Cytosolic domain of the human immunodeficiency virus envelope glycoproteins binds to calmodulin and inhibits calmodulin-regulated proteins.

S.K Srinivas,R.V Srinivas,G.M Anantharamaiah,R.W Compans,J.P Segrest

doi:10.1016/s0021-9258(18)41610-9

S.K Srinivas, R.V Srinivas + Show 3 more

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https://doi.org/10.1016/s0021-9258(18)41610-9

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Abstract

Calmodulin (CaM), the major intracellular receptor for calcium, is involved in regulation of diverse cellular functions. Positively charged amphipathic helical segments have been identified as an important structural motif in the recognition of CaM by different CaM-activated enzymes and peptides. The carboxyl-terminal domain of the envelope glycoproteins of human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV) contain regions that can fold into amphipathic helical segments, which closely resemble the amphipathic segments found in CaM-activated enzymes. We show here that synthetic peptide analogs corresponding to the two putative amphipathic helical regions of HIV-1/WMJ gp160 bind to CaM with high affinity (Kd 31-41 nM) in the presence of calcium. They also bind CaM in the absence of calcium, although with much lower affinity. The peptides inhibit CaM-regulated activation of bovine brain phosphodiesterase in vitro. The peptides also inhibit mitogen-induced lymphocyte activation, a property shared by CaM antagonists. Purified HIV-1 gp160 binds to CaM, while gp120, which lacks the putative amphipathic helical segments, does not bind CaM. In HIV-infected cells, the putative CaM-binding regions of gp160 are located intracellularly and may therefore interact with the cytosolic CaM. We postulate that CaM binding by HIV envelope proteins is likely to exert diverse modulatory effects, and the mechanism for HIV-induced cytotoxicity may involve, in part, inhibition of CaM-regulated cellular functions.

Highlights

From the Wepartments of Medicine and Biochemistry, and Atherosclerosis Research Unit, University of Alabama at Birmingham, Birmingham, Alabama 35294, the lllepartment of Microbiology and Immunology, Emory University, Atlanta, Georgia 30322, and the $Department of Infectious Diseases, St
Thecarboxyl-terminaldomain of the envelope glycoproteinosf human and simian immunodeficiency viruses (HIV-1, human immunodeficiency virus (HIV)-2,and SIV)contain regions that can fold into amphipathic helical segments,whichcloselyresemble the amphipathic segments found in CaM-activated enzymes
The HIV envelope glycoprotein is first synthesized as a precursor polyprotein, gp160; itisthen cleaved intracellularly to give rise to a surface (SUI glycoprotein designated as gp120, and a transmembrane (TM) glycoprotein designated as gp41 [3, 4].The SU and TM proteins remain associated as a complex held together by noncovalent interactions [5].The SU protein is located on the outersurfaces of the viral envelope and infected cells and functions as the viral attachment protein that binds to CD4 receptor oncell surfaces

Summary

H N Envelope Protein-Calmodulin Interactions

345 nm using an Aminco SPF 500 or a Perkin-Elmer LS spectrofluorometer. All the samples contained 50 mM MOPS in distilled water buffered to pH 7.3 at 25 “C, 200mM potassium chloride (KCl),and 1.0 m~ calcium chloride (CaCl,).In certain experiments, excess EGTA was included in thebuffer in order to determine the calcium dependenceof. Spectra were obtained at increasing concentrations of the peptides. Effectof Peptides on CaM-dependent Activationof Phosphodiesterase (PDE)-PDE assays were performed according to Johnson et al [15]. Activator-deficient bovine brain PDE (Sigma) and 8 mM mant-cGMP (Molecular Probes Inc., Eugene, OR) in 0.5 ml of 10 mM MOPS, 90 mM. Mant-cGMP undergoes a large decrease in fluorescence after hydrolysis by CaM-activated. W cGMP at 450 nm, after excitation at 280nm, was determined as a measure of PDE activity. The cells were resuspended in lymphocyte activation medium (RPMI mediumwith 20%fetal calf serum, 5 pg/ml phytohemagglutinin-P)and incubated in theabsence or presence of varying concentrationsof peptides.ARer 4 days, the number of viable cells in culture were determined by a colorimetricXTT assay [16] as a measure of lymphocyte activation. The plotswere generated using a COMBO program developed for VAXNMS workstations, which super-

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