Guangai Xue

HIV-1 Vpr Induces Interferon-Stimulated Genes in Human Monocyte-Derived Macrophages

Macrophages act as reservoirs of human immunodeficiency virus type 1 (HIV-1) and play an important role in its transmission to other cells. HIV-1 Vpr is a multi-functional protein involved in HIV-1 replication and pathogenesis; however, its exact role in HIV-1-infected human macrophages remains poorly understood. In this study, we used a microarray approach to explore the effects of HIV-1 Vpr on the transcriptional profile of human monocyte-derived macrophages (MDMs). More than 500 genes, mainly those involved in the innate immune response, the type I interferon pathway, cytokine production, and signal transduction, were differentially regulated (fold change >2.0) after infection with a recombinant adenovirus expressing HIV-1 Vpr protein. The differential expression profiles of select interferon-stimulated genes (ISGs) and genes involved in the innate immune response, including STAT1, IRF7, MX1, MX2, ISG15, ISG20, IFIT1, IFIT2, IFIT3, IFI27, IFI44L, APOBEC3A, DDX58 (RIG-I), TNFSF10 (TRAIL), and RSAD2 (viperin) were confirmed by real-time quantitative PCR and were consistent with the microarray data. In addition, at the post-translational level, HIV-1 Vpr induced the phosphorylation of STAT1 at tyrosine 701 in human MDMs. These results demonstrate that HIV-1 Vpr leads to the induction of ISGs and expand the current understanding of the function of Vpr and its role in HIV-1 immune pathogenesis.

Identification of a novel Vpr-binding compound that inhibits HIV-1 multiplication in macrophages by chemical array.

Although HIV-1 replication can be controlled by highly active anti-retroviral therapy (HAART) using protease and reverse transcriptase inhibitors, the development of multidrug-resistant viruses compromises the efficacy of HAART. Thus, it is necessary to develop new drugs with novel targets. To identify new anti-HIV-1 compounds, recombinant Vpr was purified from transfected COS-7 cells and used to screen compounds by chemical array to identify those that bound Vpr. From this screen, 108 compounds were selected as positive for Vpr binding. Among these, one structurally similar group of four compounds showed anti-HIV activity in macrophages. In particular, compound SIP-1 had high inhibition activity and reduced the levels of p24 by more than 98% in macrophages after 8 or 12 days of infection. SIP-1 had no cytotoxic effects and did not disrupt cell cycle progression or induce apoptosis of Molt-4 and HeLa cell lines as measured by MTT assay, flow-cytometry analysis, and a caspase-3 assay. In addition, SIP-1 specifically bound to Vpr as assessed by photo-cross-linked small-molecule affinity beads. These results suggest that Vpr is a good target for the development of compounds that could potentially inhibit HIV-1 replication. Collectively, our results strongly suggest that chemical array is a useful method for screening anti-viral compounds.

Synthesis and biological evaluation of deoxy-hematoxylin derivatives as a novel class of anti-HIV-1 agents.

SAR studies for the exploration a novel class of anti-human immunodeficiency virus type 1 (HIV-1) agents based on the hematoxylin structure (1) are described. The systematic deoxygenations of 1 including asymmetric synthesis were conducted to obtain a compound showing high potencies for inhibiting the nuclear import and viral replication as anti-HIV-1 agent. Among all, C-3-deoxygenated analog 16 exhibited most promising biological activities as anti-HIV-1 agent such as lower cytotoxicity (16:1; >80:40 μM), stronger inhibition of nuclear import (0.5:1.3 μM), and viral replication in HIV-1-infected TZM-bl cells (24.6:100 μM), human peripheral blood mononuclear cells (PMBCs) (30.1 μM: toxic). Different spectra of inhibitory activities against infected three healthy humans macrophages with high (donor A) and low (donor B and C) amounts of virus were also observed. Thus 16 showed 10-times stronger activity than 1 (16:1; 0.1:<1.0 μM) in the case of A, while 16 and 1 showed comparable activities in the cases of B and C (>0.01 and >0.00 1μM). The comparison of the inhibition of viral p24 antigen production was clearly indicated that compound 16 is at least twofold more potent anti-viral activity than 1. Thus, structures and actions of deoxy analogs particularly 16 could provide valuable information for the development of a novel class of anti-HIV-1 agents.

Structure of the Prion Protein and Its Gene: An Analysis Using Bioinformatics and Computer Simulation

Prion protein (PrP) gene encodes cellular PrP (PrPC), a glycosylphosphatidylinositol (GPI)-anchored cell membrane protein indispensable for infections of prion, which causes Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep. Although PrPC is known to be converted into an abnormal isoform (PrPSc) upon prion infection and play an important role in prion diseases, the mechanisms involved remain unclear, partly due to the insolubility of PrPSc, which prevents experimental biochemical and biophysical analyses. Recently, with improvements in computer power and methods, computer analyses have been contributing more to prion studies. A comparison of PrP gene sequences revealed mutations and polymorphisms in the open reading frame (ORF) of the human PrP gene related to prion diseases. In contrast, little mutations or polymorphisms related to susceptibility to BSE were found in the ORF of the bovine PrP gene, though relationships between insertion/deletion (Ins/Del) polymorphisms of the PrP gene promoter and susceptibility to BSE have been found. Our results have shown that the specific protein 1 (Sp1) plays important role in the activity of PrP gene promoter, which is influenced by polymorphisms in the Sp1 binding sites. The potential structural dynamics of PrP have been simulated by computational methods such as molecular dynamics (MD) and quantum mechanics (QM). The proposed mechanisms of conversion have revealed new insights in prion diseases. In this review, we will introduce the gene structure, polymorphisms, and potential structural dynamics of PrP revealed by basic and advanced computational analyses. The possible contribution of these methods to elucidation of the pathogenicity of prion diseases and functions of PrPC is discussed.

Novel Single Nucleotide Polymorphisms in the Specific Protein 1 Binding Site of the Bovine PRNP Promoter in Japanese Black Cattle: Impairment of Its Promoter Activity

Susceptibility to transmissible spongiform encephalopathy and different alleles of the prion protein gene (PRNP) of humans and sheep are associated. A tentative association between PRNP promoter polymorphisms and bovine spongiform encephalopathy (BSE) susceptibility has been reported in German cattle, whereas none of the known polymorphisms within the bovine PRNP-coding sequence affect BSE susceptibility. In the present study, novel single nucleotide polymorphisms located in the 5′-flanking region of bovine PRNP affecting its expression were demonstrated in Japanese Black cattle. We sequenced exon 1, and the approximately 200-bp 5′-flanking region of the PRNP translation initiation site containing the proximal promoter of PRNP was harvested. We identified 7 single nucleotide polymorphisms: –184A→G, –141T→C, –85T→G, –47C→A, –6C→T, +17C→T and +43C→T. Six segregated haplotypes in the population were cloned into luciferase-expressing plasmids, transfected into N2a cells, and their reporter activities were measured 48 h after transfection. Six haplotypes showed a decreased expression level including –6C→T in specific protein 1 binding site (p < 0.05) or –141T→C (p < 0.01) at 48 h compared with the wild-type haplotype. These results advocate that certain polymorphisms such as specific protein 1 binding site polymorphisms in the bovine PRNP promoter region in Japanese Black cattle could influence promoter activity, suggesting that breeding cattle with such substitutions may be a useful approach in reducing BSE risk.

Role of Cellular Prion Proteins in the Function of Macrophages and Dendritic Cells

The cellular isoform of prion proteins (PrPC) is expressed in hematopoietic stem cells, granulocytes, T and B lymphocyte natural killer cells, platelets, monocytes, dendritic cells, and follicular dendritic cells, which may act as carrier cells for the spread of its abnormal isoform (PrPSc) before manifesting transmissible spongiform encephalopathies (TSEs). In particular, macrophages and dendritic cells seem to be involved in the replication of PrPSc after ingestion. In addition, information on the role of PrPC during phagocytotic activity in these cells has been obtained. A recent study showed that resident macrophages from ZrchI PrP gene (Prnp)-deficient (Prnp-/-) mice show augmented phagocytotic activity compared to Prnp+/+ counterparts. In contrast, our study suggests that Rikn Prnp-/- peritoneal macrophages show pseudopodium extension arrest and up-regulation of phagocytotic activity compared to Prnp+/+ cells. Although reports regarding phagocytotic activity in resident and peritoneal macrophages are inconsistent between ZrchI and Rikn Prnp-/- mice, it seems plausible that PrPC in macrophages could contribute to maintain the immunological environment. This review will introduce the recent progress in understanding the functions of PrPC in macrophages and dendritic cells under physiological conditions and its involvement in the pathogenesis of prion diseases.

Establishment of a new glial cell line from hippocampus of prion protein gene-deficient mice.

Cellular prion protein (PrP(C)) is expressed not only in neuronal cells but also in non-neuronal cells such as astroglial cells. In the present study, the prion protein (PrP) gene (Prnp)-deficient astroglial cell line GpL1 from hippocampal cells of ZrchI Prnp(-/-) mice were established. Transfection of Prnp suppressed cell death in GpL1 cells under serum-free conditions. The PrP-expressing GpL1 cells showed increased superoxide dismutase activity compared to control GpL1 cells. These results suggest that the anti-oxidative activity of PrP(C) functions in not only neuronal cells but also astroglial cells possibly due to the increased anti-oxidative activity of astroglial cells.

The 5′ Flanking Region and Intron1 of the Bovine Prion Protein Gene (PRNP) Are Responsible for Negative Feedback Regulation of the Prion Protein

Transcription factors regulate gene expression by controlling the transcription rate. Some genes can repress their own expression to prevent over production of the corresponding protein, although the mechanism and significance of this negative feedback regulation remains unclear. In the present study, we describe negative feedback regulation of the bovine prion protein (PrP) gene PRNP in Japanese Black cattle. The PrP-expressing plasmid pEF-boPrP and luciferase-expressing plasmids containing the partial promoter fragment of PRNP incorporating naturally occurring single-nucleotide or insertion/deletion polymorphisms were transfected into N2a cells. Transfection of pEF-boPrP induced PrP overexpression and decreased the promoter activity of PRNP in the wild-type haplotype (23-bp Del, 12-bp Del, and −47C). Reporter gene assays further demonstrated that the 12- and 23-bp Ins/Del polymorphisms, which are thought to be associated with Sp1 (Specific protein 1) and RP58 (Repressor Protein with a predicted molecular mass of 58 kDa), in intron1 and the upstream region, respectively, and an additional polymorphism (−47C→A) in the Sp1-binding site responded differently to PrP overexpression. With the −47C SNP, the presence of the Del in either the 23-bp Ins/Del or the 12-bp Ins/Del allele was essential for the negative feedback caused by PrP overexpression. Furthermore, deletion mutants derived from the wild-type haplotype showed that nucleotides −315 to +2526, which include the 5′-flanking region and exon1, were essential for the response. These results indicate that certain negative feedback response elements are located in these sequences, suggesting that regulation by transcription factors such as Sp1 and RP58 may contribute to the negative feedback mechanism of PRNP.