Immunological study of the nef protein from HIV-1 by polyclonal and monoclonal antibodies

Abstract

We constructed and expressed different overlapping fusion proteins with the nef gene of HIV-1 and generated specific polyclonal rabbit and monoclonal mouse antibodies against these recombinant proteins. The rabbit antisera, one of the monoclonal antibodies as well as a serum from a HIV-1 infected patient recognized the nef protein with Mr 27 kDa in latently HIV-1 infected glioma cells in the immunoblot. In contrast, these antibodies could not detect nef in productively HIV-1 infected Molt-3 cells neither in immunoblot nor in indirect immunofluorescence assays. These results indicate the possible participation of nef in viral latency. The recombinant nef proteins were used as probes for anti-nef antibodies in human sera. We observed in 17 of 57 sera tested specific anti-nef antibodies. All of these anti-nef positive sera also contained antibodies directed against viral structural proteins. The NH2-terminal region of the recombinant nef was shown to be the major immunodominant antigenic site in the immunoblot assay.

Oligomerization of the Nef protein from human immunodeficiency virus (HIV) type 1

“…The murine mAb8 9-13/158, directed against the amino-terminal part of the Nef protein, is an IgGl isotype as reported recently (Kienzle et al, 1991).…”

“…SDSPAGE and immunoblotting were performed essentially as described previously (Kienzle et al, 1991) using the ECL-Kit. The murine mAb8 9-13/158, directed against the amino-terminal part of the Nef protein, is an IgGl isotype as reported recently (Kienzle et al, 1991).…”

Association of Human Immunodeficiency Virus Nef Protein with Actin is Myristoylation Dependent and Influences its Subcellular Localization

“…SDS/PAGE and immunoblotting were performed essentially as described previously [39] using the ECL kit from Amersham Buchler. The murine anti-Nef mAb 89-13/158 directed against the N-terminal part of the Nef protein is of IgGl isotype as reported [39]. Protein migration standards from Pharmacia were used.…”

Expression of the HIV-1 Nef protein in the baculovirus system: investigation of anti-Nef antibodies response in human sera and subcellular localization of Nef

“…For construction of recombinant baculo-transfervectors, the BamHI fragment of plasmid pUF [14] containing the complete nef gene of HIV-1LAV-BRU [30] was cloned into a pAcYM1 non-fusion vector [19] as well as into a pAc360 fusion vector [17]. The two neftransfer vectors encoded different Nef proteins: (0 a non-fused, 206 aa long Nef (AYF3) and (ii) a N-terminal fused Nef protein (D3F3), which additionally carried 16 aa from the linker-and the N-terminal polyhedrin-sequence and 9 aa of the 5' upstream vicinity of the nefsequence.…”