Monica Galvan

Human herpesvirus-8 (Kaposi's sarcoma-associated herpesvirus) ORF50 interacts synergistically with the tat gene product in transactivating the human immunodeficiency virus type 1 LTR

Human herpesvirus-8 (HHV-8) is a lymphotropic virus associated with several AIDS-related neoplasms. Two ORFs play a critical role in the regulation of virus replication: ORF50, encoding an immediate-early transcriptional activator, and ORF57, encoding a post-transcriptional regulator. We analysed their effects on the activation of the human immunodeficiency virus type 1 (HIV-1) LTR. ORF50 interacted synergically with tat, inducing a 10-fold enhancement of HIV-1 LTR transactivation. This effect occurred both in BCBL-1 cells, latently infected with HHV-8, and in HL3T1 cells, an epithelial cell line non-permissive to HHV-8 infection. Also, ORF57 enhanced tat-induced transactivation of HIV-1 LTR, but only in BCBL-1 cells, suggesting that its action was likely mediated by the induction of other viral functions. Finally, when both ORFs were expressed, the enhancement of transactivation induced by ORF50 was partially inhibited. The findings suggest that ORF57 can modulate ORF50 activity and that ORF50 may render biologically active small amounts of tat.

TEMPORAL MAPPING OF TRANSCRIPTS IN HUMAN HERPESVIRUS-7

Transcription of human herpesvirus-7 (HHV-7) in cultures of productively infected T-cells was studied. Transcription of HHV-7 was regulated by the typical herpesvirus cascade in which alpha, beta and gamma genes are sequentially transcribed. Transcripts of U10, U14, U18, U31, U39, U41, U42, U53, U73 and U89/90 were detected 3 h after infection and were not inhibited by the absence of protein synthesis and therefore were alpha functions. U19 and U18/20 were beta genes; their transcription was inhibited by cycloheximide but not by phosphonoacetate, an inhibitor of DNA synthesis. U60/66 and U98/100 were gamma genes since their spliced transcripts were not detected in cells treated with phosphonoacetate. HHV-7 transcription was regulated by complex mechanisms, which involve the temporal coordinated activation of specific viral promoters and post-transcriptional processing. Splice mechanisms were also temporally regulated. Transcription of U89/90 pre-mRNA and splice took place simultaneously in the immediate-early phase. On the other hand, U16/17 pre-mRNA was synthesized with typical alpha kinetics, but the spliced product was regulated as a beta function. Likewise, the primary transcripts of U60/66 and U98/100 were alpha and beta, respectively, but both spliced products were synthesized in the late phase of virus replication. Finally, HHV-7 supported a bona fide latent infection in the adult population, since viral transcripts were not detected in peripheral blood mononuclear cells of healthy donors infected with HHV-7.

Transient Expression of Human Herpesvirus-8 (Kaposi’s Sarcoma-Associated Herpesvirus) ORF50 Enhances HIV-1 Replication

Objective: Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi’s sarcoma, the most common neoplasm in patients with acquired immunodeficiency syndrome. Current evidence indicates that activation of viral replication may be critical to the development of the disease. A key factor in the induction of HHV-8 lytic replication is ORF50, an immediate-early gene encoding a transactivating protein necessary for viral reactivation. We recently reported that ORF50 synergizes with HIV-1 tat at a post-transcriptional level. To study the effects of these molecular interactions upon HIV replication and biology, cell lines of different origin were transiently transfected with ORF50 and subsequently infected with HIV. Methods: Jurkat, BCBL-1 and A172 cells were transfected with ORF50 and subsequently infected with different MOI of HIV. The development of infection was evaluated by analyzing p24 antigen release, production of infectious HIV particles and the presence and transcription of HIV proviral DNA. Results: ORF50 induced increased levels of HIV replication and production in CD4+ Jurkat T cells. Transfection of ORF50 into nonsusceptible B and glial cells (BCBL-1 and A172, respectively) increased cell susceptibility to infection and resulted in transient permissiveness to HIV replication. Conclusions: HIV replication can be significantly affected by the presence of HHV-8. Expression of ORF50 increases the efficiency of HIV infection in different cell types. This potentially could result in enhanced HIV spread within the infected organism and faster progression of the disease.

Long-Lasting CD3+ T-Cell Deficiency after Cord Blood Stem Cell Transplantation in a Human Herpesvirus 6-Infected Child

ABSTRACT We report a long-lasting (8-month) reactivation of human herpesvirus 6 (HHV-6) infection in child who had undergone cord blood stem cell transplantation. The reactivation was characterized by high viral loads and by immediate-early mRNA positivity. HHV-6 infection was associated with a deep depletion of CD3, while the CD4/CD8 ratio remained substantially unchanged.