Interaction of CCR5 with the HIV-1 gp120-CD4 complex involves its amino-terminal domain (Nt-CCR5) and requires sulfation of 2-4 tyrosine residues in Nt-CCR5. The conformation of a 27-residue Nt-CCR5 peptide, sulfated at Y10 and Y14, was studied in both its free form and in a ternary complex with deglycosylated-gp120 and a CD4-mimic peptide. NMR experiments revealed a helical conformation at the center of Nt-CCR5(1-27) which is induced upon gp120 binding, as well as a helical propensity for the free peptide. A well-defined structure for the bound peptide was determined for residues 7-23, increasing by two-fold the length of Nt-CCR5's known structure. Two-dimensional saturation transfer experiments and measurement of relaxation-times highlighted Nt-CCR5 residues Y3, V5, P8-T16, E18, I23 and possibly D2 as the main binding determinant. A calculated docking model for Nt-CCR5(1-27) suggests that residues 2-22 of Nt-CCR5 interact with the bases of V3 and C4 while the C-terminal segment of Nt-CCR5(1-27) points towards the target cell membrane reflecting an Nt-CCR5 orientation that differs by 180° from a previous model. A gp120 site that could accommodate CCR5Y3 in a sulfated form has been identified. The present model attributes a structural basis for binding interactions to all gp120 residues previously implicated in Nt-CCR5 binding. Moreover, the strong interaction of sulfated CCR5Tyr14 with gp120Arg440 revealed by the model and the previously found correlation between E322 and R440 mutations shed light on the role of these residues in HIV-1 phenotype conversion furthering our understanding of CCR5 recognition by HIV-1.

Chemokines constitute a large family of small proteins that regulate leukocyte trafficking to the site of inflammation by binding to specific cell-surface receptors belonging to the GPCR superfamily. The interactions between N-terminal (Nt-) peptides of these GPCRs and chemokines have been studied extensively using NMR spectroscopy. However, due to lower affinities of peptides representing the three extracellular loops (ECLs) of chemokine receptors to their respective chemokine ligands, information concerning these interactions is scarce. To overcome the low affinity of ECL peptides to chemokines, we linked two or three CCR5 extracellular domains by either biosynthesis in Escherichia coli or by chemical synthesis. Using such chimeras, CCR5 binding to RANTES was followed using 1H-15N-HSQC spectra to monitor titration of the chemokine with peptides corresponding to the extracellular surface of the receptor. Nt-CCR5 and ECL2 were found to be the major contributors to CCR5 binding to RANTES, creating a nearly closed ring around this protein by interacting with opposing faces of the chemokine. A RANTES positively charged surface involved in Nt-CCR5 binding resembles the positively charged surface in HIV-1 gp120 formed by the C4 and the base of the V3. The opposing surface on RANTES, composed primarily of β2-β3 hairpin residues, binds ECL2 and was found to be analogous to a surface in the crown of the gp120 V3. The chemical and biosynthetic approaches for linking GPCR surface regions discussed herein should be widely applicable to investigation of interactions of extracellular segments of chemokine receptors with their respective ligands.

HSV-1 vhs activity.The infection of cells with herpes simplex virus type 1 (HSV-1) is associated with a global shutoff of host protein synthesis early postinfection (p.i.) (15,25,26,38,46,48). Analyses of the infected cells have revealed the destruction of polyribosomes and the degradation of infected cell mRNAs following infection with the wild type (wt) virus (9,11,25,26,46,48,50). We have previously derived viral mutants which did not shut off host protein synthesis and degrade the mRNAs (38). Employing marker rescue analyses with fragments from the entire genome, we have mapped the virion host shutoff ("vhs") phenotype within a 265-bp segment of the UL41 gene (26). The mRNA degradation function was also found to map in this region. Sequencing analyses of the corresponding 265-bp segments within the wt and the vhs-1 mutant virus revealed a threonine-to-isoleucine mutation in amino acid 214 of the vhs protein (A. D. Kwong and N. Frenkel, unpublished results). All in all, the UL41 gene product corresponds to a 489-amino-acid phosphoprotein localized in the virion tegument, from where it is released upon viral entry into the cell (25,(38)(39)(40)(41). It is expressed as a late (␥1) gene product which is incorporated into the structural particles of progeny virus. We have shown that in the presence of actinomycin D the UL41 protein was involved in the degradation of mRNAs transcribed prior to and during infection, including the housekeeping genes ␤-actin and ␣-tubulin, which were persistently present in the cells, and the heat shock protein 70 (HSP70), induced p.i. (46). These mRNAs were stable in cells infected with the vhs-1 mutant virus.Several studies examined whether the vhs protein had an intrinsic nucleolytic activity. Read and coworkers (36) have shown that the transfection of cells with a plasmid containing the UL41 gene inhibited the expression of a cotransfecting chloramphenicol acetyltransferase reporter gene. This activity was absent in cells transfected with mutant plasmids (22,36). Furthermore, Everly and Read (12) provided genetic and biochemical evidence that the vhs protein has an mRNase activity. Smiley and coworkers (7,8) documented that the vhs protein produced in vitro employing the rabbit reticulocyte (RRL) translation system possessed an endonucleolytic activity on added exogenous RNA substrates. Furthermore, when vhs was expressed in the budding yeast Saccharomyces cerevisiae there was inhibition of colony formation, while vhs mutants tested in the system had no activity. Cell extracts of yeast expressing vhs displayed an endoribonuclease activity if the extracts were

The HIV-1 envelope glycoprotein gp41 undergoes a sequence of extensive conformational changes while participating in the fusion of the virus with the host cell. Since the discovery of its postfusion conformation, the structure and function of the protease-resistant six-helix bundle (6-HB) have been the subject of extensive investigation. In this work, we describe additional determinants (S528-Q540 and W666-N677) in the fusion peptide proximal region (FP-PR) and the membrane proximal external region (MPER) that stabilize the six-helix bundle and are involved in the interaction of T-20 (FUZEON, an anti-HIV-1 fusion inhibitor drug) with the gp41 FP-PR. Circular dichroism and sedimentation equilibrium measurements indicate that the 1:1 mixture of N′ and C′ peptides comprising residues A541-T569 and I635-K665 from the gp41 first and second helical repeats, HR1 and HR2, respectively, fail to form a stable six-helix bundle. Triglutamic acid and triarginine tags were added to these N′ and C′ peptides, respectively, at the termini distant from the FP-PR and the MPER to alter their pI and increase their solubility at pH 3.5. The tagged HR1 and HR2 peptides were elongated by addition of residues S528-Q540 from the FP-PR and residues W666-N677 from the MPER, respectively. A 1:1 complex of the elongated peptides formed a stable six-helix bundle which melted at 60 °C. These results underscore the importance of a detailed high-resolution characterization of MPER interactions, the results of which may improve our understanding of the structure-function relationship of gp41 and its role in HIV-1 fusion.The human immunodeficiency virus type 1 (HIV-1) 1 envelope protein is initially produced as a precursor glycoprotein gp160, which is proteolytically cleaved into two subunits. The resulting surface subunit (gp120) and transmembrane subunit (gp41) remain noncovalently associated and oligomerize as trimers on the surface of the virion. The transmembrane subunit gp41 mediates fusion between HIV-1 and its target cells (1). After the penetration of the gp41 † This study was supported by NIH Grants GM53329 (J.A.) and GM22086 (F. * To whom correspondence should be addressed. E-mail: Jacob.Anglister@weizmann.ac.il. . ‡ Weizmann Institute of Science. § College of Staten Island of the City University of New York.1 Abbreviations: CD, circular dichroism; DIEA, diisopropylethylamine; EDTA, ethylenediaminetetraacetic acid; EEE, Glu-Glu-Glu tag; FP-PR, fusion peptide proximal region; gp41, HIV-1 glycoprotein 41; gp120, HIV-1 glycoprotein 120; HIV-1, human immunodeficiency virus type 1; HPLC, high-pressure liquid chromatography; HR1, HIV-1 gp41 N′ heptad repeat 1; HR2, HIV-1 gp41 C′ heptad repeat 2; IPTG, isopropyl β-D-1-thiogalactopyranoside; K d , dissociation constant; MPER, membrane proximal external region; MALDI, matrixassisted laser desorption ionization; NMR, nuclear magnetic resonance; RRR, Arg-Arg-Arg tag; SIV, simian immunodeficiency virus; T-20, HIV fusion inhibitor corresponding to gp41 residues Y638-F673; TFA, trifluo-roacet...