CCL8/MCP-2 is a target for mir-146a in HIV-1-infected human microglial cells.

MicroRNA-mediated regulation of gene expression appears to be involved in a variety of cellular processes, including development, differentiation, proliferation, and apoptosis. Mir-146a is thought to be involved in the regulation of the innate immune response, and its expression is increased in tissues associated with chronic inflammation. Among the predicted gene targets for mir-146a, the chemokine CCL8/MCP-2 is a ligand for the CCR5 chemokine receptor and a potent inhibitor of CD4/CCR5-mediated HIV-1 entry and replication. In the present study, we have analyzed changes in the expression of mir-146a in primary human fetal microglial cells upon infection with HIV-1 and found increased expression of mir-146a. We further show that CCL8/MCP-2 is a target for mir-146a in HIV-1 infected microglia, as overexpression of mir-146a prevented HIV-induced secretion of MCP-2 chemokine. The clinical relevance of our findings was evaluated in HIV-encephalitis (HIVE) brain samples in which decreased levels of MCP-2 and increased levels of mir-146a were observed, suggesting a role for mir-146a in the maintenance of HIV-mediated chronic inflammation of the brain.

BAG3 protein regulates caspase-3 activation in HIV-1-infected human primary microglial cells.

BAG3, a member of the BAG co-chaperones family, is expressed in several cell types subjected to stressful conditions, such as exposure to high temperature, heavy metals, drugs. Furthermore, it is constitutively expressed in some tumors. Among the biological activities of the protein, there is apoptosis downmodulation; this appears to be exerted through BAG3 interaction with the heat shock protein (Hsp) 70, that influences cell apoptosis at several levels. We recently reported that BAG3 protein was detectable in the cytoplasm of reactive astrocytes in HIV-1-associated encephalopathy biopsies. Here we report that downmodulation of BAG3 protein levels allows caspase-3 activation by HIV-1 infection in human primary microglial cells. This is the first reported evidence of a role for BAG3 in the balance of death versus survival during viral infection.

Expression of human neurotropic polyomavirus JCV late gene product agnoprotein in human medulloblastoma.

BACKGROUND: The human neurotropic polyomavirus, JCV, contains an open reading frame within the late region of the viral genome that encodes a 71-amino-acid protein, agnoprotein. Because accumulating evidence supports an association between JCV infection and human brain tumors, including medulloblastomas, we assessed the presence of JCV Agno gene sequences and the expression of agnoprotein in a series of 20 well-characterized medulloblastomas. METHODS: Formalin-fixed, paraffin-embedded tumor tissue samples were used for Agno gene amplification and for immunohistochemical analysis. Adjacent sections were stained with an antibody to agnoprotein and with antibodies to cellular structural and regulatory proteins, including the JCV early gene product, T antigen. RESULTS: Analysis of amplified DNA from paraffin-embedded samples revealed the presence of the Agno gene in 11 (69%) of 16 samples. Immunohistochemical analysis showed cytoplasmic localization and widespread distribution of agnoprotein in the neoplastic cells in 11 (55%) of 20 samples. The JCV early gene product, T antigen, was present in the nucleus of some, but not all, of the neoplastic cells. Some medulloblastoma samples that expressed agnoprotein had no sign of T-antigen expression. p53 was detected in only six of the 11 tumors in which agnoprotein was expressed. None of the 20 samples showed expression of the viral late capsid proteins, ruling out productive infection of the tumor cells with JCV. CONCLUSIONS: Our data provide evidence that the JCV late gene encoding the auxiliary agnoprotein is expressed in tumor cells. The finding of agnoprotein expression in the absence of T-antigen expression suggests a potential role for agnoprotein in pathways involved in the development of JCV-associated medulloblastomas.

Evidence for dysregulation of cell cycle by human polyomavirus, JCV, late auxiliary protein.

The late region of the human neurotropic JC virus encodes a 71 amino acid protein, named Agnoprotein, whose biological function remains elusive. Here we demonstrate that in the absence of other viral proteins, expression of Agnoprotein can inhibit cell growth by deregulating cell progression through the cell cycle stages. Cells with constitutive expression of Agnoprotein were largely accumulated at the G2/M stage and that decline in the activity of cyclins A and B is observed in these cells. Agnoprotein showed the ability to augment p21 promoter activity in transient transfection assay and a noticeable increase in the level of p21 is detected in cells continuously expressing Agnoprotein. Results from binding studies revealed the interaction of Agnoprotein with p53 through the N-terminal of the Agnoprotein spanning residues 1-36. Co-expression of p53 and Agnoprotein further stimulated transcription of the p21 promoter. Thus, the interaction of p53 and Agnoprotein can lead to a higher level of p21 expression and suppression of cell cycle progression during the cell cycle.

Hepatitis G virus genotypes in a tertiary care centre in southern India.

BACKGROUND: The association of GBV-C/HGV in patients at high risk of parenteral infection and those with chronic liver disease in southern India is unknown. OBJECTIVES: To study the occurrence of GBV-C/HGV in three patient groups attending a tertiary care centre in Vellore and identify the prevailing genotype(s). STUDY DESIGN: Two hundred and twenty-seven individuals were studied using an RT-PCR assay with primers specific for the 5'NCR and the NS5a-coding regions. Ten randomly selected samples were sequenced in the 5'NCR region of the GBV-C/HGV genome. RESULTS: GBV-C/HGV RNA was detected in 37/70 (52.9%) of renal transplant recipients, 18/70 (25.7%) of patients with haematological disorders and in 9/87 (10.3%) patients with chronic liver disease. Nine of the ten sequences could be assigned to genotype 2a while the remaining sequence could not be assigned. CONCLUSIONS: In this centre, GBV-C/HGV is widely prevalent in patients with high parenteral risk and in those with chronic liver disease. The predominantly circulating genotype among study patients is genotype 2a.

Distribution of the different genotypes of HCV among patients attending a tertiary care hospital in south India.

BACKGROUND: Genotyping of the hepatitis C virus (HCV) and assessment of viral load is important for designing therapeutic strategies and region specific diagnostic assays. OBJECTIVES: To determine the distribution of HCV genotypes among patients attending a tertiary care hospital in south India, and to correlate this with viral load. STUDY DESIGN: Ninety HCV RNA positive patients were recruited for the study. HCV genotyping was carried out using type-specific primers from the core region of the viral genome [J. Clin. Microbiol. 35 (1997) 201]. Viral load estimations were carried out using the Amplicor HCV Monitor (Versions 1.5 and 2, Roche Diagnostics, Branchburg, NJ, USA). Clinical details were elicited from patients' hospital records. RESULTS: Genotype 3 was detected most frequently (62.2%) followed by infection with HCV genotype 1 (18.8%). There was no significant difference seen in alanine aminotransferase (ALT) values between the two genotypes. Genotype 1 was associated with a significantly higher viral load as compared with genotype 3 (P=0.001). Parenteral transmission accounted for 61% of all infection caused. Infection with genotype 1 was significantly associated with a history of haemodialysis (P=0.01). Genotype 3 was detected more frequently in patients from east India, as compared with its detection in patients from south India (P=0.004). Similarly, genotype 1 was detected with greater frequency in individuals from south India as compared with patients from east India (P=0.004). The concordance between Ohno's genotyping assay and nucleotide sequencing, for genotypes 1 and 3, was 75%. CONCLUSIONS: HCV genotypes 1 and 3 accounted for 81% of HCV infections in patients from this geographical region. HCV genotype distribution showed regional differences and genotype 1 was associated with higher viral loads. Parenteral transmission was the major route for acquisition of HCV infection. Ohno's type-specific primer based genotyping assay can be used for distinguishing between HCV genotype 1 and non-1 HCV genotypes in laboratories that do not possess nucleotide sequencing facilities.

GB virus C/hepatitis G virus and TT virus infections among high risk renal transplant recipients in India.

BACKGROUND: GB virus C/hepatitis G virus (GBV-C/HGV) and TT virus (TTV) have been widely reported in patients with high parenteral risk such as haemodialysis and renal transplant recipients. The occurrence of these agents in association with hepatitis B virus (HBV) and hepatitis C virus (HCV), in Indian renal transplant recipients, is yet unreported. STUDY DESIGN: Molecular and serological markers of GBV-C/HGV and TTV were examined in addition to those for HBV, HCV and hepatitis D virus (HDV) in a selected group of seventy renal transplant recipients. HGV RNA detection was achieved using primers specific for the 5'NCR and NS5a regions of the genome. Anti-GBV-C/HGV antibody was detected using the mu plate anti-HG env kit (Roche, Germany). TTV DNA PCR was performed using primers specific for the coding region (method A) of the genome. In 50% of patients, TTV DNA was also tested for using primers specific for the non-coding region (method B). Host related factors such as age, alanine aminotransferase (ALT) levels, number of transfusions, haemodialysis sessions, and months following transplantation were also studied. RESULTS: Exposure rates to GBV-C/HGV, TTV (method A), HBV, HCV and HDV were 58.6, 32.9, 52.9, 54.3 and 2.9%, respectively. 'Active' infection as measured by viraemia and/or virus-specific antigenaemia for GBV-C/HGV, TTV, HBV and HCV was 52.9, 32.9, 15.7 and 52.9%, respectively. The majority of GBV-C/HGV and TTV infections were seen as co-infections with other hepatitis viruses. Single infection with GBV-C/HGV and TTV was seen in ten (14.2%) and eight (11.4%) patients, and was not associated with ALT elevation when compared to uninfected blood donors. Using univariate analysis, GBV-C/HGV RNA was significantly associated with > or =20 haemodialysis sessions. TTV DNA occurrence was not associated with any risk factors. CONCLUSIONS: There is a high occurrence of GBV-C/HGV and TTV in this select group of renal transplant recipients in India. These viruses mostly occurred in the context of co-infections with other hepatitis viruses. Long term effects of multiple hepatotropic viral infections need to be carefully documented in such transplant populations.

Comparative clinical evaluation of combination anorganic bovine-derived hydroxyapatite matrix (ABM)/cell binding peptide (P-15) and open flap debridement (DEBR) in human periodontal osseous defects: a 6 month pilot study.

A synthetic cell binding peptide (P-15) combined with anorganic bovine-derived hydroxyapatite bone matrix (ABM) was compared to open flap debridement (DEBR) in human periodontal osseous defects. Following initial preparation procedures, two osseous defects per patient were treated randomly with one of the two procedures after surgical preparation. Appropriate periodontal maintenance schedules were followed, and at 6 months clinical and radiographic assessments for soft tissue and hard tissue were performed for documentation and finalization of treatment. Statistical analysis using Student's paired t-test analyses of patient mean value from 10 patients revealed that the combination ABM/P-15 grafts demonstrated significantly better mean defect fill of 3.4+/-1.7 mm (73.2%) versus a mean defect fill of 0.6 mm (15.8%) for defects treated with DEBR. Soft tissue findings showed significant differences among treatments with ABM/P-15 compared to DEBR. These results suggest that the use of the P-15 synthetic cell-binding peptide combined with ABM yields better clinical results than DEBR.

Effects of JC virus infection on anti-apoptotic protein survivin in progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system resulting from the productive infection of oligodendrocytes by the opportunistic polyomavirus JC virus (JCV). Apoptosis is a host defense mechanism to dispose of damaged cells; however, certain viruses have the ability to deregulate apoptotic pathways to complete their life cycles. One such pathway involves survivin, a member of the inhibitor of apoptosis family, which is abundantly expressed during development in proliferating tissues but should be absent in normal, terminally differentiated cells. Immunohistochemistry performed in 20 cases of PML revealed the presence of survivin in JCV-infected oligodendrocytes and bizarre astrocytes within demyelinated plaques. Survivin up-regulation was also found in oligodendroglial and astrocytic cultures infected with JCV. Cell cycle analysis and DNA laddering demonstrated a significantly lower number of cells undergoing apoptosis on JCV infection compared with noninfected cultures; small interfering RNA inhibition of survivin resulted in a dramatic increase in apoptotic cells in JCV-infected cultures. This is the first report describing the activation of survivin by JCV infection in vitro and in PML clinical cases. These observations provide new insights into the anti-apoptotic mechanisms used by JCV to complete its lytic cycle and may suggest new therapeutic targets for PML.

Alterations of DNA damage repair pathways resulting from JCV infection.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disorder of the CNS caused by infection of glial cells with the polyomavirus, JCV. Here we report that genomic stability and DNA repair are significantly dysregulated by JCV infection of human astrocytes. Metaphase spreads exhibited increased ploidy correlating with duration of infection. Increased micronuclei formation and phospho-Histone2AX expression also indicated DNA damage. Western blot analysis revealed perturbation in expression of some DNA repair proteins including a large elevation of Rad51. Immunohistochemistry on clinical samples of PML showed robust labeling for Rad51 in nuclei of bizarre astrocytes and inclusion body-bearing oligodendrocytes that are characteristic of JCV infection. Finally, in vitro end-joining DNA repair was altered in extracts prepared from JCV-infected human astrocytes. Alterations in DNA repair pathways may be important for the life cycle of JCV and the pathogenesis of PML.

Cross-interaction between JC virus agnoprotein and human immunodeficiency virus type 1 (HIV-1) Tat modulates transcription of the HIV-1 long terminal repeat in glial cells.

The human polyomavirus JC virus (JCV) is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), which is commonly seen in AIDS patients. The bicistronic viral RNA, which is transcribed at the late phase of infection, is responsible for expressing the viral capsid proteins and a small regulatory protein, agnoprotein. Immunohistochemical analysis of brain tissue from subjects with AIDS/PML revealed colocalization of the human immunodeficiency virus type 1 (HIV-1) transactivator, Tat, and JCV agnoprotein in nucleus and cytoplasm of "bizarre" astrocytes. In accord with this observation, we detected the copresence of agnoprotein and Tat in human astrocytes upon infection with JCV and HIV-1 or in astrocytic cells expressing these proteins after transfection. Interestingly, results from infection of human astrocytes with HIV-1 and JCV showed a decrease in the level of HIV-1 replication in cells that are coinfected with JCV. Conversely, a slight increase in the level of JCV replication was observed in the presence of HIV-1. The copresence of JCV and HIV-1 in astrocytes prompted us to investigate the possible cross-interaction of agnoprotein with Tat and its impact on HIV-1 gene transcription. Our results demonstrate that agnoprotein through its N-terminal domain associates with Tat and the interaction causes the suppression of Tat-mediated enhancement of HIV-1 promoter activity in these cells. Results from RNA and protein binding assays showed that agnoprotein can inhibit the association of Tat with its target RNA sequence, TAR, and with cyclin T1. Furthermore, agnoprotein is able to interfere with cross-interaction of Tat with the p65 subunit of NF-kappaB and Sp1, whose functions are critical for Tat activation of the long terminal repeat. These observations unravel a new pathway for the molecular interaction of these two viruses in biologically relevant cells in the brains of AIDS/PML patients.

Intracellular approach for blocking JC virus gene expression by using RNA interference during viral infection.

The human polyomavirus, JC virus (JCV), encodes two regulatory proteins at the early (T antigen) and the late (agnoprotein) phases of viral infection whose activities are important for the production of the viral capsid proteins and the dysregulation of several host factors and their functions. For this study, we designed and utilized an RNA interference strategy via small interfering RNAs (siRNAs) that targeted the expression of T antigen and agnoprotein in human astrocytic cells. The treatment of cells with specific siRNA oligonucleotides targeting a conserved region of T antigen, nucleotides (nt) 4256 to 4276 (Mad-1 strain), caused a >50% decline in the level of T antigen and in its transcriptional activity upon the viral capsid genes as well as a significant reduction in viral DNA replication in infected cells. Similarly, a single siRNA that aimed at nt 324 to 342 of agnoprotein noticeably reduced early and late viral protein production. A combined treatment of the infected cells with both T-antigen and agnoprotein siRNAs completely abolished viral capsid protein production, indicative of the ability of the siRNAs to effectively halt multiplication of the virus in infected cells. These observations provide a new avenue for possible treatments of patients with the JCV-induced demyelinating disease progressive multifocal leukoencephalopathy.

JC virus-induced changes in cellular gene expression in primary human astrocytes.

Cell-type-specific transcription of the JC virus (JCV) promoter in glial cells initiates a series of events leading to viral replication in the brain and the development of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in patients with neurologic complications due to infection with human immunodeficiency virus type 1. Here we employed an in vitro infection of primary cultures of human astrocytes to compare the transcriptional profile of cellular genes after JCV infection by using an oligonucleotide-based microarray of 12600 genes. Transcription of nearly 355 genes was enhanced and expression of 130 genes was decreased to various degrees. Many transcripts that were increased upon JCV infection were found to encode proteins with properties that suggest their involvement in cell proliferation, including cyclin A and cyclin B1; signaling pathways, such as transforming growth factor beta receptor 1, platelet-derived growth factor receptor and fibroblast growth factor family receptor; and other regulatory events, such as inflammatory responses, including cyclo-oxygenase-2 (Cox-2). Microarray-based data for several cell cycle-regulatory genes were further examined by using Western blot analysis of in vitro infected astrocytes harvested early and late during the infection. Results demonstrate that protein levels of all upregulated genes were found to increase at some point during the infection time course. In parallel, immunohistochemical assessment of cell cycle proteins, including cyclins A, B1, E, and Cdk2, showed positive staining of astrocytes within PML lesions of brain tissue from patients with neuro-AIDS. Microarray analysis was found to be a useful predictor of gene expression in infected cells; however, it may not directly correlate with protein levels during infection with JCV.