A small molecule CXCR4 inhibitor that blocks T cell line-tropic HIV-1 infection.

Several members of the chemokine receptor family have been shown to function in association with CD4 to permit human immunodeficiency virus type 1 (HIV-1) entry and infection. The CXC chemokine receptor CXCR4/fusin is a receptor for pre-B cell growth stimulating factor (PBSF)/stromal cell-derived factor 1 (SDF-1) and serves as a coreceptor for the entry of T cell line-tropic HIV-1 strains. Thus, the development of CXCR4 antagonists or agonists may be useful in the treatment of HIV-1 infection. T22 ([Tyr5,12,Lys7]-polyphemusin II) is a synthesized peptide that consists of 18 amino acid residues and an analogue of polyphemusin II isolated from the hemocyte debris of American horseshoe crabs (Limulus polyphemus). T22 was found to specifically inhibit the ability of T cell line-tropic HIV-1 to induce cell fusion and infect the cell lines transfected with CXCR4 and CD4 or peripheral blood mononuclear cells. In addition, T22 inhibited Ca2+ mobilization induced by pre-B cell growth stimulating factor (PBSF)/SDF-1 stimulation through CXCR4. Thus, T22 is a small molecule CXCR4 inhibitor that blocks T cell line-tropic HIV-1 entry into target cells.

Critical contribution of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to apoptosis of human CD4+ T cells in HIV-1-infected hu-PBL-NOD-SCID mice.

Apoptosis is a key for CD4+ T cell destruction in HIV-1-infected patients. In this study, human peripheral blood lymphocyte (PBL)-transplanted nonobese diabetic (NOD)-severe combined immunodeficient (SCID) (hu-PBL-NOD-SCID) mice were used to examine in vivo apoptosis after HIV-1 infection. As the hu-PBL-NOD-SCID mouse model allowed us to see extensive infection with HIV-1 and to analyze apoptosis in human cells in combination with immunohistological methods, we were able to quantify the number of apoptotic cells with HIV-1 infection. As demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), massive apoptosis was predominantly observed in virus-uninfected CD4+ T cells in the spleens of HIV-1-infected mice. A combination of TUNEL and immunostaining for death-inducing tumor necrosis factor (TNF) family molecules indicated that the apoptotic cells were frequently found in conjugation with TNF-related apoptosis-inducing ligand (TRAIL)-expressing CD3+CD4+ human T cells. Administration of a neutralizing anti-TRAIL mAb in HIV-1-infected mice markedly inhibited the development of CD4+ T cell apoptosis. These results suggest that a large number of HIV-1-uninfected CD4+ T cells undergo TRAIL-mediated apoptosis in HIV-infected lymphoid organs.

Humanized mice for human retrovirus infection.

Inbred mice with specific genetic defects have greatly facilitated the analysis of complex biological events. Several humanized mouse models using the C.B.-17 scid/scid mouse (referred to as the SCID mouse) have been created from two transplantation protocols, and these mice have been utilized for the investigation of human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type I (HTLV-I) pathogenesis and the evaluation of antiviral compounds. To generate a more prominent small animal model for human retrovirus infection, especially for examination of the pathological process and the immune reaction, a novel immunodeficient mouse strain derived from the NOD SCID mouse was created by backcrossing with a common gamma chain (gamma(c))-knockout mouse. The NOD-SCID gamma(c)null (NOG) mouse has neither functional T and B cells nor NK cells and has been used as a recipient in humanized mouse models for transplantation of human immune cells particularly including hematopoietic stem cells (HSC). From recent advances in development of humanized mice, we are now able to provide a new version of the animal model for human retrovirus infection and human immunity.

Infection of HTLV-III/LAV in HTLV-I-carrying cells MT-2 and MT-4 and application in a plaque assay.

The human T-cell lines MT-2 and MT-4 carry the human T-cell leukemia virus type I (HTLV-I). When MT-2 and MT-4 were infected with HTLV-III, the probable etiologic agent of the acquired immune deficiency syndrome (AIDS), rapid cytopathogenic effects and cytotoxicity were observed that made it possible to titrate the biologically active virus in a plaque-forming assay. The cytopathogenic effects were preceded by the rapid induction and increase of HTLV-III antigens as revealed by immunofluorescence and immunoprecipitation. Activities of HTLV-III were neutralized by the human antibodies against the virus when immunofluorescence and plaque assays were used. Essentially the same results were obtained with the lymphadenopathy-associated virus (LAV1).

Transformation of human leukocytes by cocultivation with an adult T cell leukemia virus producer cell line.

The transmission of adult T cell leukemia virus, a human retrovirus, into fresh leukocytes from normal humans was examined. One of three virus-carrying cell lines, tested after being subjected to lethal x-irradiation, consistently transformed leukocytes from adult peripheral blood and umbilical cord blood. All the transformed cell lines expressed adult T cell leukemia virus-associated antigen, but transformed lines originating from adult and umbilical cord blood exhibited T cell and non-T, non-B cell surface natures, respectively. Efforts to transform human leukocytes with cell-free virus were unsuccessful.

Cytokines alter production of HIV-1 from primary mononuclear phagocytes.

Some strains of human immunodeficiency virus type 1 (HIV-1) can infect primary monocytes and monocyte-derived macrophages in vitro. In this report, the effect of cytokines on the production of one of these strains that shows a tropism for mononuclear phagocytes, designated HIV-1JR-FL, was studied. Primary peripheral blood mononuclear phagocytes infected with HIV-1JR-FL were treated with the hematopoietic factors: granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), macrophage colony-stimulating factor (M-CSF), and gamma-interferon (gamma-IFN). The M-CSF, GM-CSF, IL-3, and gamma-IFN were able to alter HIV-1 production under different conditions.

Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms.

Human immunodeficiency virus (HIV) is the causative agent of the acquired immune deficiency syndrome (AIDS). A large number of AIDS patients show evidence of neurologic involvement, known as AIDS-related subacute encephalopathy, which has been correlated with the presence of HIV in the brain. In this study, two genetically distinct but related viruses were isolated from one patient from two different sources in the central nervous system: brain tissue and cerebrospinal fluid. Both viruses were found to replicate in peripheral blood lymphocytes, but only virus from brain tissue will efficiently infect macrophage/monocytes. The viruses also differ in their ability to infect a brain glioma explant culture. This infection of the brain-derived cells in vitro is generally nonproductive, and appears to be some form of persistent or latent infection. These results indicate that genetic variation of HIV in vivo may result in altered cell tropisms and possibly implicate strains of HIV with glial cell tropism in the pathogenesis of some neurologic disorders of AIDS.

Mutational analysis of the 5' noncoding region of human immunodeficiency virus type 1 genome.

Retrovirus particles are released by budding from the membranes of infected cells. In the course of virus production, particularly during the late stage, viral genomic RNA is incorporated specifically into virion particles. This specific incorporation of the genomic RNA requires a packaging signal sequence. A region that functions as the packaging signal was mapped to a location upstream of the gag open reading frame on the HIV-1 viral genome. In addition of this packaging signal, other cis-acting elements that are scattered throughout the genome are also required for efficient packaging. The region upstream of the splice donor site is probably important for dimer formation. Therefore, we focused on one region located between the 3' end of the primer binding site and the 5' splice donor site of HIV-1. Experiments were conducted to investigate how deletions or point mutations in this region affect both dimerization in vitro and the production of infectious virus particles. A series of RNAs of varying lengths containing the 5' noncoding region were generated, and genomic dimerization of the altered viral RNA was analyzed in vitro. One RNA construct which consisted of 112 nucleotides (nt) from nt 639 to nt 750 formed a heterodimeric complex with the RNA which consisted of 200 nucleotides from nt 551 to nt 750. We then constructed proviruses with mutations in the 639 to 750 nt region and assayed for virus production. Several mutants that lacked the complementarity necessary to form a possible stem-loop structure in this region showed decreased production of infectious virus particles. Moreover, both deletion of this region and randomization of its nucleotide sequence completely impaired infectious virus production. Thus, the way that this region affects infectious virus production may be through its RNA secondary structure.

High levels of viremia in hu-PBL-NOD-scid mice with HIV-1 infection.

We studied the compatibility of human lymphocyte engraftment and susceptibility to HIV-1 infection in 2 new immunodeficient mice. NOD/Shi-scid mice were generated by backcrossing of the scid mutation into NOD mice while C57BL/6-RAG2(0/0) were generated by knocking out the RAG-2 gene. Human T lymphocytes were reconstituted in new immunodeficient mouse strains. We found that the new immunodeficient mouse strains accepted human PBL engraftment and HIV-1 infection more efficiently than conventional C.B-17-scid mice. Especially in the hu-PBL-NOD/Shi-scid strain, we reproduced the high levels of HIV-1 viremia comparable to or at significantly higher levels than after HIV-1 primary infection. These results indicate that our hu-PBL-NOD-scid animal is useful for investigations of the activation mechanism in HIV-1 replication in vivo and after primary infection.

Transcriptional regulation of HIV-1 LTR during antigen-dependent activation of primary T cells by dendritic cells.

Numerous factors are known to bind human immunodeficiency virus (HIV) long terminal repeat (LTR) and activate viral transcription, but little is known as to how they function in naturally activated T cells and to what extent their binding is relevant to HIV replication in vivo. To characterize the HIV LTR-binding factors responsible for antigen-dependent activation of HIV, we examined replication of LTR mutant viruses in CD4+ T cells activated by different stimuli. NF-kappaB or Sp1 mutant virus replicated well in CD4+ T cells activated by phorbol ester and calcium ionophore. When they were activated by antigen-pulsed dendritic cells, the replication of the Sp1-deleted virus was severely impaired in CD45RA+, but not in CD45RO+ T cell subsets that dominantly produce interleukin-2 (IL-2). Stimulation via CD3/CD28 induced a high level of IL-2 production in both T cell subsets, but Sp1-deleted virus poorly replicated in CD45RA+ subset. The level of NF-kappaB and Sp1-binding factors did not differ between these subsets. Our results suggest that additional cofactors distinct from IL-2-inducing signaling molecules are important for LTR activation during antigen-dependent T cell activation.

Presynaptic cell type-dependent regulation of GABAergic synaptic transmission by nitric oxide in rat insular cortex.

Nitric oxide (NO) is a key retrograde messenger that regulates synaptic transmission in the cerebral cortex. However, little is known about NO-induced modulatory effects and their mechanisms relative to inhibitory synaptic transmission. The present study aimed to examine the effects of NO on unitary inhibitory postsynaptic currents (uIPSCs) and to postulate the synaptic location of NO action. We performed multiple whole-cell patch-clamp recordings from rat insular cortex and divided recorded cells into three subtypes: pyramidal cells (Pyr), fast-spiking interneurons (FS), and non-FS GABAergic interneurons. In the connections from FS to Pyr (FS→Pyr), the application of S-nitroso-N-acetyl-dl-penicillamine (SNAP, 100 µM), an NO donor, suppressed uIPSC amplitudes in 31% of the connections, whereas 39% of the connections showed uIPSC facilitation. The remaining FS→Pyr connections showed little effect of SNAP on uIPSCs. An analysis of paired-pulse ratio (PPR) implied the involvement of presynaptic mechanisms in SNAP-induced effects on uIPSCs. Similar effects of SNAP were observed in FS→FS/non-FS connections; 33%, 54%, and 13% of the connections were facilitated, suppressed, and unchanged, respectively. In contrast, non-FS→Pyr or FS/non-FS showed constant uIPSC suppression by SNAP. PPR analysis supports the hypothesis that these SNAP-induced effects are mediated by presynaptic mechanisms in FS→FS/non-FS and non-FS→Pyr/FS/non-FS connections. The NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazolineoxyl-1-oxyl-3-oxide (PTIO), or the inhibitor of guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), abolished the SNAP-induced uIPSC modulation. These results suggest that NO regulation of inhibitory synaptic transmission is dependent on presynaptic cell subtypes and that, at least in part, the effects are mediated by presynaptic mechanisms.

Quantitative evaluations of the effect of UV irradiation on the infectivity of HTLV-III (AIDS virus) with HTLV-I-carrying cell line, MT-4.

The effect of UV irradiation on HTLV-III was quantitatively studied to evaluate the dosage of UV irradiation which inactivates the virus for sterilization of blood products and for laboratory decontamination. In order to estimate the biologic activity and quantitation of the virus, induction of HTLV-III-specific antigens and inhibition of DNA synthesis in MT-4 cells infected by UV-irradiated HTLV-III were detected by indirect immunofluorescence technique and proliferation assay using [3H]thymidine uptake, respectively. Furthermore, plaque-forming assay was performed to count the infectious viral particles. Results showed that HTLV-III was completely inactivated by 5000 J/m2 UV irradiation. Cloned UV-irradiated HTLV-III (UV-1) was obtained from a plaque that was formed by 2000 J/m2 UV-irradiated virus. When MT-4 cells were infected by the clone UV-1, ballooning degeneration of cells was predominantly induced. These ballooning cells were not usually observed in MT-4 cells infected by unirradiated HTLV-III. The resistance to UV was not different between clone UV-1 and unirradiated HTLV-III.

Sensitive assay for neutralizing antibodies against AIDS-related viruses (HTLV-III/LAV).

A sensitive assay for neutralizing antibodies (NA) against AIDS-related viruses (HTLV-III and LAV) was developed, using human T-cell lymphotropic virus type-I (HTLV-I)-bearing and HTLV-III-susceptible MT-4 cells. NA to HTLV-III in 21 patients with acquired immune deficiency syndrome (AIDS), 10 individuals with AIDS-related complex (ARC), 20 healthy male homosexuals, and 10 healthy male controls were titrated. Antibodies to HTLV-III were also detected by indirect immunofluorescence (IF). The assay was sensitive up to a dilution of 1:10 000. Sera from patients with AIDS showed a geometric mean titer (GMT) of NA of 1:475, whereas much higher GMTs (1:1318 and 1:1009) were observed in patients with ARC and healthy male homosexuals, respectively. Moreover, titers of NA significantly correlated with the levels of anti-HTLV-III antibodies detected by IF.

Establishment of a high production system for AIDS retroviruses with a human T-leukemic cell line Molt-4.

A cell culture system was developed for the continuous and efficient production of acquired immune deficiency syndrome (AIDS) retrovirus. After infection of a human T-cell line Molt-4 with HTLV-III and LAV the cells grow permanently and produce large amounts of virus continuously. The yields of production of virus were assessed either with reverse transcriptase activity or a newly established biological quantitation assay of active virus. The amounts of virus with this cell system were much higher than those of the H9 cell system. This procedure enabled us first to compare the two viral isolates HTLV-III and LAV directly in the same cell line. Establishment of the culture system, allowing efficient production of AIDS retroviruses, provides a useful tool for the isolation of the virus from patients with AIDS and for more basic research, such as the mechanisms of immune destruction caused by the virus leading to the occurrence of various malignancies.

Binding of adult T-cell leukemia virus to various hematopoietic cells.

A newly found human retrovirus, adult T-cell leukemia virus (ATLV) was shown by means of membrane immunofluorescence to bind to various hematopoietic cells including T-, B- and non-T, non-B-cell lines. Partially purified viral gp46 from culture fluids of ATL virus producer lines also bound efficiently to an ATLV-negative T-cell line, CCRF-CEM cells. When the viruses were pre-incubated with anti-ATLV-positive human sera, ATLV binding to the cells was clearly inhibited but not by pre-incubation with anti-ATLV-negative sera. These data suggest that: (1) ATLV binds not only to T-cells but also to multiple types of cells of hematopoietic origin; (2) anti-ATLV antibody-positive human sera have the blocking antibody for the binding of ATLV to lymphoid cells.

Epitope mapping of rat neutralizing monoclonal antibody against human immunodeficiency virus type-1 by a phage peptide library: comparison with ELISA using synthetic peptides.

We generated a rat monoclonal antibody (mAb W#10) with the ability to neutralize human immunodeficiency virus type 1IIIB (HIV-1IIIB) infection. The epitope recognized by mAb W#10 was defined as R-I-Q-R-G-P-G by enzyme-linked immunosorbent assay (ELISA) with the use of synthetic peptides. The filamentous phage clones displaying random 15-amino-acid peptides on the amino terminus of the pIII coat protein reacting with mAb W#10 were identified with affinity and immunological selection procedures. Thirteen out of 16 selected phage clones contained the G-X-G-R-X-F sequence in the coat protein region representing significant homology to a part of conserved G-P-G-R-A-F sequence in the V3 loop of various HIV-1 strains. In addition, the phage clones included the G-X-G sequence in the sequence detected by synthetic peptides as the recognition site. The selected phage clones were stained by mAb W#10 specifically and were able to compete with mAb binding to cells expressing viral antigens.

In vitro infection of human B lymphocytes with adult T-cell leukemia virus.

Experimental transmission of adult T-cell leukemia (ATL) virus (ATLV) into human B lymphocytes was attempted. Cocultivation of B-cell rich fraction of peripheral blood from a healthy adult with X-ray irradiated ATLV producer MT-2 cells resulted in the establishment of OKA(B) cell line co-infected with both Epstein-Barr virus (EBV) and ATLV. OKA(B) cells and its subclones contained: (1) B cell markers exclusively; (2) both EBV-specific antigen, EBNA and ATLV-specific antigen, ATLA detected by immunofluorescence test; (3) ATLV-specific polypeptides, p24 and p19; (4) ATLV-specific mRNA in ATLA-positive clones; (5) ATLV and EBV particles detectable by electron microscopy. These data clearly show that human B lymphocytes are susceptible to ATLV infection.

Pertussis toxin enhances human immunodeficiency virus type 1 replication.

Pertussis toxin (PTX) has been used as a reagent to identify involvement of the G protein-mediated signal transduction pathway. In this study, we found that PTX enhanced HIV-1 replication in acute infection systems at a high dose (1-10 microg/ml) in vitro. PTX treatment enhanced the infectivity of HIV-1-based pseudovirus enveloped with HIV-1 or amphotropic murine leukemia virus (A-MuLV), but not with vesicular stomatitis virus (VSV). This high dose of PTX treatment did not affect HIV-1 gene expression. These data suggested that the effect was virus envelope dependent and that PTX acted on an early stage of viral infection. Treatment with B-oligomer, a nonenzymatic subunit of PTX, mimicked this enhancing effect of PTX. However, desialylation of viral and cellular surface glycoproteins, which are receptors for B-oligomer, did not affect the augmentation induced by PTX. These results indicate that the enhancement of HIV-1 replication is mediated through an unknown biological function of B-oligomer.

Acquisition of HIV type 1 resistance by beta-chemokine-producing CD4+ T cells.

The CD4+ T cell is a major target cell type for human immunodeficiency virus type 1 (HIV-1) infection. In this study, we provide evidence that the susceptibility to HIV-1 infection is variable in individual CD4+ T cells. Five CD4+ T cell clones were isolated from an HIV-1-seronegative donor and were investigated for their susceptibility to HIV-1 infection. Four CD4+ T cell clones were resistant to infection by a macrophage-tropic (R5) HIV-1 isolate whereas one clone was fully permissive. The level of susceptibility to HIV-1 correlated inversely with beta-chemokine production, including RANTES (regulated on activation, normally T cell expressed and secreted), macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta. Resistance to HIV-1 infection was abrogated by the combined use of neutralizing antibodies against these three beta-chemokines. Interestingly, a complete inhibition of HIV-1 infection was observed in peripheral blood mononuclear cells on infection induced by adding the culture supernatant or a small number of HIV-1-resistant cell clones. Our results suggest the presence of a clonal self-defense mechanism within the CD4+ T cell population in vivo that involves the secretion of beta-chemokines.

Isolation and characterization of a full-length molecular DNA clone of Ghanaian HIV type 1 intersubtype A/G recombinant CRF02_AG, which is replication competent in a restricted host range.

We have isolated a replication-competent, full-length molecular clone of HIV-1 CRF02_AG, designated p97GH-AG1, by reconstituting two separately amplified genomic regions of an HIV-1 provirus of a 1997 Ghanaian isolate. The phylogenetic and recombination breakpoint analyses revealed that 97GH-AG1 had an A/G recombinant structure similar to that of prototype Nigerian isolate IbNG. The 17-nucleotide insertion downstream of the primer-binding site appeared to be a common sequence signature specific to most CRF02_AG strains, including 97GH-AG1. 97GH-AG1 showed an R5 phenotype and exerted productive infection in both HOS and NP2 cell infectivity assays, whereas it failed to show a detectable level of progeny production in peripheral blood mononuclear cells (PBMCs). The data may suggest the presence of unknown determinant(s) that dictate efficient replication in PBMCs, but that are not required for replication in immortalized cell lines.