Ayalew Mergia

A Kaposi's Sarcoma-Associated Herpesvirus-Encoded Ortholog of MicroRNA miR-155 Induces Human Splenic B-Cell Expansion in NOD/LtSz-scid IL2Rγnull Mice

ABSTRACT MicroRNAs (miRNAs) are small noncoding RNA molecules that function as posttranscriptional regulators of gene expression. Kaposis sarcoma (KS)-associated herpesvirus (KSHV), a B-cell-tropic virus associated with KS and B-cell lymphomas, encodes 12 miRNA genes that are highly expressed in these tumor cells. One viral miRNA, miR-K12-11, shares 100% seed sequence homology with hsa-miR-155, an oncogenic human miRNA that functions as a key regulator of hematopoiesis and B-cell differentiation. So far, in vitro studies have shown that both miRNAs can regulate a common set of cellular target genes, suggesting that miR-K12-11 may mimic miR-155 function. To comparatively study miR-K12-11 and miR-155 function in vivo, we used a foamy virus vector to express the miRNAs in human hematopoietic progenitors and performed immune reconstitutions in NOD/LtSz-scid IL2Rγnull mice. We found that ectopic expression of miR-K12-11 or miR-155 leads to a significant expansion of the CD19+ B-cell population in the spleen. Subsequent quantitative PCR analyses of these splenic B cells revealed that C/EBPβ, a transcriptional regulator of interleukin-6 that is linked to B-cell lymphoproliferative disorders, is downregulated when either miR-K12-11 or miR-155 is ectopically expressed. In addition, inhibition of miR-K12-11 function using antagomirs in KSHV-infected human primary effusion lymphoma B cells resulted in derepression of C/EBPβ transcript levels. This in vivo study validates miR-K12-11 as a functional ortholog of miR-155 in the context of hematopoiesis and suggests a novel mechanism by which KSHV miR-K12-11 induces splenic B-cell expansion and potentially KSHV-associated lymphomagenesis by targeting C/EBPβ.

Reactivity of primate sera to foamy virus Gag and Bet proteins.

In order to establish criteria for the serodiagnosis of foamy virus infections we investigated the extent to which sera from infected individuals of human and primate origin react with structural and non-structural virus proteins in immunoblot assays. Using lysates from infected cells as the source of virus antigen, antibodies were preferentially detected against the Gag proteins and the non-structural Bet protein. Both the Gag precursor molecules of 70 and 74K apparent M(r) and the cytoplasmic 60K M(r) Bet protein were found to be phosphorylated, the latter being synthesized in large amounts in infected cells. Rabbit antiserum raised against recombinant human foamy virus (HFV) Gag major capsid protein cross-reacted with foamy viruses of chimpanzee, gorilla, orang-utan, rhesus monkey and African green monkey origin. This was reflected by a broad cross-reactivity of the respective monkey sera to the Gag proteins of the various foamy virus isolates. Cross-reactivity of antisera against the Bet protein was restricted to viruses from man and the great apes. Recombinant Gag and Bet proteins expressed in prokaryotes or in insect cells were readily recognized by foamy virus-positive primate sera. Screening serum samples from chimpanzees with HFV Gag and Bet proteins expressed by recombinant baculoviruses revealed that 18 out of 35 (52%) were positive for Gag antibodies. Of these, 13 (72%) showed antibodies against the Bet protein, indicating that Bet antigen is of value in serological screening for foamy virus infections.

Cell tropism of the simian foamy virus type 1 (SFV-1)

Abstract: Several cell lines representing different species and cell types were tested for simian foamy virus type 1 (SFV‐1) infection. SFV‐1 infections were monitored by polymerase chain reaction, reverse transcriptase, cytopathology, and immunofluorescent assays. All cells tested were permissive for SFV‐1, demonstrating that SFV‐1 has a broad host range with respect to species and cell types. Infected fibroblasts, epithelial cells, and neural cells all showed extensive cytopathology that is characteristic of foamy virus infection. No cytopathology was induced in T cell‐derived Jurkats and Hut‐78 cell lines. The cytopathic effects in B and macrophage originated cells were delayed by several days. Cytopathology in these cell lines was not as dramatic as seen in the infected fibroblast and epithelial cells. The reverse transcriptase values in the SFV‐1 infected lymphoid and macrophage cell lines were severalfold lower than that of the fibroblasts and epithelial cells. Therefore, SFV‐1 appears to establish a low level persistent infection in lymphoid and macrophage cell lines.

Foamy virus vectors.

Gene therapy is a promising novel treatment for a variety of human diseases. Successful application of gene therapy requires the availability of vehicles with the ability to efficiently deliver and express genes. Viral vectors are efficient means of transferring a gene of interest into target cells. Current available vehicles for gene transfer are either inefficient or potentially unsafe for human gene therapy applications. Foamy viruses offer a fresh alternative vector system for gene transfer with the potential to overcome the concerns of the current vectors. Foamy viruses are nonpathogenic and have a broad host range with the ability to infect various types of cells from different species. Foamy virus replication is distinct and may provide an edge for foamy virus vector usage over other retroviral vectors. These features offer the foamy vectors unique opportunities to deliver several genes into a number of different cell types in vivo safely and efficiently. The principal problems for the design of foamy virus vectors have been solved, and several foamy virus vectors that efficiently transduce a variety of cell types are available. This chapter reviews specific features of foamy virus vector systems and recent advances in the development and use of these vectors.

Accuracy estimation of foamy virus genome copying.

BackgroundFoamy viruses (FVs) are the most genetically stable viruses of the retrovirus family. This is in contrast to the in vitro error rate found for recombinant FV reverse transcriptase (RT). To investigate the accuracy of FV genome copying in vivo we analyzed the occurrence of mutations in HEK 293T cell culture after a single round of reverse transcription using a replication-deficient vector system. Furthermore, the frequency of FV recombination by template switching (TS) and the cross-packaging ability of different FV strains were analyzed.ResultsWe initially sequenced 90,000 nucleotides and detected 39 mutations, corresponding to an in vivo error rate of approximately 4 × 10-4 per site per replication cycle. Surprisingly, all mutations were transitions from G to A, suggesting that APOBEC3 activity is the driving force for the majority of mutations detected in our experimental system. In line with this, we detected a late but significant APOBEC3G and 3F mRNA by quantitative PCR in the cells. We then analyzed 170,000 additional nucleotides from experiments in which we co-transfected the APOBEC3-interfering foamy viral bet gene and observed a significant 50% drop in G to A mutations, indicating that APOBEC activity indeed contributes substantially to the foamy viral replication error rate in vivo. However, even in the presence of Bet, 35 out of 37 substitutions were G to A, suggesting that residual APOBEC activity accounted for most of the observed mutations. If we subtract these APOBEC-like mutations from the total number of mutations, we calculate a maximal intrinsic in vivo error rate of 1.1 × 10-5 per site per replication. In addition to the point mutations, we detected one 49 bp deletion within the analyzed 260000 nucleotides.Analysis of the recombination frequency of FV vector genomes revealed a 27% probability for a template switching (TS) event within a 1 kilobase (kb) region. This corresponds to a 98% probability that FVs undergo at least one additional TS event per replication cycle. We also show that a given FV particle is able to cross-transfer a heterologous FV genome, although at reduced efficiency than the homologous vector.ConclusionOur results indicate that the copying of the FV genome is more accurate than previously thought. On the other hand recombination among FV genomes appears to be a frequent event.

Caveolin-1 Modulates HIV-1 Envelope-Induced Bystander Apoptosis through gp41

ABSTRACT Human immunodeficiency virus (HIV) envelope (Env)-mediated bystander apoptosis is known to cause the progressive, severe, and irreversible loss of CD4+ T cells in HIV-1-infected patients. Env-induced bystander apoptosis has been shown to be gp41 dependent and related to the membrane hemifusion between envelope-expressing cells and target cells. Caveolin-1 (Cav-1), the scaffold protein of specific membrane lipid rafts called caveolae, has been reported to interact with gp41. However, the underlying pathological or physiological meaning of this robust interaction remains unclear. In this report, we examine the interaction of cellular Cav-1 and HIV gp41 within the lipid rafts and show that Cav-1 modulates Env-induced bystander apoptosis through interactions with gp41 in SupT1 cells and CD4+ T lymphocytes isolated from human peripheral blood. Cav-1 significantly suppressed Env-induced membrane hemifusion and caspase-3 activation and augmented Hsp70 upregulation. Moreover, a peptide containing the Cav-1 scaffold domain sequence markedly inhibited bystander apoptosis and apoptotic signal pathways. Our studies shed new light on the potential role of Cav-1 in limiting HIV pathogenesis and the development of a novel therapeutic strategy in treating HIV-1-infected patients.

HIV Infection Upregulates Caveolin 1 Expression To Restrict Virus Production

ABSTRACT Caveolin 1 (Cav-1) is a major protein of a specific membrane lipid raft known as caveolae. Cav-1 interacts with the gp41 of the human immunodeficiency virus (HIV) envelope, but the role of Cav-1 in HIV replication and pathogenesis is not known. In this report, we demonstrate that HIV infection in primary human monocyte-derived macrophages (MDMs), THP-1 macrophages, and U87-CD4 cells results in a dramatic upregulation of Cav-1 expression mediated by HIV Tat. The activity of p53 is essential for Tat-induced Cav-1 expression, as our findings show enhanced phosphorylation of serine residues at amino acid positions 15 and 46 in the presence of Tat with a resulting Cav-1 upregulation. Furthermore, inhibition of p38 mitogen-activated protein kinase (MAPK) blocked phosphorylation of p53 in the presence of Tat. Infection studies of Cav-1-overexpressing cells reveal a significant reduction of HIV production. Taken together, these results suggest that HIV infection enhances the expression of Cav-1, which subsequently causes virus reduction, suggesting that Cav-1 may contribute to persistent infection in macrophages.

The Foamy Virus Genome Remains Unintegrated in the Nuclei of G1/S Phase-Arrested Cells, and Integrase Is Critical for Preintegration Complex Transport into the Nucleus

ABSTRACT Foamy viruses are a member of the spumavirus subfamily of retroviruses with unique mechanisms of virus replication. Foamy virus replication is cell cycle dependent; however, the genome is found in the nuclei of cells arrested in the G1/S phase. Despite the presence of genome in the nuclei of growth-arrested cells, there is no viral gene expression, thus explaining its dependency on cell cycle. This report shows that the foamy virus genome remains unintegrated in G1/S phase-arrested cells. The foamy virus genome is detected by confocal microscopy in the nuclei of both dividing and growth-arrested cells. Alu PCR revealed foamy virus-specific DNA amplification from genomic DNA isolated in cycling cells at 24 h postinfection. In arrested cells no foamy virus DNA band was detected in cells harvested at 1 or 7 days after infection, and a very faint band that is significantly less than DNA amplified from cycling cells was observed at day 15. After these cells were arrested at the G1/S phase for 1, 7, or 15 days they were allowed to cycle, at which time foamy virus-specific DNA amplification was readily observed. Taken together, these results suggest that the foamy virus genome persists in nondividing cells without integrating. We have also established evidence for the first time that the foamy virus genome and Gag translocation into the nucleus are dependent on integrase in cycling cells, implicating the role of integrase in transport of the preintegration complex into the nucleus. Furthermore, despite the presence of a nuclear localization signal sequence in Gag, we observed no foamy virus Gag importation into the nucleus in the absence of integrase.

Caveolin 1 inhibits HIV replication by transcriptional repression mediated through NF-κB.

ABSTRACT Caveolin 1 (Cav-1), the scaffold protein of a specific membrane lipid raft called caveolae, has been reported to suppress HIV-1 replication. However, the mechanism by which Cav-1 inhibits HIV replication remains unclear. In this study, we investigated the mechanism by which Cav-1 inhibits HIV replication at the level of gene expression. Our results show that Cav-1 represses viral gene expression and that this suppression involves the NF-κB pathway. We used several approaches in different cell types, including primary CD4+ T cells and macrophages, to demonstrate the role of nuclear factor κB (NF-κB) in Cav-1-mediated inhibition of viral expression. A mutational analysis of the cis-acting element shows that the two NF-κB sites in the U3 region of the long terminal repeat (LTR) are critical for Cav-1-mediated inhibition of viral expression. In the presence of Cav-1, phosphorylation of IKKβ, IKKα, IκBα, and NF-κB p65 is dramatically reduced, while viral gene expression is suppressed. In addition, translocation of NF-κB p65 to the nucleus decreases substantially in the presence of Cav-1. Furthermore, significant inhibition of NF-κB activation and binding to target DNA are evident in the presence of Cav-1. These results establish evidence that Cav-1 inhibits HIV replication by transcriptional repression of viral gene expression and contributes to HIVs persistent infection of macrophages.

Tissue Dynamics of CD8 Lymphocytes That Suppress Viral Replication in Cats Infected Neonatally with Feline Immunodeficiency Virus

The purpose of this study was to determine the tissue distribution and antiviral activity of the CD8 lymphocytes that suppress the replication of feline immunodeficiency virus (FIV). Cell-associated FIV load, CD8alpha(+)beta(low) cells, and CD8 cell-mediated suppression of FIV were measured serially in the blood, thymus, and peripheral lymph nodes after neonatal inoculation. Between 6 and 10 weeks, relative numbers of CD8alpha(+)beta(low) cells increased, whereas CD8alpha(+)beta(high) cells declined in the thymus and blood of infected cats. By 12-16 weeks, the lymph nodes were enlarged because of an absolute expansion of all CD8beta subpopulations. The strength of CD8 cell-mediated FIV suppression in vitro, but not CD8alpha(+)beta(low) cell content, was correlated inversely with virus load in the thymus and blood. Thus, after neonatal FIV inoculation, CD8alpha(+)beta(low) cells first occupy the thymus and blood, where strong CD8 cell-mediated antiviral activity is linked to reduced virus load in multiple lymphoid tissues.