To identify specific points of impact on the viral replication cycle, we carried out one-round infection and one-round viral gene expression assays. The HIV-1 replication cycle can be divided into two broad trajectories: (1) a process of infection in which the viral RNA genome is introduced into the cell and reverse transcribed into a DNA provirus, which then integrates into the host cell's chromosomes, and (2) a process of viral gene expression in which cellular activation induces transcription and translation of viral genes, followed by assembly and budding of new virus particles.
To determine whether norepinephrine might increase cellular vulnerability to infection, we carried out one-round infection assays in which cells were pretreated with either medium or norepinephrine and then exposed to a fixed concentration of HIV-1 virions. Proviral HIV-1 DNA was assayed by PCR 12 h later, and cells were cultured in the antiretroviral drug Indinivir to prevent production of infectious virions and subsequent rounds of infection. Exposure to micromolar concentrations of norepinephrine enhanced HIV-1 proviral penetrance by three- to fivefold (Fig. 9.3A). Subsequent studies identified the primary molecular mechanism of this effect in increased expression of the chemokine receptors CCR5 and CXCR4 that collaborate with CD4 to mediate HIV-1 entry into human cells (Cole et al., 1999a) (Fig. 9.3A). Activation of the PKA signaling pathway by norepinephrine or cAMP increased the cell surface density of CXCR4 by 6- to 10-fold on quiescent and activated lymphocytes, and norepinephrine also upregulated cell surface expression of CCR5 (Cole et al., 2001). Chemokine receptors constitutively traffic between the cell surface and endosomal compartments within the cell. Flow cytometric analyses demonstrated that PKA can inhibit CXCR4 internalization and thus enhance the fraction of the total receptor pool localized to the cell surface. HIV-1 binding assays showed that PKA-induced externalization of CXCR4 enhanced virion binding to the cell membrane. PKA-induced externalization of CXCR4 also
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Figure 9.3. Norepinephrine increases HIV-1 infectivity and gene expression.Treatment of PBMC with norepinephrine increased cell-surface expression of the CXCR4 and CCR5 chemokine receptors (A, top), resulting in enhanced vulnerability to viral infection as measured by PCR detection of proviral DNA (copies per ß-globin) at 12 h after exposure to virus (A, bottom). The PKA activator db-cAMP also enhanced cell-surface CXCR4 density on CD3/CD28 costimulated lymphocytes, resulting in enhanced chemotaxis in response to the chemokine SDF1a (B). Norepinephrine also enhanced activity of the HIV-1 promoter (LTR), as measured by expression of a murine CD24 reporter gene in activated T lymphocytes (C).
enhanced cellular chemotaxis in response to CXCR4's cognate ligand SDF-1a (Bleul et al., 1996; Oberlin et al., 1996) (Fig. 9.3B). Norepinephrine signaling enhanced CXCR4 surface levels in both CD4+ and CD8+ T lymphocytes as well as CD19+ B cells. In CD14+ monocytes, PKA suppressed cell surface expression of CXCR4. These data suggest that cate-cholamine activation of the PKA signaling pathway might modulate normal cellular trafficking and maturation processes in addition to enhancing cellular vulnerability to HIV-1 infection (Cole et al., 1999a).
To determine whether catecholamines might influence the viral gene expression phase of the life cycle, we also carried out one-round expression assays in which a population of primary PBMCs was infected with an HIV-1 reporter virus bearing the murine CD24 gene (Jamieson and Zack, 1998). Cells were then activated with antibodies to CD3 and CD28 in the presence of Indinivir to prevent subsequent rounds of infection. When these stimulation conditions were supplemented with micromolar concentrations of norepinephrine, flow cytometry showed a three- to fivefold increase in cell surface expression of the reporter gene product (Cole et al., 2001) (Fig. 9.3C). Thus, norepinephrine can enhance expression of genes under the control of the HIV-1 promoter—the viral long terminal repeat (LTR).
In addition to the direct effects on the viral replication cycle, cate-cholamines can also exert indirect or permissive effects on viral replication by undermining cellular production of antiviral cytokines. Immunore-gulatory cytokines such as IL-10 can suppress HIV replication, whereas proinflamatory cytokines enhance HIV replication by activating NF-kB (Finnegan et al., 1996; Badou et al., 2000; Weissman et al., 1995). Analysis of supernatant cytokine concentrations from CD3/CD28 costimulated T lymphocytes showed that norepinephrine could substantially suppress the production of both IL-10 and IFN-y (Fig. 9.4A). Addition of exogenous IL-10 or IFN-y to the cultures abrogated the replication-enhancing effects of norepinephrine (Fig. 9.4B), indicating that simultaneous suppression of both cytokines is required for norepinephrine-mediated enhancement of HIV-1 replication. Cytokine modulation might represent an overarching distal influence on more virus-proximal mechanisms such as LTR activity or chemokine receptor trafficking.
In a series of studies on type I interferon responses to HIV-1 or Toll-like receptor (TLR) ligands, we have also found that norepinephrine can suppress production of this crucial innate antiviral cytokine by both myeloid and lymphoid dendritic cells. These effects are mediated via the P-AR/ cAMP/PKA signaling cascade, which inhibits expression of both IFN-a and IFN-P genes in response to activation of either TLR-3 or TLR-9 (Fig. 9.4C). Using the IFN-P gene as a model system, we traced PKA's effects to the modulation of transcription control processes in a specific positive regulatory domain (PRD) in the promoter of the IFNB gene (Collado-Hidalgo et al., 2006). Type I interferons are known to suppress HIV-1 replication (Baca-Regen et al., 1994; Pitha, 1994; Agy et al., 1995; Korth et al., 1998), so
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