Cellular localization of Nef expressed in persistently HIV-1-infected low-producer astrocytes.
OBJECTIVE
The characterization and localization of HIV-1 Nef highly expressed in permanently infected astrocytes (TH4-7-5) as a model for latent infection of human brain cells.


METHODS
Immunochemical methods are an appropriate tool to investigate expression and localization of cellular proteins.


METHODS
Nef expression was analysed by Western blot and immunoperoxidase staining using a panel of monoclonal and polyclonal antibodies. Cellular localization studies were performed by indirect immunofluorescence and subcellular fractionation of TH4-7-5 cells. Myristoylation of Nef was investigated by immunoprecipitation of [3H]myristic acid-labelled cell extract. TH4-7-5 nef gene was cloned and amplified by polymerase chain reaction and the nef nucleotide sequence analysed.


RESULTS
Reactivities of various Nef-specific antibodies with Nef antigen in TH4-7-5 cells were demonstrated by Western blot analysis. Immunofluorescence revealed cytoplasmic perinuclear staining of Nef with most antibodies. However, one monoclonal antibody against amino acids 168-175 of Nef showed intense homogeneous nuclear staining in TH4-7-5 cells. Reactivity of this Nef antibody was blocked with recombinant Nef derived from TH4-7-5 cells. After subcellular fractionation, Nef was detected in nuclear, membrane and cytosolic fractions of TH4-7-5 cells. No myristoylated Nef antigen was detectable, perhaps because of a serine residue at position 2 of the TH4-7-5 nef gene instead of the glycine residue required for myristoylation.


CONCLUSIONS
Chronically HIV-1-infected astrocytoma cells with restricted virus production express different antigenic forms of Nef, which can be distinguished by their subcellular localization. Variant subcellular targeting of Nef suggests the existence of multiple activities of Nef within HIV-infected cells.
Kohleisen
B
B
Institut für Molekulare Virologie, GSF Forschungszentrum für Umwelt und Gesundheit, Neuherberg, Germany.
Neumann
M
M
Herrmann
R
R
Brack-Werner
R
R
Krohn
K J
KJ
Ovod
V
V
Ranki
A
A
Erfle
V
V
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral / Q000276:immunology / Q000302:isolation & purification*; D001253:Astrocytes / Q000382:microbiology*; D002451:Cell Compartmentation; D015693:Gene Products, nef / Q000276:immunology / Q000302:isolation & purification*; D014644:Genetic Variation; D015497:HIV-1 / Q000254:growth & development*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D009685:Nuclear Envelope / Q000737:chemistry / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000737:chemistry / Q000276:immunology; D014407:Tumor Cells, Cultured; D054311:nef Gene Products, Human Immunodeficiency Virus
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D015693:Gene Products, nef; D009227:Myristic Acids; D010446:Peptide Fragments; D054311:nef Gene Products, Human Immunodeficiency Virus; D019814:Myristic Acid