Journal of human virology最新文献

Objective: A high prevalence of GB virus C (GBV-C)/hepatitis G virus (HGV) RNA and E2 antibodies is observable in human immunodeficiency virus type 1 (HIV-1)-infected individuals in Belgium, including intravenous drug users (IDUs), in whom the highest prevalence is observed. A molecular analysis of GBV-C/HGV could give indications on the origin of this infection in IDUs.

Methods: We directly sequenced 7 GBV-C/HGV isolates from this IDU population and performed a phylogenetic analysis comparing the results to known GBV-C/HGV sequences.

Results: All 7 isolates were GBV-C/HGV genome type 2. Three were found to be subtype 2a, and 4 belonged to the 2b subtype. No specific clustering was observed for strains obtained from IDUs in Belgium, and they were interspersed between other sequences with long branch lengths.

Conclusion: Based on our results, it is unlikely that the IDUs were infected recently by GBV-C/HGV from a common ancestral virus.

Objectives: To investigate some of the cellular consequences of HIV-1 Tat expression in human astrocytic cells. This study is based on evidence that cellular factors play a critical role in facilitating transcriptional activation by Tat through its interaction with the trans-acting-response (TAR) RNA element and upstream HIV-1 long terminal repeat (LTR) promoter binding site. STUDY DESIGN-METHODS: Using the previously established astrocytic cell line of human origin stably transfected with Tat cDNA, we analyzed the formation of a nucleoprotein complex consisting of three cellular proteins associated with TAR RNA using ultraviolet (UV) crosslinking and glutathione-S-transferase (GST) pull-down assays.

Results: UV crosslinking experiments reveal that the molecular masses of the proteins range from 50 to 62 kd. Transient transfection studies demonstrate that the presence of these proteins correlates with the ability of Tat to transactivate the HIV-1 LTR in the absence of the trinucleotide bulge, a region within TAR that has been shown to be important for Tat-TAR interaction. A combination of GST pull-down assays and RNA binding studies demonstrates that the 50-kd protein interacts with both Tat and TAR and is likely to be NF-kappa B p50.

Conclusions: Taken together, these data suggest that in the absence of a functional Tat binding site such as TAR (which tethers the viral protein to the RNA), cellular protein NF-kappa B p50 may be able to bring Tat into the RNA binding complex Tat has been shown to activate expression of a variety of cellular genes that may not contain a binding site for Tat but do contain binding sites for NF-kappa B family members. The results presented in this study may be relevant for Tat-mediated transactivation of cellular as well as viral genes, both of which might contribute to the central nervous system damage associated with HIV-1 infection.

Objective: Because of the important medical consequences of human cytomegalovirus (HCMV) infection in human immunodeficiency virus (HIV)-infected individuals, we wanted to understand the molecular interactions that occur during co-infection. Specifically, in this study, we wanted to identify the transactivating target sequences on the HIV long terminal repeat (LTR) that responded to HCMV infection.

Study design/methods: In this study, we transfected the HIV-LTR into human fibroblasts and then mapped the regulation of this promoter following HCMV infection and co-transfection with the HCMV immediate-early (IE) gene product IE2-86. In addition, we examined IE2-86 binding to specific sequences in the HIV-LTR by electrophoretic mobility shift assay.

Results: Our results documented that HCMV and IE2-86 could transactivate the HIV-LTR. In mapping the regions of the HIV-LTR that IE2-86 transactivates, we identified discrete target sequences between -120 and -20 that are the major transactivating regions for the IE2-86-mediated effects and determined that IE2-86 could specifically bind to several discrete sequences within this region of the HIV-LTR.

Conclusions: Our discovery of the binding of IE2-86 to the HIV-LTR, coupled with its ability to transactivate the HIV-LTR and induce cellular transcription factors, points to potential molecular mechanisms used by HCMV to upregulate the HIV life cycle and, consequently, exacerbate the conditions observed in individuals co-infected with HCMV and HIV.

Objective: To identify and characterize an open reading frame (ORF) in association with its mRNA and a coding protein recognized by anti-human herpesvirus 8 (HHV-8) monoclonal antibody B291.

Study design/methods: The antigen detected by B291 was characterized by immunofluorescence method, Western blot analysis, laser confocal microscopy, and immunohistology cDNA of the protein was identified by immunoscreening the HHV-8 cDNA library with B291 and sequenced. Structure and kinetics of the mRNA expression was investigated by 5',3' rapid amplification of cDNA ends (RACE) and Northern blot analysis.

Results: Viral interferon regulatory factor (vIRF; approximately 50 kd) was a protein recognized by B291. It was detected in 12-O-tetradecanoyl phorbol-13 acetate (TPA)-induced, but not uninduced, HHV-8-infected cell lines (BCBL-1 and BCP-1) and the tumor cells obtained from a SCID mouse injected with uninduced BCBL-1. The expression of vIRF was predominantly in the nucleus and was partially diminished by phosphonoformic acid (PFA) treatment, whereas the mRNA (approximately 2.4 kb) was accumulated.

Conclusions: vIRF is an early protein expressed in the nucleus and may be involved in Kaposi's sarcoma tumor formation.

Objectives: To study the cellular localization of human herpesvirus 8 (HHV-8) in rare cases of HHV-8 infection from Italy that are associated neither with human immunodeficiency virus (HIV) infection nor Kaposi's sarcoma (KS).

Methods: The presence and distribution of HHV-8-infected cells was investigated by direct in situ polymerase chain reaction (PCR) in the lymph node tissues from 2 patients with reactive lymphadenopathies with florid follicular hyperplasia and increased vascularity and in the lung tissue from 1 patient with chronic interstitial pneumonitis.

Results: HHV-8 was localized in lymphoid and monocyte-macrophage cells scattered in the interfollicular regions of both lymph nodes but not in endothelial cells. In the lung tissue, HHV-8 was found in the inflammatory cells infiltrating the interalveolar interstitium, in endothelial cells of the pulmonary vasculature, and in rare pneumocytes.

Conclusions: HHV-8 can infect nonneoplastic lymph nodes of immunocompetent subjects, and the distribution of infected cells outside of the germinal centers resembles that of Epstein-Barr virus (EBV)-infected cells in the lymph nodes in the course of infectious mononucleosis. Endothelial cells and pneumocytes may be a target of HHV-8 infection out of the KS setting, at least in the presence of a chronic inflammatory process.

Objective: To explore and compare the relations between proviral DNA load and CD4+ lymphocyte counts in both HIV-2 monotypic and HIV dual infection.

Study design/methods: In Dakar, Senegal, where the HIV-1 and HIV-2 epidemics overlap, serum and peripheral blood mononuclear cell (PBMC) DNA samples were collected from registered female sex workers and hospitalized patients. Sera were evaluated for reactivity to antigens of HIV-1 and HIV-2 by immunoblot; dual reactivity was confirmed with recombinant envelope peptides for HIV-1 and HIV-2. These samples were then subjected to HIV-1 and HIV-2 proviral DNA polymerase chain reaction (PCR). To evaluate the HIV-2 cellular proviral DNA loads, a quantitative competitive PCR (QC-PCR) was developed using nested primers to amplify the gag region of HIV-2. This assay used an internal competitor generated by inserting 25 bp in the first-round PCR target sequence. T-lymphocyte subset counts were estimated by flow cytometry for both HIV-2 monotypic and dually infected persons.

Results: 35 HIV-2-infected and 33 dually seroreactive samples were evaluated in this study. The CD4+ lymphocyte counts were similar in both groups, with mean values of 449 +/- 390 cells/mm3 for the HIV-2 monotypic infected persons and 476 +/- 308 cells/mm3 among the dually infected persons. However, the median proviral loads differed significantly, with those in the HIV-2 group ranging from 63.2 to 669.8 copies/10(5) CD4+ cells and demonstrating an inverse correlation with CD4+ lymphocyte count. The HIV dually infected persons showed less variation in viral load, ranging from 9.9 to 43.3 copies/10(5) CD4+ cells. Among the HIV dually infected persons, low HIV-2 proviral load was correlated with low CD4+ lymphocyte counts.

Conclusions: The HIV-2 proviral loads in HIV dually infected persons were significantly lower than those in HIV-2 monotypically infected individuals (P < .0001), despite comparable CD4+ lymphocyte counts. These results suggest that different HIV-2 proviral dynamics prevail in HIV dual infection.

Objective: Human herpesvirus 8/Kaposi's sarcoma herpesvirus (HHV-8/KSHV) contains, in addition to genes required for viral replication, an unique set of nonstructural genes which may be part of viral mimicry and contribute to viral replication and pathogenesis in vivo. Among these, HHV-8 encodes four open reading frames (ORFs) that show homology to the transcription factors of the interferon regulatory factor (IRF) family. In this study we demonstrate that one of these ORFs (vIRF-2) encodes a protein with mobility of 18 kd which has distinct pattern of expression and properties from the cellular IRFs and the previously characterized vIRF-1.

Methods: We cloned vIRF-2 by polymerase chain reaction (PCR) and studied its expression by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR). Biologic activities were tested by chloramphenicol acetyltransferase (CAT) assay in transiently transfected mammalian cells. We characterized its DNA binding specificity by electrophoretic mobility shift analysis (EMSA) and its protein-protein interactions by in vitro pull-down assay.

Results: Although low levels of vIRF-2 mRNAs can be detected in the HHV-8-positive BCBL-1 tumor cell line, 12-0-tetradecanoylphorbol-13-acetate (TPA) treatment does not stimulate expression of vIRF-2 gene together with primary lytic cycle genes. Recombinant vIRF-2, which can form homodimers, does not bind specifically to the oligodeoxynucleotide repeats corresponding to the interferon-stimulated response element (ISRE), but it does bind to the NF-kappa B binding site. The fusion protein generated from vIRF-2 and the RelA (p65) activation domain stimulates transcriptional activity of HIV LTR, which contains two NF-kappa B sites, but does not stimulate the interferon-beta (IFNB) promoter, which contains only one NF-kappa B site. Interaction between recombinant vIRF-2 and cellular IRFs such as IRF-1, IRF-2, and ICSBP was detected by in vitro binding assay, but no interaction between IRF-3 and vIRF-2 was found. Interaction of vIRF-2 with RelA (p65) and the carboxy-terminal part of p300 was also observed. In a transient transfection assay, vIRF-2 inhibits the IRF-1- or IRF-3-mediated transcriptional activation of interferon-alpha (IFNA) gene promoter in infected cells and downmodulates RelA (p65)-stimulated activity of HIV LTR.

Conclusions: These results suggest that, by interacting with cellular transcription factors and cofactors, vIRF-2 may modulate the expression of the early inflammatory genes and potentially deregulate the immune system.