HIV-1 Rev escorts unspliced viral mRNAs out of the nucleus of infected cells, which allows formation of infectious HIV-1 virions. We have identified a putative DEAD box (Asp-Glu-Ala-Asp) RNA helicase, DDX1, as a cellular co-factor of Rev, through yeast and mammalian two-hybrid systems using the N-terminal motif of Rev as "bait". DDX1 is not a functional homolog of HIV-1 Rev, but down-regulation of DDX1 resulted in an alternative splicing pattern of Rev-responsive element (RRE)-containing mRNA, and attenuation of Gag p24 antigen production from HLfb rev- cells rescued by exogenous Rev. Co-transfection of a DDX1 expression vector with HIV-1 significantly increased viral production. DDX1 binding to Rev, as well as to the RRE, strongly suggest that DDX1 affects Rev function through the Rev-RRE axis. Moreover, down-regulation of DDX1 altered the steady state subcellular distribution of Rev, from nuclear/nucleolar to cytoplasmic dominance. These findings indicate that DDX1 is a critical cellular co-factor for Rev function, which maintains the proper subcellular distribution of this lentiviral regulatory protein. Therefore, alterations in DDX1-Rev interactions could induce HIV-1 persistence and targeting DDX1 may lead to rationally designed and novel anti-HIV-1 strategies and therapeutics.

The human CXC chemokine receptor 4 (CXCR4) is a receptor for the chemokine stromal cell-derived factor (SDF-1␣) and a co-receptor for the entry of specific strains of human immunodeficiency virus type I (HIV-1). CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus type VIII. SDF-1␣ or vMIP-II binding to CXCR4 can inhibit HIV-1 entry via this co-receptor. An approach combining protein structural modeling and site-directed mutagenesis was used to probe the structure-function relationship of CXCR4, and interactions with its ligands SDF-1␣ and vMIP-II and HIV-1 envelope protein gp120. Hypothetical threedimensional structures were proposed by molecular modeling studies of the CXCR4⅐SDF-1␣ complex, which rationalize extensive biological information on the role of CXCR4 in its interactions with HIV-1 envelope protein gp120. With site-directed mutagenesis, we have identified that the amino acid residues Asp (D20A) and Tyr (Y21A) in the N-terminal domain and the residue Glu (E268A) in extracellular loop 3 (ECL3) are involved in ligand binding, whereas the mutation Y190A in extracellular loop 2 (ECL2) impairs the signaling mediated by SDF-1␣. As an HIV-1 co-receptor, we found that the Nterminal domain, ECL2, and ECL3 of CXCR4 are involved in HIV-1 entry. These structural and mutational studies provide valuable information regarding the structural basis for CXCR4 activity in chemokine binding and HIV-1 viral entry, and could guide the design of novel targeted inhibitors.Chemokines are a family of 8 -10-kDa small proteins that act as chemoattractants of various types of leukocytes to sites of inflammation and to secondary lymphoid organs. Based on the positions of two conserved cysteine residues in their N termini, chemokines can be divided into four subfamilies: CC, CXC, CX3C, and C (1, 2). The stromal cell-derived factor-1 (SDF-1␣) 1 is one of the CXC chemokines, which plays critical roles in the migration, proliferation, and differentiation of leukocytes. SDF-1␣ is the only known natural ligand of CXCR4 receptor (3, 4). CXCR4 can also be recognized by an antagonistic ligand, the viral macrophage inflammatory protein-II (vMIP-II) encoded by the Kaposi's sarcoma-associated herpesvirus (5). vMIP-II acts as a selective chemoattractant for T helper 2 cells and monocytes and is an agonist for CCR8 (6). vMIP-II displays a broader spectrum of receptor activities than any mammalian chemokine, as it binds with high affinity to a number of both CC and CXC chemokine receptors including CXCR4 and CCR5 and inhibits cell entry of human immunodeficiency virus type I (HIV-1) mediated by these receptors (7,8).CXCR4 belongs to the family of seven transmembrane Gprotein-coupled receptors that transduce signals via heterotrimeric G-proteins (9). Recent studies with knockout mice of CXCR4 have demonstrated that this molecule plays an important role in immunomodulation, organogenesis, hematopoiesis, and derailed cerebellar neuron migration (10 -12). CXCR4 has...

The chemokine receptor CXCR4 is critical for many biological functions, such as B-cell lymphopoiesis, regulation of neuronal cell migration, and vascular development (1-3). In addition, CXCR4 together with another chemokine receptor CCR5 are two principal co-receptors for the cellular entry of the human immunodeficiency virus type 1 (HIV-1) 1 (4 -7). The stromal cell-derived factor-1 (SDF-1␣) is the only known natural ligand of CXCR4 and plays important roles in migration, proliferation, and differentiation of leukocytes (8, 9). The viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus 8 (10) is an antagonistic chemokine ligand of CXCR4 (11, 12). vMIP-II also interacts with other chemokine receptors such as CCR5 and CCR3 and inhibits HIV-1 entry mediated by these co-receptors.CXCR4 and other chemokine receptors belong to the superfamily of seven transmembrane G-protein-coupled receptors (GPCRs) (13). These membrane proteins transmit signals from extracellular ligands to intracellular biological pathways via heterotrimeric G-proteins and have been a major class of therapeutic targets for a wide variety of human diseases (14). As such, characterizing the mechanism of biological recognition between these receptors and their ligands is essential for understanding the physiological or pathological processes that they mediate and devising novel strategies for clinical intervention. For CXCR4, studies have been carried out by a number of laboratories using chimeric chemokine receptors and site-specific mutants to study multiple domains of CXCR4 that are important for interacting with chemokine ligands and HIV-1 (15-23). However, because there is no high resolution crystal structure available for CXCR4 (or any other chemokine receptor) alone or complexed with ligands, the structural and biochemical basis of ligand binding and signaling through these important membrane receptors remains poorly understood.To further define the structure-function relationship of the chemokine receptor-ligand interaction, theoretical computer modeling and site-directed mutagenesis were combined to predict plausible structural models for chemokine receptors and their complexes with ligands, such as interleukin-8 receptor ␤ (24) and CCR5 (25,26). Structural models of CXCR4 and its complex with ligands were also proposed (27, 28). Complementary to modeling and mutational analyses of the receptors,

The APJ receptor is widely expressed in the human central nervous system (CNS). Apelin was recently identified as the endogenous peptidic ligand for human APJ. Studies with animal models suggested that APJ and apelin play an important role in the hypothalamic regulation of water intake and the endocrine axis, in the regulation of blood pressure, and in cardiac contractility. Apelin has been found to block the activity of APJ as a human immunodeficiency virus type I (HIV-1) coreceptor. In this study, we combined chemical synthetic approaches with alanine substitution to evaluate the structural requirements for interactions with the APJ receptor. We demonstrated that apelin peptides in aqueous solution adopt a random conformation, and the positive charge and hydrophobic residues of apelin-13 play important roles in interactions with the APJ receptor. We have observed an important correlation between receptor binding affinity and cell-cell fusion inhibitory activity. The elucidation of structural requirements of apelin-13 in its interaction with the APJ receptor is critical for further investigation of apelin-APJ functions in vivo and in the design of small molecular inhibitors for potential treatment of HIV-1 infection in the CNS.