Genetic and Functional Diversity of Human Immunodeficiency Virus Type 1 Subtype B Nef Primary Isolates

Abstract

We have characterized the functional integrity of seven primary Nef isolates: five from a long-term nonprogressing human immunodeficiency virus (HIV)-infected individual and one each from two patients with AIDS. One of the seven Nefs was defective for CD4 downregulation, two others were defective for PAK-2 activation, and one Nef was defective for PAK-2 activation and major histocompatibility complex (MHC) class I downregulation. Five of the Nefs were tested and found to be functional for the enhancement of virus particle infectivity. The structural basis for each of the functional defects has been analyzed by constructing a consensus nef, followed by mutational analysis of the variant amino acid residues. Mutations A29V and F193I were deleterious to CD4 downregulation and PAK-2 activation, respectively, while S189R rendered Nef defective for both MHC class I downregulation and PAK-2 activation. A search of the literature identified HIVs from five patients with Nefs predominantly mutated at F193 and from one patient with Nefs predominantly mutated at A29. A29 is highly conserved in all HIV subtypes except for subtype E. F193 is conserved in subtype B (and possibly in the closely related subtype D), but none of the other HIV group M subtypes. Our results suggest that functional distinctions may exist between HIV subtypes.Human immunodeficiency virus (HIV) Nef has multiple, well-defined functions, including the downregulation of cell surface CD4 (18), downregulation of cell surface major histocompatibility complex (MHC) class I (55), the enhancement of viral infectivity (14), and the activation of a 62-kDa protein kinase that has been recently identified as PAK-2 (3, 49). Determining which Nef functions are crucial for high rates of virus replication and pathogenesis in HIV disease will provide new targets for anti-HIV drug development (29).HIV replicates actively and mutates rapidly; therefore, any mutation that prevents an optimal rate of replication would be rapidly selected against (10,22,60). Conversely, some Nef functions may be dispensable after the establishment of infection and of little significance in disease progression. Therefore, finding primary isolate nef genes that are functionally defective suggests that the lost functions may not be required for efficient viral replication in vivo. We have previously characterized a primary isolate of Nef (233 Nef) for CD4 downregulation, PAK-2 activation, and enhancement of infectivity. 233 Nef was found to be deficient in PAK-2 activation (36). Primary isolate nef genes defective in CD4 downregulation have also been reported (40,42).To further explore the possibility that some in vitro-defined Nef functions are not required for efficient replication in vivo, we have characterized the functional integrity of five primary isolate Nefs from a long-term nonprogressing HIV-infected individual (patient D [24]). These Nefs exhibit considerable functional diversity despite a high degree of structural similarity. The structural basis for each of the functional defects ...

Dynamic Evolution of the Human Immunodeficiency Virus Type 1 Pathogenic Factor, Nef

“…S1, top, in the supplemental material). Mutations of the SH3 binding domain (13,22,53) or the dimerization domain have consistently resulted in Nefs defective for multiple activities (5,12,17,26,35,37,(51)(52)(53). Specifically, mutations of the SH3 domain and dimerization domain result in Nefs defective for MHC class I downregulation, PAK-2 activation, and enhancement of virus particle infectivity, with the dimerization domain also defective in CD4 downregulation (5,35).…”

“…To this end, in a previous report we characterized seven primary isolate Nefs for each of the four genetically separable functions listed above (17). In this way we hoped to utilize the structural integrity of functioning Nefs to discover mechanistically interpretable structure-function relationships.…”

“…These are PAK-2 activation, CD4 downregulation, major histocompatibility complex (MHC) class I downregulation, and enhancement of viral particle infectivity (17,36). One significant aspect of these Nef activities is the remarkable fact that all four of these activities are genetically independent of each other (17,36). How Nef accomplishes these four tasks presents a difficult problem of mechanistic analysis because of the complications of multiple, overlapping functional domains in the context of high sequence variability.…”

“…To study this functionally and structurally complex protein, we have focused on four well-characterized activities proposed to be pathologically significant (6). These are PAK-2 activation, CD4 downregulation, major histocompatibility complex (MHC) class I downregulation, and enhancement of viral particle infectivity (17,36). One significant aspect of these Nef activities is the remarkable fact that all four of these activities are genetically independent of each other (17,36).…”

Activation of p21-Activated Kinase 2 by Human Immunodeficiency Virus Type 1 Nef Induces Merlin Phosphorylation

“…We have previously demonstrated that a conserved phenylalanine in the C-terminal region of Nef (position 195 of SF2 Nef) is essential for PAK2 activation but not for CD4 downregulation, MHC class I downregulation, or the enhancement of viral infectivity (9). When the SF2 Nef F195 was mutated to isoleucine or arginine, the mutant lost its ability to activate PAK2 but retained the activities of CD4 or MHC class I downregulation and enhancement of infectivity (9) (data not shown).…”