Kaori Otake

Soluble Nef antigen of HIV-1 is cytotoxic for human CD4+ T cells

We have previously shown that Nef‐gene 10 fusion protein induces marked growth arrest of human primary CD4+ T cells. Here, in vitro cytostatic and cytotoxic activities of human immunodeficiency virus type 1 (HIV‐1) Nef against CD4+ T cells were extensively investigated. Growth of human CD4+ cells was inhibited significantly just by the addition of purified full‐length Nef to cultures. When Nef was cross‐linked by anti‐Nef antibodies, it became very cytocidal for CD4+ T cells. A high percentage of sera from HIV‐1‐infected individuals contained soluble Nef. Thus, soluble Nef in vivo may play an important role in immunodysfunction of CD4+ T lymphocytes in HIV‐1 infection.

Double‐Stranded nef RNA Interferes with Human Immunodeficiency Virus Type 1 Replication

RNA interference (RNAi) has been reported to be post‐transcriptional gene silencing (PTGS) by approximately 500 nucleotide‐(nt)‐long double‐stranded (ds) RNA that specifically targets homologous sequences of messenger RNA. In this report, we describe inhibition of HIV‐1 transcription by synthetic dsRNAs constructed with mutated nef genes (nef dsRNAs) derived from long‐term non‐progressors (LTNPs) using cotransfection of the target gene‐expressing plasmid and dsRNA. The effects of nef dsRNAs were examined with luciferase (Luc) reporter which is combined with the HIV‐1 (SF2) LTR in persistently HIV‐1‐infected T cell and macrophage cell lines. At 48 hr, a defective nef dsRNA (556 nt) suppressed Luc activity more potently than did SF2 full‐length nef dsRNA (744 nt), suggesting that approximately 500 nt‐long nef dsRNA could interfere with the HIV‐1 transcription.

HIV-1 Nef protein in the nucleus influences adipogenesis as well as viral transcription through the peroxisome proliferator-activated receptors.

Background: Although the HIV-1 Nef protein (27 kDa) localizes primarily in cytoplasm, there is considerable evidence suggesting its occasional localization in the nucleus. Nef is known to play an important role in transcriptional events and viral replication, but the actual target of Nef in the nucleus remains to be identified. Objective: To examine the functional roles of Nef in the nucleus and its possible interactions with other unknown factors in the nucleus. Methods: High-density microarray analysis was used to screen directly the unique functions of Nef on host gene transcription. The nuclear localization of Nef and its effects on the expression of peroxisome proliferator-activated receptors (PPAR) was examined using PPAR promoter/reporter assay and immunoblotting. A long terminal repeat/reporter assay was used to investigated the effects of Nef and PPAR on viral transcription. Results: Nef in the nucleus suppressed PPARγ expression and reduced fatty acid levels in human T and macrophage cell lines. Expression of Nef or PPAR suppressed viral replication; the effect of PPARγ or retinoid X receptor-α on viral replication were reduced by coexpression of Nef in MT(−)4 T cells. Conclusion: Nef may be involved in both viral replication and the wasting syndrome associated with AIDS.

Human immunodeficiency virus type 1 Nef protein on the cell surface is cytocidal for human CD4+ T cells

We have previously shown that the carboxyl‐terminal region of human immunodeficiency virus type 1 (HIV‐1) Nef antigen present on the outer surface of virus‐infected cells has affinity for uninfected T cells. Here, the in vitro cytotoxic potential of HIV‐1 Nef on the T cell surface against CD4+ T cells was investigated in detail. Human T cells expressing Nef on the cell surface by transfection with non‐infectious mutant HIV‐1 proviruses were demonstrated to kill CD4+ T cells effeciently. Furthermore, it was shown that the carboxyl‐terminal portion of Nef was cytotoxic for CD4+ T cells and that monoclonal antibody against the carboxyl‐terminal region of Nef inhibited Nef induced‐cytolysis. Thus, we concluded that Nef protein on CD4+ T cells may play an important role in the specific loss of CD4+ T lymphocytes during HIV‐1 infection.

Clustered localization of oligomeric Nef protein of human immunodeficiency virus type 1 on the cell surface

We studied human immunodeficiency virus type 1 (HIV‐1) Nef protein biochemically and histologically. HIV‐1 Nef, derived from baculosystem and from cells infected with HIV‐1, formed homomeric monomers, dimers, trimers, and further polymers. These oligomers were non‐covalently associated. In cells infected with HIV‐1, Nef molecules were clustered at the cell surface as well as cytoplasm. Our previous results have indicated that the Nef on the surface of cells infected with HIV‐1 is cytotoxic against uninfected CD4+ T cells. Thus, it is very likely that the HIV‐1‐mediated cytotoxic reaction is due, at least in part, to the clustered localization of oligomeric Nef on the cell surface.

In vitro cytocidal effects of human immunodeficiency virus type 1 Nef on unprimed human CD4+ T cells without MHC restriction

We have previously shown that the C-terminal region of human immunodeficiency virus type 1 (HIV-1) Nef antigen present on the outer surface of virus-infected cells has an affinity for uninfected T cells and that the Nef protein is responsible for T cell death. To exclude completely the possibility of MHC restriction of this cytotoxic activity, the in vitro cytotoxic potential of HIV-1 Nef against various CD4+ T cell lines as well as naive T lymphocytes was investigated using a baculovirus expression system. Insect cells expressing myristoylated Nef on their cell surface were shown to kill a proportion of CD4+ T cells within 8 h. However, N-terminal truncated and unmyristoylated Nef proteins were not present on the outer surface of insect cell membranes and failed to show any killing activity. Monoclonal antibodies against the C-terminal region of Nef inhibited cytolysis. Thus, we conclude that specific Nef-mediated cytolysis is induced by contact with unprimed CD4+ T lymphocytes without MHC restriction.

Isolation and sequencing of infectious clones of feline foamy virus and a human/feline foamy virus Env chimera

Full-length DNAs of the Coleman and S7801 strains (pSKY3.0, pSKY5.0) of infectious feline foamy viruses (FFVs) were cloned and sequenced. Parental viruses, designated SKY3.0 and SKY5.0, were secreted following transfection of Crandell feline kidney (CRFK) cells. Production of the rescued parental viruses was enhanced in the presence of trichostatin A. Amino acid sequence similarities between FFV and human foamy virus (HFV) are extremely low for the envelope protein and capsid antigen, as predicted from the two clones. However, a chimeric FFV clone was constructed with the HFV Env substituted for the FFV Env. The chimeric virus (HFFV, SKY4.0) was able to infect and replicate in CRFK cells as well as in peripheral blood mononuclear cells of cats in vivo. Consequently, the chimeric HFFV may be useful for the creation of FV vectors for gene transfer strategies.

HIV-1 Nef impairs multiple T-cell functions in antigen-specific immune response in mice

The viral protein Nef is a key element for the progression of HIV disease. Previous in vitro studies suggested that Nef expression in T-cell lines enhanced TCR signaling pathways upon stimulation with TCR cross-linking, leading to the proposal that Nef lowers the threshold of T-cell activation, thus increasing susceptibility to viral replication in immune response. Likewise, the in vivo effects of Nef transgenic mouse models supported T-cell hyperresponse by Nef. However, the interpretation is complicated by Nef expression early in the development of T cells in these animal models. Here, we analyzed the consequence of Nef expression in ovalbumin-specific/CD4(+) peripheral T cells by using a novel mouse model and demonstrate that Nef inhibits antigen-specific T-cell proliferation and multiple functions required for immune response in vivo, which includes T-cell helper activity for the primary and memory B-cell response. However, Nef does not completely abrogate T-cell activity, as defined by low levels of cytokine production, which may afford the virus a replicative advantage. These results support a model, in which Nef expression does not cause T-cell hyperresponse in immune reaction, but instead reduces the T-cell activity, that may contribute to a low level of virus spread without viral cytopathic effects.