Biomedical subjects
T M Niederman
Publications and source records attributed to T M Niederman.
Myristoylation-enhanced binding of the HIV-1 Nef protein to T cell skeletal matrix.
The negative factor, Nef, of HIV-1 was found to associate to an extent of 16-42% with the detergent insoluble cytoskeletal fraction of T lymphocytes. Furthermore, Escherichia coli expressed Nef protein was found to bind during in vitro reactions with the cytoskeletal matrix to an extent of 30-50%. Cytoskeletal association of Nef was significantly enhanced by myristoylation. The specificity of the myristoylation-enhanced binding was demonstrated by the lack of an effect of myristoylation on binding of the HIV-1 Gag protein to the cytoskeleton. Cytoskeletal binding was saturable, and inhibited by high concentrations of sodium chloride, or with SDS or urea. Binding of Nef to the cytoskeletal matrix may be important in mediating its effects on HIV-1 replication.
Human immunodeficiency virus type 1 Nef protein inhibits NF-kappa B induction in human T cells.
Human immunodeficiency virus type 1 (HIV-1) can establish a persistent and latent infection in CD4+ T lymphocytes (W. C. Greene, N. Engl. J. Med. 324:308-317, 1991; S. M. Schnittman, M. C. Psallidopoulos, H. C. Lane, L. Thompson, M. Baseler, F. Massari, C. H. Fox, N. P. Salzman, and A. S. Fauci, Science 245:305-308, 1989). Production of HIV-1 from latently infected cells requires host cell activation by T-cell mitogens (T. Folks, D. M. Powell, M. M. Lightfoote, S. Benn, M. A. Martin, and A. S. Fauci, Science 231:600-602, 1986; D. Zagury, J. Bernard, R. Leonard, R. Cheynier, M. Feldman, P. S. Sarin, and R. C. Gallo, Science 231:850-853, 1986). This activation is mediated by the host transcription factor NF-kappa B [G. Nabel and D. Baltimore, Nature (London) 326:711-717, 1987]. We report here that the HIV-1-encoded Nef protein inhibits the induction of NF-kappa B DNA-binding activity by T-cell mitogens. However, Nef does not affect the DNA-binding activity of other transcription factors implicated in HIV-1 regulation, including SP-1, USF, URS, and NF-AT. Additionally, Nef inhibits the induction of HIV-1- and interleukin 2-directed gene expression, and the effect on HIV-1 transcription depends on an intact NF-kappa B-binding site. These results indicate that defective recruitment of NF-kappa B may underlie Nef's negative transcriptional effects on the HIV-1 and interleukin 2 promoters. Further evidence suggests that Nef inhibits NF-kappa B induction by interfering with a signal derived from the T-cell receptor complex.
Simian immunodeficiency virus negative factor suppresses the level of viral mRNA in COS cells.
The nef gene is conserved among all human and simian lentiviruses. However, the amino acid similarity between simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 NEF is only 38%. To assess the role of SIV NEF on virus replication and compare its activity with that of its human immunodeficiency virus type 1 counterpart, we examined the activity of an intact nef gene from proviral clone pSIV 102, an isolate from SIV-MAC-251-infected cells. Proviral clone pSIV BA was constructed by introducing a premature termination codon at codon 40 of the nef gene without altering the predicted amino acid sequence of the overlapping env gene. These two clones were transfected into CD4- COS cells, and virus replication was monitored by p27 enzyme-linked immunosorbent assay kits. In seven independent experiments, clone pSIV BA afforded two- to sixfold greater levels of viral antigen compared with those in clone pSIV 102 and two- to sixfold-increased levels of viral mRNAs as indicated with Northern (RNA) blot and S1 nuclease protection analyses. Nuclear run-on assays demonstrated a two- to threefold increased rate of RNA synthesis with nuclei isolated from cells transfected with pSIV BA compared with that from cells transfected with pSIV 102. In contrast, there was no apparent destabilization of SIV mRNAs by NEF, as measured in dactinomycin-treated cells. This study demonstrates that SIV NEF is a negative regulator of virus replication and acts by suppressing the level of mRNA synthesis and accumulation in COS cells.
Human immunodeficiency virus type 1 negative factor is a transcriptional silencer.
The negative factor (nef) of human immunodeficiency virus (HIV) type 1 acts to down-regulate virus replication. To decipher the step in the virus life cycle affected by nef, functional proviral clones with (pHIV F-) or without (pHIV F+) a deletion mutation in the nef gene were constructed. In CD4+ cells, 30- to 50-fold more virus was produced over the course of 18-20 days with cultures infected with F- compared to F+ virus. In CD4- cell lines, 2- to 10-fold greater virus production was found from cultures transfected with pHIV F- than those transfected with pHIV F+. The negative regulatory effects of nef on pHIV F- could be supplied in trans with a plasmid expressing only the nef gene product. Virus produced by COS-1 cells transfected with pHIV F- or pHIV F+ showed similar binding, uptake, uncoating, and reverse transcription. Analysis of HIV-1 RNA and structural protein levels and rates of viral RNA synthesis in CD4- cells also showed 2- to 10-fold higher levels in cells transfected with pHIV F- compared to pHIV F+. The activity of a HIV-1-chloramphenicol acetyltransferase (CAT) plasmid was also suppressed by nef, whereas other CAT plasmids were unaffected. These findings demonstrate that nef acts as a specific silencer of HIV-1 transcription. This activity may be critical for maintenance of HIV-1 latency in vivo.
Functional analysis of HIV1 and SIV Nef proteins.
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