A carboxy-terminally truncated human CPSF6 lacking residues encoded by exon 6 inhibits HIV-1 cDNA synthesis and promotes capsid disassembly.

Since HIV-1 replication is modulated at multiple stages by host cell factors, identification and characterization of those host cell factors are expected to contribute to the development of novel anti-HIV therapeutics. Previous studies showed that a C-terminally truncated cytosolic form of cleavage and polyadenylation-specific factor 6 (CPSF6-358) inhibits HIV-1 infection through interference with HIV-1 trafficking to the nucleus. Here we identified and characterized a different configuration of C-terminally truncated human CPSF6 (hCPSF6-375) through cDNA expression cloning coupled with ganciclovir-mediated lethal selection. Notably, hCPSF6-375, but not mouse CPSF6-358 (mCPSF6-358) as previously reported, remarkably interfered with viral cDNA synthesis after HIV-1 infection. Moreover, we found that hCPSF6-375 aberrantly accelerated the disassembly of the viral capsid in target cells, while CPSF6-358 did not. Sequence comparison of CPSF6-375 and CPSF6-358 cDNAs showed a lack of exon 6 and additional coding sequence for 54 amino acid residues in the C terminus of hCPSF6-375. Mutational analyses revealed that the residues encoded by exon 6, but not the C-terminal 54 residues in hCPSF6-375, is responsible for impaired viral cDNA synthesis by hCPSF6-375. This is the first report demonstrating a novel mode of HIV-1 inhibition by truncated forms of CPSF6 that involves rapid capsid disassembly and inhibition of viral cDNA synthesis. These findings could facilitate an increased understanding of viral cDNA synthesis in light of the viral capsid disassembly.

Follicular dendritic cells activate HIV-1 replication in monocytes/macrophages through a juxtacrine mechanism mediated by P-selectin glycoprotein ligand 1.

Follicular dendritic cells (FDCs) are located in the lymphoid follicles of secondary lymphoid tissues and play a pivotal role in the selection of memory B lymphocytes within the germinal center, a major site for HIV-1 infection. Germinal centers are composed of highly activated B cells, macrophages, CD4(+)T cells, and FDCs. However, the physiological role of FDCs in HIV-1 replication remains largely unknown. We demonstrate in our current study that FDCs can efficiently activate HIV-1 replication in latently infected monocytic cells via an intercellular communication network mediated by the P-selectin/P-selectin glycoprotein ligand 1 (PSGL-1) interaction. Upon coculture with FDCs, HIV-1 replication was significantly induced in infected monocytic cell lines, primary monocytes, or macrophages. These cocultures were found to synergistically induce the expression of P-selectin in FDCs via NF-kappaB activation and its cognate receptor PSGL-1 in HIV-1-infected cells. Consistent with this observation, we find that this response is significantly blocked by antagonistic Abs against PSGL-1 and almost completely inhibited by PSGL-1 small interfering RNA. Moreover, a selective inhibitor for Syk, which is a downstream effector of PSGL-1, blocked HIV-1 replication in our cultures. We have thus elucidated a novel regulatory mechanism in which FDCs are a potent positive bystander that facilitates HIV-1 replication in adjacent infected monocytic cells via a juxtacrine signaling mechanism.

Selective transmission of R5 HIV-1 over X4 HIV-1 at the dendritic cell-T cell infectious synapse is determined by the T cell activation state.

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of T cell immunity against HIV. On the other hand, due to the susceptibility of DCs to HIV infection, virus replication is strongly enhanced in DC-T cell interaction via an immunological synapse formed during the antigen presentation process. When HIV-1 is isolated from individuals newly infected with the mixture of R5 and X4 variants, R5 is predominant, irrespective of the route of infection. Because the early massive HIV-1 replication occurs in activated T cells and such T-cell activation is induced by antigen presentation, we postulated that the selective expansion of R5 may largely occur at the level of DC-T cell interaction. Thus, the immunological synapse serves as an infectious synapse through which the virus can be disseminated in vivo. We used fluorescent recombinant X4 and R5 HIV-1 consisting of a common HIV-1 genome structure with distinct envelopes, which allowed us to discriminate the HIV-1 transmitted from DCs infected with the two virus mixtures to antigen-specific CD4(+) T cells by flow cytometry. We clearly show that the selective expansion of R5 over X4 HIV-1 did occur, which was determined at an early entry step by the activation status of the CD4(+) T cells receiving virus from DCs, but not by virus entry efficiency or productivity in DCs. Our results imply a promising strategy for the efficient control of HIV infection.

Pycnogenol, a procyanidin-rich extract from French maritime pine, inhibits intracellular replication of HIV-1 as well as its binding to host cells.

A procyanidin-rich extract from French maritime pine, Pycnogenol(R) (PYC), is known as an antioxidant that exerts a variety of physiological activities and is widely used in human beings. We report here that PYC inhibits not only human immunodeficiency virus type-1 (HIV-1) binding to host cells, but also its replication after entry in susceptible cells in vitro. Prominent biochemical alterations induced by PYC were the elevated expression of an intracellular antioxidant protein, manganese superoxide dismutase (Mn-SOD), and the inhibition of phosphorylation of the ribosomal S6 protein. Interestingly, ectopic expression of Mn-SOD inhibited HIV-1 replication as well. Inhibition of HIV-1 replication associated with induced expression of Mn-SOD in cells treated with PYC suggests the potential of this natural antioxidant inducer as a new anti-HIV-1 agent.

Analysis of human immunodeficiency virus type 1 integration by using a specific, sensitive and quantitative assay based on real-time polymerase chain reaction.

A novel real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type-1 (HIV-1) DNA with high specificity and sensitivity. This assay reproducibly allowed the detection of three copies of integrated HIV DNA in a background of 100,000 cell equivalents of human chromosomal DNA. The non-specific amplification of unintegrated HIV-1 DNA was significantly inhibited in this assay and the specificity of this assay was much higher than the previously reported method. This assay showed that kinetics in viral DNA synthesis was cell-type dependent and that the kinetics of HIV-1 DNA integration was very rapid in Jurkat T cell line. This method may provide new insights into the integration processes and be useful in evaluating future integrase inhibitors.

Highly potent anti-HIV-1 activity isolated from fermented Polygonum tinctorium Aiton.

A water-soluble extract of fermented Polygonum tinctorium Aiton (Polygonaceae) called Sukumo, exhibited a potent inhibitory activity against HIV type 1 in vitro. The extract potently suppressed acute HIV-1 (IIIB) infection in MT-4 cells with EC50 values of 0.5 microg/ml but exhibited low cytotoxicity to MT-4 cells even at a high concentration (CC50 > 1000 microg/ml). It also inhibited giant cell formation in co-cultures of HIV-infected cells and uninfected Molt-4 cells. Sukumo extract was found to interact with both the viral envelope glycoprotein and cellular receptors, thus blocking virus-cell binding and virus-induced syncytium formation. There was a good correlation between the extract's anti-HIV-1 activity and its inhibitory effects on HIV-1 binding. It also suppressed replication of herpes simplex virus type 1 in Vero cells with an EC 50 of 11.56 microg/ml. On the other hand, there was no appreciable activity against influenza A virus, poliovirus or SARS corona virus when tested at concentrations ranging from 3.2-400 microg/ml as shown by microscopic image analysis for cytopathic effect (CPE). Physico-chemical studies revealed that the anti-HIV activity in the extract was essentially maintained after boiling at 100 degrees C in 1N HCl or 1N NaOH, and after treatment with 100 mM NaIO4. The inhibitory activity of the extract was also not reduced after pronase digestion. The active factor in the extract is likely to be a novel compound(s) having a polyanionic substructure and a molecular weight of 10,000-50,000.

Intracellular expression of antisense RNA transcripts complementary to the human immunodeficiency virus type-1 vif gene inhibits viral replication in infected T-lymphoblastoid cells.

The human immunodeficiency virus type-1 (HIV-1)-encoded vif protein is essential for viral replication, virion production, and pathogenicity. HIV-1 vif interacts with the endogenous human APOBEC3G protein (an mRNA editor) in target cells to prevent its virions from encapsidation. Although some studies have established targets within the HIV-1 vif gene that are important for its biologic function, it is however important to further screen for effective therapeutic targets in the vif gene that could interfere with the HIV-1 vif-dependent infectivity and pathogenicity. This report demonstrates that HIV-1 vif antisense RNA fragments constructed within mid-3' region, notably the region spanning nucleic acid positions 5561-5705 (M-3'-AS), significantly inhibited HIV-1 replication in MT-4 and H9-infected cells and reduced the HIV-1 vif mRNA transcripts. These data clearly suggest that the above vif fragment, which corresponds to amino acid residues 96-144, could be an effective novel therapeutic target site for gene therapy applications, for the control and management of HIV-1 infection, due to its strong inhibition of HIV-1 replication in cells.

Tumor necrosis factor-related apoptosis-inducing ligand induces neuronal death in a murine model of HIV central nervous system infection.

HIV-1 infection in the brain induces neuronal apoptosis leading to HIV-associated dementia. To explore the underlying mechanism, we developed a murine model by using human peripheral blood mononuclear cell (PBMC)-transplanted nonobese diabetic (NOD)-severe combined immunodeficiency (SCID) (hu-PBMC-NOD-SCID) mice. Administration of lipopolysaccharide (LPS) to HIV-1-infected hu-PBMC-NOD-SCID mice induced infiltration of HIV-1-infected human cells into the perivascular region of the brain and neuronal apoptosis was found in macrophage (M)-tropic but not T cell (T)-tropic HIV-1-infected brains. The apoptotic neurons were frequently colocalized with the HIV-1-infected macrophages that expressed tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Administration of a neutralizing antibody against human TRAIL but not human TNF-alpha or Fas ligand (FasL) blocked the neuronal apoptosis in the HIV-1-infected brain. These results strongly suggest a critical contribution of TRAIL expressed on HIV-1-infected macrophages to neuronal apoptosis.

Down regulation of human immunodeficiency virus type-1 (HIV-1) expression by Vif antisense RNA expression vectors in transfected cells.

The HIV-1 vif gene is a potential candidate in the quest for anti-retroviral interventions, due to its unique role in the target cell infection. We employed the antisense RNA strategy to determine the antiviral activity of intracellularly expressed anti-sense RNAs of various lengths complementary to the targeted HIV-1 vif gene. Expression vectors mediating the delivery of the vif-ORF, 5'-Vif, M-vif, and 3'-vif antisense RNAs under the CMV promoter were constructed using pcDNA 3.1. The COS cells transfected with the antisense vectors showed a steady-state of antisense RNA expression levels. In contrast, those co-transfected with the Infectious molecular clone, pNL-E, exhibited a significant reduction in the steady-state antisense RNA levels, which correlated with a significant reduction in p24 antigen production. Thus, this expression method for these antisense RNAs provides a promising gene therapy strategy for HIV-1.

Analysis of the molecules involved in human T-cell leukaemia virus type 1 entry by a vesicular stomatitis virus pseudotype bearing its envelope glycoproteins.

Cellular entry of human T-cell leukaemia virus type 1 (HTLV-1) was studied by a quantitative assay system using vesicular stomatitis virus (VSV) pseudotypes in which a recombinant VSV (VSVDeltaG*) containing the gene for green fluorescent protein instead of the VSV G protein gene was complemented with viral envelope glycoproteins in trans. Most of the cell lines tested showed susceptibility to VSVDeltaG* complemented with either HTLV-1 envelope glycoproteins (VSVDeltaG*-Env) or VSV G protein (VSVDeltaG*-G), but not to VSVDeltaG* alone, indicating that cell-free HTLV-1 could infect many cell types from several species. High concentration pronase treatment of cells reduced their susceptibility to VSVDeltaG*-Env, while trypsin treatment, apparently, did not. Treatment of the cells with sodium periodate, heparinase, heparitinase, phospholipase A2 or phospholipase C reduced the susceptibility of cells to VSVDeltaG*-Env, but not to VSVDeltaG* complemented with measles virus (Edmonston strain) H and F proteins (VSVDeltaG*-EdHF), which was used as a control. Purified phosphatidylcholine also inhibited the infectivity of VSVDeltaG*-Env, but not VSVDeltaG*-G. These findings indicated that, in addition to cell surface proteins, glycosaminoglycans and phospholipids play an important role in the process of cell-free HTLV-1 entry.