Structural constraints on human immunodeficiency virus type 1 Nef function

Abstract

HIV-1 Nef is a multifunctional protein that exerts its activities through interactions with multiple cellular partners. Nef uses different domains and mechanisms to exert its functions including cell surface down-modulation of CD4 and MHC-I receptors and activation of the serine/threonine kinase PAK-2. We inserted tags at the C-terminus and proximal to the N-terminus of Nef and the effects on Nef's structure/function relationships were examined. We discovered significant defects in MHC-I down-modulation with the insertion of HA/FLAG tags at either region. We also found impaired PAK-2 activation with a C-terminal fusion with GFP. Interestingly, Nef-GFP and Nef-GH(7) induced MHC-I down-modulation, suggesting that the negative charge of the HA/FLAG tag could contribute to the observed defect. Together, these observations highlight elements of Nef's functional complexity and demonstrate previously unsuspected structural requirements for PAK-2 activation and MHC-1 down-modulation in Nef's flexible N- and C-terminal regions.

FIG. 1. Diagram of Nefs Modified with Exogenous Sequences

(A) SF2-HF or NA7-HF Nefs; (B) SF2-GFP or NA7-GFP Nefs; (C) SF2-GH7 Nef; (D) HA-SF2, HA/HA-SF2 or HA/FL/HA-SF2 Nefs. The dark line represents the coding sequence of HIV-1 SF2 or NA7 Nef.

FIG. 2. Preferential co-immunoprecipitation of activated PAK-2 with C-terminal HA/FLAG (HF) tagged SF2 and NA7 Nefs with anti-Nef antibodies

Plasmids expressing SF2 or NA7 Nefs or Nefs tagged at the C-terminus with HA/FLAG were transfected into 293T cells. Cellular lysates were immunoprecipitated with the indicated antibodies and PAK-2 binding and activation was assayed by in vitro kinase assay (A) or immunoprecipitates were analyzed by Western blot with the mouse monoclonal anti-Nef antibody (EH1) to monitor the efficiency of immunoprecipitation (B). The positions of the heavy and light chains of the mouse antibodies used for immunoprecipitation are indicated to the right of the blots. Protein expression levels and transfection efficiency were assayed by Western blot of whole cell lysates using anti-Nef or anti-GFP antibodies (C).

FIG. 3. CD4 and MHC-I down-modulation with C-terminal HA/FLAG (HF) tagged SF2 and NA7 Nefs

CEM cells expressing SF2, SF2-HF, NA7 or NA7-HF Nefs were analyzed for down-modulation of cell-surface MHC-I and CD4 using two-color flow cytometry (left panels). Nef expression in the transduced CEM cells was confirmed by Western blotting of cellular lysates with anti-Nef antibodies (right panel).

FIG. 4. Functional Analysis of C-terminal Nef-GFP fusion proteins

(A) Empty vector (pCGCG) or plasmids expressing SF2, SF2-GFP, NA7, and NA7-GFP were transfected into 293T cells. Cellular lysates were immunoprecipitated with polyclonal anti-Nef antibodies and PAK-2 binding and activation was assayed by in vitro kinase assay (upper left panel). The efficiency of immunoprecipitation of the different Nefs using polyclonal anti-Nef antibodies was determined by Western blot of immunoprecipitates (lower left panel). Protein expression levels and transfection efficiency were assayed by Western blot of whole cell lysates using mouse anti-Nef and anti-GFP antibodies (right upper and lower panels). Note that the pCGCG vector constitutively expresses GFP (30 kD). (B) CEM cells transduced with LXSN or LnefSN (expressing Nef or Nef-GFP fusion proteins) were analyzed for down-modulation of cell-surface MHC-I and CD4 using two-color flow cytometry. Nef expression levels in the transduced CEM cells were determined by Western blotting of cellular lysates with anti-Nef antibodies.

FIG. 5. Functional Analysis of Nef containing a positive amino acid C-terminal tag (SF2 Nef-GH₇)

(A) Empty vector (pcDNA3.1) or plasmids expressing SF2 or SF2-GH₇ were transfected into 293T cells. Cellular lysates were immunoprecipitated with polyclonal anti-Nef antibodies and PAK-2 binding and activation was assayed by in vitro kinase assay (upper left panel). The efficiency of immunoprecipitation of the Nefs using polyclonal anti-Nef antibodies was determined by Western blot of separate immunoprecipitates (lower left panel). Protein expression levels and transfection efficiency were assayed by Western blot of the whole cell lysate using mouse anti-Nef and anti-GFP antibodies (right upper and lower panels respectively). (B) CEM cells expressing SF2 or SF2-GH₇ were analyzed for down-modulation of cell-surface MHC-I and CD4 using two-color flow cytometry. Nef expression levels in the transduced CEM cells were determined by Western blotting of cellular lysates with anti-Nef antibodies.

FIG. 6. Co-immunoprecipitation of activated PAK-2 with Nefs containing HA, HA/HA or HA/FLAG/HA tags inserted at position 24 with anti-Nef and anti-tag antibodies

Empty vector (pcDNA3.1) or plasmids expressing SF2 or SF2 containing the internally inserted tags HA, HA/HA or HA/FLAG/HA were transfected into 293T cells. Cellular lysates were immunoprecipitated with the indicated antibodies and PAK-2 binding and activation was assayed by in vitro kinase assay (A), or anti-tag immunoprecipitates were analyzed by Western blot with mouse anti-Nef antibodies (EH1) to monitor the efficiency of immunoprecipitation (B). The positions of the heavy and light chains of the mouse antibodies used for immunoprecipitation are indicated to the right of the blots. Protein expression levels and transfection efficiency were assayed by Western blot of the whole cell lysate using mouse anti-Nef and anti-GFP antibodies (C).

FIG. 7. CD4 and MHC-I down-modulation with Nefs containing HA, HA/HA or HA/FLAG/HA tags inserted at position 24

CEM cells transduced with LXSN or LnefSN (expressing SF2 or SF2 Nefs with internal tags) were analyzed for down-modulation of cell-surface MHC-I and CD4 using two-color flow cytometry (left panel). Nef expression levels in the transduced CEM cells were determined by Western blotting of cellular lysates with anti-Nef antibodies (right panel).