Stimulation of Cell Surface CCR5 and CD40 Molecules by Their Ligands or by HSP70 Up-Regulates APOBEC3G Expression in CD4+ T Cells and Dendritic Cells

Abstract

Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like-3G (A3G) is an intracellular innate antiviral factor that deaminates retroviral cytidine to uridine. In an attempt to harness the anti-HIV effect of A3G, we searched for an agent that would up-regulate A3G and identify the receptors involved. Stimulation of cell surface CCR5 with CCL3 and CD40 with CD40L or both molecules with microbial 70-kDa heat shock protein (HSP)70 up-regulated A3G mRNA and protein expression in human CD4+ T cells and monocyte-derived dendritic cells (DC), demonstrated by real-time PCR and Western blots, respectively. The specificity of CCR5 and CD40 stimulation was established by inhibition with TAK 779 and mAb to CD40, as well as using human embryonic kidney 293 cells transfected with CCR5 and CD40, respectively. A dose-dependent increase of A3G in CCL3- or HSP70-stimulated CD4+ T cells was associated with inhibition in HIV-1 infectivity. To differentiate between the inhibitory effect of HSP70-induced CCR5 binding and that of A3G, GFP-labeled pseudovirions were used to infect human embryonic kidney 293 cells, which showed inhibition of pseudovirion uptake, consistent with A3G being responsible for the inhibitory effect. Ligation of cell surface CCR5 receptors by CCL3 or CD40 by CD40L activated the ERK1/2 and p38 MAPK signaling pathways that induced A3G mRNA expression and production of the A3G protein. These in vitro results were corroborated by in vivo studies in rhesus macaques in which A3G was significantly up-regulated following immunization with SIVgp120 and p27 linked to HSP70. This novel preventive approach may in addition to adaptive immunity use the intracellular innate antiviral effect of A3G.

Innate and adaptive immune correlates of vaccine and adjuvant-induced control of mucosal transmission of SIV in macaques

“…This evidence is in agreement with a previous study showing in vitro up-regulation of A3G can prevent cells from HIV/SIV infection in vitro (41). A number of observations have implicated innate immunity in affecting HIV replication and AIDS progression; however, direct experimental evidence was lacking.…”

A heat shock protein gene (Hsp70.1) is critically involved in the generation of the immune response to myelin antigen

“…Intriguingly, in hsp70.1 -/-mice this might represent a specific effect on CD4 + T cells since we saw no similar deficit in anti-CD3-induced proliferation or any increased apoptosis of CD8 + T cells in hsp70.1 -/-mice, and ConA stimulation of T cells was unaltered in these mice. Interestingly, a similar discrepancy between CD4 + and CD8 + T cell responses was reported for hsp70-induced T cellspecific up-regulation of the intracellular innate antiviral factor, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptidelike-3G (APOBEC3G) [56]. Thus, our results might suggest that hsp70 acts differentially on CD4 + and CD8 + T cells, especially with regard to cell survival.…”

Calreticulin requires an ancillary adjuvant for the induction of efficient cytotoxic T cell responses

“…However, Gao et al reported that some of the pro-inflammatory effects prescribed to hsp60 and hsp70 were likely due to endotoxin contamination (Gao and Tsan, 2003a;Gao and Tsan, 2003b;Gao and Tsan, 2004); likewise, flagellin (a TLR-2 ligand) has also been cited as a contaminant of hsp70 responsible for some of the observed stimulatory effects (Ye and Gan, 2007). Despite these reports, recent studies indicate that hsp70 produced in cell free systems, or expressed ectopically on the cell surface, can lead to activation of APCs, with CCR5 recently reported to be a candidate receptor (Korbelik et al, 2005;Pido-Lopez et al, 2007;Quintana and Cohen, 2005). Of note is that previous studies showed that endotoxin-depleted CRT and gp96 (up to 40 μg/ml) did not stimulate NF-κB activation nor did they induce nitric oxide synthesis by APCs: these are characteristic of TLR-mediated APC activation (Reed et al, 2003).…”