Interactions of HIV-1 nef with the mu subunits of adaptor protein complexes 1, 2, and 3: role of the dileucine-based sorting motif.

HIV-1 Nef interacts with cellular adaptor protein (AP) complexes and their medium (mu) subunits. However, the role of the dileucine-based sorting motif within Nef in these interactions has been incompletely characterized. Here, yeast two-hybrid assays indicated that HIV-1 Nef interacted not only with the mu subunits of AP-1 and AP-2, but also with that of AP-3. The interactions with mu1 and mu3 were markedly stronger than the interaction with mu2. Leucine residues of the sorting motif were required for the interactions with mu3 and mu2 and contributed to the interaction with mu1. Confocal immunofluorescence microscopy indicated that Nef, AP-1, and AP-3 (but not AP-2) were concentrated in a juxtanuclear region near the cell center, potentially facilitating interaction between Nef and the mu1 and mu3 subunits. However, leucine residues of the sorting motif were not required for this subcellular localization of Nef. These data suggest that the dileucine motif, required for optimal viral replication, functions through interactions with a variety of AP complexes, including AP-3, potentially by recruiting adaptor complexes to subcellular locations specified by additional determinants in the Nef protein.

Two independent regions of HIV-1 Nef are required for connection with the endocytic pathway through binding to the mu 1 chain of AP1 complex.

The Nef protein from the human immunodeficiency virus (HIV) induces down-regulation of the CD4 and major histocompatibility complex class I molecules from the cell surface by interfering with the endocytic machinery. This work focuses on the interaction of HIV-1 Nef with the mu 1 chain of adaptor protein type 1 (AP1) complex and its contribution to the Nef-induced alterations of membrane trafficking. Two independent regions surrounding a disordered loop located in the C-terminal part of Nef are involved in mu 1 binding. Each region can separately interact with mu 1, and simultaneous point mutations within both regions are needed to abolish binding. We used CD8 chimeras in which the cytoplasmic tail was replaced by Nef mutants to show that these mu 1-binding sites contain determinants required to induce CD4 down-regulation and to target the chimera to the endocytic pathway by promoting AP1 complex recruitment. Ultrastructural analysis revealed that the CD8-Nef chimera provokes morphological alterations of the endosomal compartments and co-localizes with AP1 complexes. These data indicate that the recruitment by Nef of AP1 via binding to mu 1 participates in the connection of Nef with the endocytic pathway.

Analysis of the SH3-binding region of HIV-1 nef: partial functional defects introduced by mutations in the polyproline helix and the hydrophobic pocket.

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.

The dileucine-based sorting motif in HIV-1 Nef is not required for down-regulation of class I MHC.

A dileucine-based protein sorting motif has recently been identified within the C-terminal, solvent-exposed loop of HIV-1 Nef and has been shown to be required for Nef-mediated down-regulation of CD4 and for optimal viral infectivity. Here, we report that mutation of the dileucine motif has no effect on Nef-mediated down-regulation of class I MHC heavy chain. Instead, deletion of an acidic domain just N-terminal of the polyproline helix of the SH3-binding domain significantly impairs this function. These data indicate that down-regulation of class I MHC and CD4 are mechanistically distinct processes. The data also suggest that protein interactions mediated by the acidic domain, rather than by the dileucine motif, may contribute to this function of Nef.

Interaction of HIV-1 Nef with the cellular dileucine-based sorting pathway is required for CD4 down-regulation and optimal viral infectivity.

The HIV-1 Nef protein is important for pathogenesis, enhances viral infectivity, and regulates the sorting of at least two cellular transmembrane proteins, CD4 and major histocompatibility complex (MHC) class I. Although several lines of evidence support the hypothesis that the Nef protein interacts directly with the cellular protein sorting machinery, the sorting signal in HIV-1 Nef has not been identified. By using a competition assay that functionally discriminates between dileucine-based and tyrosine-based sorting signals, we have categorized the motif through which Nef interacts with the sorting machinery as dileucine-based. Inspection of diverse Nef proteins from HIV-1, HIV-2, and simian immunodeficiency virus revealed a well-conserved sequence in the central region of the C-terminal, solvent-exposed loop of Nef (E/DXXXLphi) that conforms to the consensus sequence of the dileucine-based sorting motifs found in cellular transmembrane proteins. This sequence in NefNL4-3, ENTSLL, functioned as an endocytosis signal when appended to the cytoplasmic tail of a heterologous protein. The leucine residues in this motif were required for the interaction of full-length Nef with the dileucine-based sorting pathway and were required for Nef-mediated down-regulation of CD4. These leucine residues were also required for optimal viral infectivity. These data indicate that a dileucine-based sorting signal in Nef is utilized to address the cellular sorting machinery. The data also suggest that an influence on the distribution of cellular transmembrane proteins may mechanistically unite two previously distinct properties of Nef: down-regulation of CD4 and enhancement of viral infectivity.

Producer-cell modification of human immunodeficiency virus type 1: Nef is a virion protein.

Type 1 human immunodeficiency viruses encoding mutated nef reading frames are 10- to 30-fold less infectious than are isogenic viruses in which the nef gene is intact. This defect in infectivity causes nef-negative viruses to grow at an attenuated rate in vitro. To investigate the mechanism of Nef-mediated enhancement of viral growth rate and infectivity, a complementation analysis of nef mutant viruses was performed. To provide Nef in trans upon viral infection, a CEM derivative cell line (designated CLN) that expresses Nef under the control of the viral long terminal repeat was constructed. When nef-negative virus was grown in CLN cells, its growth rate was restored to wild-type levels. However, the output of nef-negative virus during the first 72 h after infection of CLN cells was not restored, suggesting that provision of Nef within the newly infected cell does not enhance the productivity of a nef-negative provirus. The genetically nef-negative virions produced by the CLN cells, however, were restored to wild-type levels of infectivity as measured in a syncytium formation assay in which CD4-expressing HeLa cells were targets. These trans-complemented, genetically nef-negative virions yielded wild-type levels of viral output following a single cycle of replication in primary CD4 T cells as well as in parental CEM cells. To define the determinants for producer cell modification of virions by Nef, the role of myristoylation was investigated. Virus that encodes a myristoylation-negative nef was as impaired in infectivity as was virus encoding a deleted nef gene. Because myristoylation is required for both membrane association of Nef and optimal viral infectivity, the possibility that Nef protein is included in the virion was investigated. Wild-type virions were purified by filtration and exclusion chromatography. A Western blot (immunoblot) of the eluate fractions revealed a correlation between peak Nef signal and peak levels of p24 antigen. Although virion-associated Nef was detected in part as the 27-kDa full-length protein, the majority of immunoreactive protein was detected as a 20-kDa isoform. nef-negative virus lacked both 27- and 20-kDa immunoreactive species. Production of wild-type virions in the presence of a specific inhibitor of the human immunodeficiency virus type 1 protease resulted in virions which contained only 27-kDa full-length Nef protein. These data indicate that Nef is a virion protein which is processed by the viral protease into a 20-kDa isoform within the virion particle.

Prevalence of Type A behavior in untreated hypertensive individuals.

Type A behavior has been associated with coronary heart disease as well as high cholesterol and smoking, major risk factors for coronary heart disease, but the data indicating a similar association with hypertension are inconsistent. Since past studies have usually based hypertension on a single blood pressure assessment or have often included treated hypertensive patients, this inconsistency is not surprising. The current study compared the prevalence of Type A behavior (assessed by Rosenman's structured interview) between 109 untreated hypertensive subjects and 109 age-, sex-, ethnic-, and occupation-matched normotensive subjects. Hypertension status was based on five repeated assessments over a 5-month period. Results indicated that Type A behavior is more prevalent in untreated, mildly hypertensive employed individuals than occupationally matched normotensive subjects. Type A component analysis confirmed the importance of hostility and certain vigorous voice stylistics in predicting cardiovascular conditions. These findings, taken together with the evidence linking Type A behavior with high cholesterol and cigarette smoking, further support the view that this behavior pattern is associated with increased risk of coronary heart disease.

Accuracy of indirect measures of medication compliance in hypertension.

The purpose of this study was to identify the most accurate indirect measure of medication compliance in primary hypertension through comparison with a recently developed direct measure of the antihypertensive agent, hydrochlorothiazide. A convenience sample of 40 subjects was seen by the investigator twice in an office setting and once in their homes. Data were collected by an interview schedule, blood pressure measurement, pill counts, urine analysis, and hospital record review. Patient interview was the most sensitive and accurate measure of compliance; this measure correctly classified 85% of patients as to compliant or noncompliant.

Adaptation in chronic illness: an eclectic model for nurses.

The growing prevalence of chronic illness is due, in large part, to the control and/or eradication of infectious diseases as well as, in North America, the increasingly aged population and overall lengthening life span. Although the trajectory of chronic illness is in a generally downward direction, the rate of progression may vary as plateaux an remissions occur. As these changes occur, the individual and his family move through a process of continual adjustment. This paper presents a conceptual model of the process of adaptation in chronic illness. The primary concept in the model is appraisal (Lazarus, 1966) and the thought that continuous appraisal/reappraisal of the individual's progress toward the goal of adaptation is required by the individual, his family, and the nurse. The focus of the model is the chronically ill individual and his family. The goal is movement toward adaptation to the illness and its ramifications. The process is caring, which facilitates movement towards the goal. The model and its interpretation are outlined in the paper. Finally, some strategies for nurses, and others, are suggested as ways in which the concepts may be applied and implemented in practice.