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Updated: Jan 25, 2026
Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals
Published on: January 5, 2016
Structural and functional correlates between HIV-1 and SIV Nef isolates
1Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, USA. victor.garcia@stjude.org
Abstract:
The nef genes of HIV-1 and SIV encode 27-34 kDa myristoylated proteins which have been shown to induce cell surface CD4 downregulation and bind to a cellular protein kinase. To identify regions of Nef important for function, structure-function correlates of HIVSF2 nef (Nef) and SIVmm239open nef (SNef) were sought by constructing Nef/SNef hybrids. Metabolic labeling with 35[S]methionine/cysteine demonstrated similar amounts of 35[S] incorporation into all but one hybrid, SNeftll, in which the C-terminus of SNef was replaced by that of Nef. The weak protein expression of SNeftll was attributable to its short half-life of approximately 45 min. Nef, SNef, and SHSNef, a hybrid containing the internal sequences of Nef and the N- and C-terminal sequences from SNef, downregulated CD4 in human CEM cells. Only Nef and SHSNef downregulated CD4 in mouse AKR1-G1 cells. Nef, SNef, and SHSNef also effectively bound phosphoproteins of MW = 62,000 and 78,000 in CEM cells. Two additional hybrids, in which the Nef sequences of SHSNef were replaced with additional SNef sequences, were essentially ineffective in both assays. Thus, in two different assays of Nef function, swapping the SIV and HIV internal nef sequences were shown to be greatly deleterious to Nef function while SHSNef remained functional.
Insights
The nef genes of Human Immunodeficiency Virus type 1 (HIV-1) and Simian Immunodeficiency Virus (SIV) are crucial for viral function. Swapping internal nef sequences between HIV-1 and SIV impaired protein function, but a specific hybrid retained activity.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- The nef genes of HIV-1 and SIV encode proteins involved in viral pathogenesis.
- These Nef proteins are known to downregulate cell surface CD4 and bind cellular protein kinases.
Purpose of the Study:
- To identify critical regions within Nef proteins essential for their function.
- To investigate structure-function relationships by creating and analyzing Nef/SNef hybrids.
Main Methods:
- Construction and analysis of hybrid nef genes combining sequences from HIV-1 Nef and SIV SNef.
- Metabolic labeling with 35S-methionine/cysteine to assess protein expression and stability.
- Functional assays including CD4 downregulation in human and mouse cell lines and binding to cellular phosphoproteins.
Main Results:
- Most Nef/SNef hybrids were expressed similarly, except one with a truncated SNef C-terminus, which showed reduced expression due to a short half-life.
- Nef, SNef, and a specific hybrid (SHSNef) downregulated CD4 in human cells; only Nef and SHSNef did so in mouse cells.
- Nef, SNef, and SHSNef bound specific cellular phosphoproteins; hybrids with swapped internal sequences were largely non-functional.
Conclusions:
- Internal sequences of HIV-1 Nef and SIV SNef are critical for their respective functions.
- The SHSNef hybrid, retaining internal Nef sequences, demonstrates conserved functionality across species-specific assays.
- These findings highlight the importance of specific domains within the Nef protein for its biological activities.
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