Analysis of the SH3-Binding Region of HIV-1 Nef: Partial Functional Defects Introduced by Mutations in the Polyproline Helix and the Hydrophobic Pocket

Abstract

An SH3-binding domain within the Nef protein of primate lentiviruses has been reported to be important to viral replication and infectivity and dispensable for CD4 downregulation, but its precise role remains unclear. This study investigates the effects of mutations in both the polyproline helix and in the hydrophobic pocket that constitute the SH3-binding domain of Nef. The data demonstrate that the well-studied mutation of the central prolines is only partially disruptive to viral infectivity and replication. The central prolines also make a subtle contribution to the efficiency of CD4 downregulation, detectable only using low levels of Nef expression. Mutation of a conserved arginine in the polyproline helix abrogated more completely Nef-mediated enhancement of viral infectivity; this mutation also adversely affected CD4 downregulation at low levels of Nef expression. Only the R77A mutation substantially impaired downregulation of class I MHC. However, mutation of the central prolines and of R77 yielded proteins that were expressed less efficiently than wild-type Nef. The R77A mutant was expressed most poorly, compatible with its defective phenotypes in all assays. Mutations of the hydrophobic pocket were minimally detrimental to both the virologic and the receptor modulatory functions of Nef. Taken together, this analysis suggests that mutations in the SH3-binding domain do not abrogate fully any Nef-associated phenotype in the absence of detrimental effects on protein expression. We suggest that mutations in this domain can introduce incomplete effects caused by subtle impairments to protein expression; these effects may appear selective under certain experimental conditions due to different sensitivities of the assays to the level of Nef expression.

Dynamin 2 is required for the enhancement of HIV-1 infectivity by Nef

“…3C). The PP 72,75 AA mutation significantly impaired infectivity enhancement by Nef in one study (25), but had only a 2-fold effect in another study (34), in agreement with our results (Fig. 3C).…”

Nef Stimulates Human Immunodeficiency Virus Type 1 Replication in Primary T Cells by Enhancing Virion-Associated gp120 Levels: Coreceptor-Dependent Requirement for Nef in Viral Replication

“…Multiple functions of Nef, including CD4 downregulation and interaction with cellular signaling pathways, are important for optimal HIV replication in activated PBMC and CD4 ϩ primary T lymphocytes (18,29,43,60,72). Although the large defects in the infectivity and gp120 content of X4-tropic, Nef-defective HIV virions produced in primary T cells likely result from the incapacity of these viruses to downregulate cell surface CD4, it is entirely possible that the absence of another function of Nef is responsible for these defects.…”

Human Immunodeficiency Virus Type 1 Nef: Adapting to Intracellular Trafficking Pathways

“…It has been reported that binding of the SH3 domains of either c-Src or Hck to Nef has a major impact on the conformation of the N-terminal anchor domain of Nef (5). Thus, the polyproline domain in Nef may have an important structural role that could also affect protein stability (36). Because higher Nef levels are required for Nef to affect MHC-I compared with CD4 (96), structural defects may affect these activities differently.…”