediated HIV-1 suppression were different. Unlike HHV-6, which affects R5 HIV-1 by upregulating RANTES, HHV-7 upregulates neither CCR5-binding chemokines nor other tested cytokines. Rather, the inhibition of R5 HIV-1 by HHV-7 was associated with a marked downregulation of CD4, the cellular receptor shared by HHV-7 and HIV-1. HHV-7-triggered CD4 downregulation was sufficient for HIV-1 inhibition, since comparable downregulation of CD4 with cyclotriazadisulfonamide, a synthetic macrocycle that specifically modulates expression of CD4, resulted in suppression of HIV infection similar to that seen in HHV-7-infected tissues. In contrast to R5 HIV-1, CXCR4-tropic (X4) HIV-1 was only minimally, if at all, suppressed by HHV-7 coinfection. This selectivity in suppression of R5 and X4 HIV-1 is explained by a suppression of HHV-7 replication in X4- but not in R5-coinfected tissues. Conclusions: The results suggest that HIV-1 and herpesviruses may interfere with each other in lymphoid tissue in vivo, thus potentially affecting the progression of HIV-1 disease. This conclusion is supported by the difference in the biological properties of
simian immunodeficiency virus (SIV) isolated from singly infected macaques and from animals coinfected with HHV-6. Knowledge of the mechanisms of interaction of HIV-1 with HHV-6 and HHV-7 as well as with other pathogens that modulate HIV-1 replication may provide new insights into HIV pathogenesis and lead to the development of new anti-HIV therapeutic strategies. 13 Anti-HHV-6B DR7 antibody production in rabbit: application to DR7B oncoprotein detection in hodgkin’s disease A. Lacroix1 *, A. Jaccard2 , D. Bordessoule2 , S. Ranger-Rogez1 . 1 Microbiologie, Facult´ e de Pharmacie and CHU Dupuytren, 2 H´ ematologie clinique, CHU Dupuytren, Limoges, France E-mail address: [email protected] Background: Human herpesvirus-6 (HHV-6) DR7 sequences were previously demonstrated to possess transforming, transactivating and oncogenic properties. Objectives: The aim of this study was to obtain anti-DR7 antibodies after protein production to look for DR7 expression in tissues from patients suffering from Hodgkin’s disease (HD). DR7B was studied because of its greater frequency than DR7A in HD patients. Study Design: DR7B protein was expressed using a system based on Semliki Forest Virus (SFV). Briefly, poly-His tagged DR7B was cloned into pSFV1 vector before ClaI linearization and RNA polymerase SP6 transcription. Recombinant RNAs were then transfected into BHK-21 cells by electroporation. Protein expression was checked by immunofluorescence and western-blot using anti-poly-His tag antibodies. A rabbit was then immunized with the purified fraction and sera were collected and tested by immunofluorescence on HHV-6B infected cells. Immunohistochemistry was performed on 51 HHV-6 DNA positive lymph nodes from HD patients initially with a commercial antibody directed against the viral gp116/gp64/gp54 complex, and then with the anti-DR7B antibody. Results: Using the gp116/gp64/gp54 complex, most samples were positive on Reed-Sternberg (RS) cells and also on environmental cells, although with the DR7B antibody RS cells were found more frequently and were almost exclusively positive. Conclusion: This study reveals the widespread expression of DR7B in RS cells confirming probable oncogenic properties of DR7 in vivo in HD. 14 Functional interactions between human herpesvirus-6 (HHV-6) immediate-early 2 (IE2) protein and ubiquitin-conjugating enzyme 9 (Ubc9) in the absence of sumoylation A. Tomoiu, A. Gravel, L. Flamand *. Laboratory of Virology, Rheumatology and Immunology Research Center, Centre de Recherche du CHUL and Faculty of Medicine, Laval University, Quebec, Qc, Canada E-mail address: louis.fl[email protected] Background: HHV-6 IE2 protein is a potent transactivator of cellular and viral promoters. Currently there are no studies describing interactions between IE2 and host cell proteins in relation with promoter transactivation. Objectives: To understand the biology of IE2 by identifying proteins interacting with IE2. Study Design: We generated a LexA-IE2 fusion protein and screened, using the yeast two-hybrid system, a Jurkat T-cell cDNA library for IE2-interacting proteins. Results: The most frequently isolated IE2-interacting protein was Ubc9, a protein involved in the small ubiquitin-like modifier (SUMO) conjugation pathway. Using mutants of IE2, we mapped the IE2-Ubc9 interacting region to residues 989-1037. The IE2-Ubc9 interaction was of functional significance as Ubc9 overexpression repressed promoter activation by IE2. The C93S Ubc9 mutant exhibited similar effects on IE2, indicating that the SUMO conjugating function of Ubc9 is not required for its repressive action on IE2. No consensus
S99
7 Differential tropism of human herpesvirus 6 (HHV-6) variants and induction of latency by HHV-6A in oligodendrocytes
9 Human herpesvirus 6 variants A and B (HHV-6A and HHV-6B) exhibit different tropism for human fibroblasts
J. Ahlqvist *, J. Fotheringham, N. Akhyani, K. Yao, A. Fogdell-Hahn, S. Jacobson. Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA E-mail address: [email protected]
D. Boutolleau1,2 *, P. Bonnafous1 , H. Agut1 , A. Gautheret-Dejean1,3 . de Virologie, Universit´e Pierre et Marie Curie-Paris 6, EA 2387, Groupe Hospitalier Piti´e-Salpˆetri`ere, Paris, France, 2 Laboratoire de Bact´ eriologie-Virologie, Facult´e de M´edecine Paris Sud, CHU de Bicˆetre, Le Kremlin-Bicˆetre, France, 3 Laboratoire de Microbiologie, Facult´e des Sciences Pharmaceutiques et Biologiques, Paris, France E-mail address: [email protected]
Background: Human herpesvirus 6A and 6B (HHV-6A and HHV-6B) are associated with a number of clinical disorders. HHV-6B causes the common childhood disease exhanthem subitum, while the pathologic characteristics for HHV-6A are less well defined it has been suggested that HHV-6A is more neurotropic. Objectives: To study tropisms and growth characteristics of HHV-6A and 6B in glial cells. Study Design: The human oligodendrocyte cell line MO3.13 was infected with HHV-6A and 6B. Results: HHV-6A infection induced significantly more cytopathic effect than infection with HHV-6B. HHV-6B induced an abortive infection associated with a decrease of the initial viral DNA load over time, early RNA expression, and no expression of viral antigen. In contrast, infection with HHV-6A DNA persisted in cells for at least 62 days. During the acute phase of infection with HHV-6A, intracellular and extra cellular viral load increased and cells expressed the viral protein IE-2 and gp116/54/64. No HHV-6A RNA or protein was expressed after 30 days post infection, suggesting that HHV-6A formed a latent infection. Conclusions: Our results suggest that HHV-6A and HHV-6B have different tropism in MO3.13 cells. Differences between HHV-6A and -6B infection in different neural cell types may be associated with different neurological diseases. 8 P53 exon 4 polymorphisms and the susceptibility to herpesvirus type 6 and type 1 infection in renal transplant recipients J.L. Leite *, J.A. Manfrinatto, M. Mazalli, L.S. Ward. FCM-UNICAMP, Campinas, Brazil E-mail address: [email protected] Background: Herpesviruses, like every infectious agent that requires the replication of its own genome in the host nucleus, have to overcome the barrier presented by p53. The ability of p53 inducing apoptosis is significantly reduced by 2 polymorphisms, at codons 47 and 72 of exon 4. Objectives and Study Design: In order to investigate the influence of this germline inheritance on the susceptibility to HHV6 and HHV1 infection, we examined 78 renal transplant recipients and 151 controls. Results: HHV6, but not HHV1, infection was more frequent among the renal transplant patients (35.89%) than in the control population (11.25%) (p < 0.001). There was no difference in the number of HHV6 or HHV1 infected patients between S47 and wild type codon 47 of p53 cases. Also, HHV1 infection rate was similar in all 72 p53 genotype cases. However, HHV6 positive cases were more frequent among renal transplant patients with 72 variants (17 out of the 28 HHV6 positive cases, 60.71%) than in patients presenting the wild-type Arg/Arg genotype (11 out of the 39 positive cases, 28.20%) (F; p = 0.001). In fact, the presence of a germline P72 genotype increased the risk for HHV-6 infection more than 5 times (OR = 5.479; 95% CI: 1.992– 15.069). Conclusions: Our data suggest that infection of cells presenting an impaired apoptotic system may favor survival and represent an evolutionary developed advantage for HHV6. Genotyping for 72 p53 polymorphism may be useful to screen for patients at higher risk for post-transplant infections hence identifying individuals that could benefit from preventive treatment.
1 Laboratoire
Background: HHV-6 is a ubiquitous betaherpesvirus for which two variants, A and B, have been described on the basis of different genetic, antigenic, and biological characteristics. Nevertheless, HHV-6A and HHV-6B are increasingly recognized as distinct pathogens, with specific epidemiologic and pathogenic properties. Indeed, HHV-6B accounts for the majority of HHV-6 primary infections, whereas the clinical expression of HHV-6A primary infection remains unknown, and HHV-6A seems to be preferentially associated with central nervous system (CNS) disorders. The differential tropism of HHV-6 variants may be one of the key factors to explain these different pathologic spectra. Objectives: To study HHV-6A and -6B tropisms for human embryonic lung fibroblasts (HELFs). Study Design: HELF cultures were infected with different strains of both variants and monitored for various virologic parameters during 8 days. Results: No cytopathic effect was observed in HHV-6-infected HELFs. Nevertheless, in HHV-6A-infected HELFs, the increase of intracellular viral load, the detection of both viral nuclear antigens and the late viral transcript of U100 gene were consistent with an efficient replication of HHV-6A. Moreover, the use of the monoclonal antibody J4.48 assessed that CD46 was used as a viral receptor. Conversely, HHV-6B was not able to replicate efficiently in HELFs, since viral load decreased continuously over the study period, and neither viral antigen nor late U100 gene transcript could be detected in infected cells. Conclusion: Only HHV-6A is able to establish an active infection in HELFs, as previously reported for different types of cells from CNS. This variant-specific tropism may help to better understand the variantspecific role of HHV-6 in pathology.
Molecular Biology 10 Analysis of HHV-6 envelope glycoproteins Y. Mori *. Laboratory of Virology and Vaccinology, Division of Biomedical Research, National Institute of Biomedical Innovation. Ibaraki, Osaka, Japan E-mail address: [email protected] Background: HHV-6 isolates can be classified into variants A and B (HHV-6A and HHV-B). The two variants have different cell tropisms, and human CD46 has been reported to be a cellular receptor for HHV-6. In the herpesvirus family, the envelope glycoproteins play critical roles in viral infection, including attachment, penetration, cellto-cell spread, and the envelopment and maturation of nascent viral particles. Objectives: The aim of this study was to analyze the roles of HHV-6 glycoproteins in virus entry. Study Design and Results: We found that HHV-6 gH/gL complex associates with glycoprotein Q1 and gQ2 (gQ1 and gQ2) on the viral envelope, and the gH/gL/gQ1/gQ2 complex is a viral ligand for its cellular receptor, CD46. Furthermore, we found that HHV-6 U47 gene which is a homolog of HCMV glycoprotein O (gO) gene, encoded another third component of the HHV-6 gH/gL-containing envelope complex. And the gH/gL/gO complex did not bind to human CD46, indicating that the complex is not a ligand for CD46.
S100 Abstracts, 5th Int. Conf. on HHV-6 & 7, 1–3 May 2006, Barcelona, Spain / Journal of Clinical Virology 37 Suppl. 1 (2006) S97–118 Conclusions: These results suggest that the viral envelope contains at least two kinds of gH/gL complexes, gH/gL/gQ1/gQ2 and gH/gL/gO, and that the gH/gL/gO complex may play different roles from gH/gL/ gQ1/gQ2 during viral infection. 11 Biological characterization of HHV-6 U94 D. Di Luca *. Department of Experimental and Diagnostic Medicine, University of Ferrara, Italy E-mail address: [email protected] Background: Human herpesvirus 6 (HHV-6) is the only human herpesvirus encoding U94/rep. HHV-6 U94 is homologous to REP68/78, a human parvovirus non-structural protein with pleiotropic effects. Parvoviral REP68/78 regulated viral gene expression, inhibits HIV LTR transcription and down regulates cellular oncogenes. Objectives: With the aim to characterize the biological activity of HHV-6 U94 we produced and purified the protein. Study Design: HHV-6 U94 was cloned in prokaryotic expression vectors, produced in bacteria, recovered from the medium and purified by chromatography. The purified protein was added to cell cultures infected with human beta-herpesviruses. Results: U94 was internalized and localized in the cell nucleus. U94 did not affect cell viability or cell replication. The replication of HHV-6, HHV-7 and HCMV was significantly inhibited by the presence of U94. The inhibitory effect was associated to ss-DNA binding activity. Conclusions: U94/REP plays a key role in the regulation of viral replication, and might be a promising candidate for the development of new specific antiviral agents. 12 Interactions of herpesviruses 6 and 7 with HIV-1 in human lymphoid tissue L. Margolis *. National Institute of Child Health and Human Development Bethesda, MD, USA E-mail address: [email protected] Background: HIV infection is often accompanied by infection with other pathogens that worsen the clinical course of HIV disease. However, several viruses have been shown to suppress HIV-1 replication in vivo and in vitro. Objectives: To study the effect of two herpesviruses, 6 and 7 (HHV-6 and HHV-7) on HIV-1 replication in human lymphoid tissue, where critical events of HIV disease occur. Study Design: Isolated blocks of human tonsillar tissue were coinfected with HIV-1 and HHV-6 or HHV-7 and viruses’ replication, cytokines production and cell surface markers expression were monitored. Results: Both herpesviruses selectively suppressed the replication of CCR5-tropic (R5) HIV-1 in coinfected tissue. However, the molecular mechanisms of HHV-7- and HHV-6-mediated HIV-1 suppression were different. Unlike HHV-6, which affects R5 HIV-1 by upregulating RANTES, HHV-7 upregulates neither CCR5-binding chemokines nor other tested cytokines. Rather, the inhibition of R5 HIV-1 by HHV-7 was associated with a marked downregulation of CD4, the cellular receptor shared by HHV-7 and HIV-1. HHV-7-triggered CD4 downregulation was sufficient for HIV-1 inhibition, since comparable downregulation of CD4 with cyclotriazadisulfonamide, a synthetic macrocycle that specifically modulates expression of CD4, resulted in suppression of HIV infection similar to that seen in HHV-7-infected tissues. In contrast to R5 HIV-1, CXCR4-tropic (X4) HIV-1 was only minimally, if at all, suppressed by HHV-7 coinfection. This selectivity in suppression of R5 and X4 HIV-1 is explained by a suppression of HHV-7 replication in X4- but not in R5-coinfected tissues. Conclusions: The results suggest that HIV-1 and herpesviruses may interfere with each other in lymphoid tissue in vivo, thus potentially affecting the progression of HIV-1 disease. This conclusion is supported by the difference in the biological properties of
simian immunodeficiency virus (SIV) isolated from singly infected macaques and from animals coinfected with HHV-6. Knowledge of the mechanisms of interaction of HIV-1 with HHV-6 and HHV-7 as well as with other pathogens that modulate HIV-1 replication may provide new insights into HIV pathogenesis and lead to the development of new anti-HIV therapeutic strategies. 13 Anti-HHV-6B DR7 antibody production in rabbit: application to DR7B oncoprotein detection in hodgkin’s disease A. Lacroix1 *, A. Jaccard2 , D. Bordessoule2 , S. Ranger-Rogez1 . 1 Microbiologie, Facult´ e de Pharmacie and CHU Dupuytren, 2 H´ ematologie clinique, CHU Dupuytren, Limoges, France E-mail address: [email protected] Background: Human herpesvirus-6 (HHV-6) DR7 sequences were previously demonstrated to possess transforming, transactivating and oncogenic properties. Objectives: The aim of this study was to obtain anti-DR7 antibodies after protein production to look for DR7 expression in tissues from patients suffering from Hodgkin’s disease (HD). DR7B was studied because of its greater frequency than DR7A in HD patients. Study Design: DR7B protein was expressed using a system based on Semliki Forest Virus (SFV). Briefly, poly-His tagged DR7B was cloned into pSFV1 vector before ClaI linearization and RNA polymerase SP6 transcription. Recombinant RNAs were then transfected into BHK-21 cells by electroporation. Protein expression was checked by immunofluorescence and western-blot using anti-poly-His tag antibodies. A rabbit was then immunized with the purified fraction and sera were collected and tested by immunofluorescence on HHV-6B infected cells. Immunohistochemistry was performed on 51 HHV-6 DNA positive lymph nodes from HD patients initially with a commercial antibody directed against the viral gp116/gp64/gp54 complex, and then with the anti-DR7B antibody. Results: Using the gp116/gp64/gp54 complex, most samples were positive on Reed-Sternberg (RS) cells and also on environmental cells, although with the DR7B antibody RS cells were found more frequently and were almost exclusively positive. Conclusion: This study reveals the widespread expression of DR7B in RS cells confirming probable oncogenic properties of DR7 in vivo in HD. 14 Functional interactions between human herpesvirus-6 (HHV-6) immediate-early 2 (IE2) protein and ubiquitin-conjugating enzyme 9 (Ubc9) in the absence of sumoylation A. Tomoiu, A. Gravel, L. Flamand *. Laboratory of Virology, Rheumatology and Immunology Research Center, Centre de Recherche du CHUL and Faculty of Medicine, Laval University, Quebec, Qc, Canada E-mail address: louis.fl[email protected] Background: HHV-6 IE2 protein is a potent transactivator of cellular and viral promoters. Currently there are no studies describing interactions between IE2 and host cell proteins in relation with promoter transactivation. Objectives: To understand the biology of IE2 by identifying proteins interacting with IE2. Study Design: We generated a LexA-IE2 fusion protein and screened, using the yeast two-hybrid system, a Jurkat T-cell cDNA library for IE2-interacting proteins. Results: The most frequently isolated IE2-interacting protein was Ubc9, a protein involved in the small ubiquitin-like modifier (SUMO) conjugation pathway. Using mutants of IE2, we mapped the IE2-Ubc9 interacting region to residues 989-1037. The IE2-Ubc9 interaction was of functional significance as Ubc9 overexpression repressed promoter activation by IE2. The C93S Ubc9 mutant exhibited similar effects on IE2, indicating that the SUMO conjugating function of Ubc9 is not required for its repressive action on IE2. No consensus