D similar effects. The inhibitory effect on viral replication was not dose-dependent within the range of tested concentrations of VIP and PACAP between 1 nM and 100 nM, with the optimal inhibitory Title Loaded From File concentration at 10 nM for both peptides. The finding that VIP treatment at a concentration 5-fold that of the optimal HIV-1 inhibitory concentration and PACAP treatment at a 10-fold ratio did not influence viral production may be explained by receptor desensitization. On the other hand, theVIP and PACAP Inhibit HIV-1 Infectionobserved increase in HIV-1 production induced by 100 10457188 nM VIP could be explained by the property of inverse agonism that occurs for some ligands at saturating concentrations, a phenomenon that has already been described for a variety of G-coupled receptor ligands [37]. Alternatively, taking into account that the sole activation of VPAC1 facilitates HIV-1 infection in CD4+ T cell lines (27), one could explain the HIV-1 promoting effect by VIP at 100 nM due to a possible preferential engagement of VPAC1 at high VIP concentrations, although it has been described that the affinity of VIP for its receptors VPAC1 and VPAC2 is similar [4?7]. Importantly, the concentration of both neuropeptides that effectively inhibited HIV-1 production (10 nM) also possesses a variety of immunomodulatory roles [38?1], suggesting that the ability of these peptides to influence HIV-1 replication in macrophages is associated with their immunoregulatory activities on these cells. The ability of VIP and PACAP to down-regulate HIV-1 production became even more evident in light of the experiment testing their additive and synergistic activity on infected macrophages. Both neuropeptides presented an additive effect at 5 nM concentration, and showed potent synergy at 1 nM concentration, and their combined effects at these concentrations were the same as those observed following treatment with each molecule individually at 10 nM concentration (Fig. 2). Furthermore, the addition of sub-optimal concentrations of the receptor agonists to HIV-1-infected macrophages restricted the viral growth in a similar manner to treatment with equivalent doses of VIP and PACAP (Fig. 5). Thus, even taking into account that the activation of the VPAC1 receptor may favor HIV-1 replication, we believe that the concomitant engagement of the three receptors by the native neuropeptides, as presumably occurs in lymphoid tissues, will indeed enhance the macrophage resistance to HIV-1 growth. VIP promoted HIV-1 inhibition Title Loaded From File through stimulation of the receptors VPAC1 and VPAC2 but not through stimulation of PAC1. This effect likely occurred due to the high affinity of VIP for VPAC1 and VPAC2 and its low affinity for PAC1. The ability of PACAP to diminish HIV-1 replication, on the other hand, resulted from its ligation of all three receptors because its effect was only abrogated when all three receptors were blocked. Because the PACAP affinity for PAC1 is higher than its affinity for VPAC1 or VPAC2 [8], it is conceivable that it inhibits HIV-1 upon preferential binding to PAC1, but can also exert an inhibitory effect following ligation to VPAC1 and VPAC2 in the absence or hindrance of PAC1, or in a situation of excess PACAP concentration. We defined the receptors preference for the HIV-1 inhibitory activity of VIP and PACAP using specific receptor agonists. The combination of these molecules not only mimicked the effects of the natural neuropeptides but also established the receptors VP.D similar effects. The inhibitory effect on viral replication was not dose-dependent within the range of tested concentrations of VIP and PACAP between 1 nM and 100 nM, with the optimal inhibitory concentration at 10 nM for both peptides. The finding that VIP treatment at a concentration 5-fold that of the optimal HIV-1 inhibitory concentration and PACAP treatment at a 10-fold ratio did not influence viral production may be explained by receptor desensitization. On the other hand, theVIP and PACAP Inhibit HIV-1 Infectionobserved increase in HIV-1 production induced by 100 10457188 nM VIP could be explained by the property of inverse agonism that occurs for some ligands at saturating concentrations, a phenomenon that has already been described for a variety of G-coupled receptor ligands [37]. Alternatively, taking into account that the sole activation of VPAC1 facilitates HIV-1 infection in CD4+ T cell lines (27), one could explain the HIV-1 promoting effect by VIP at 100 nM due to a possible preferential engagement of VPAC1 at high VIP concentrations, although it has been described that the affinity of VIP for its receptors VPAC1 and VPAC2 is similar [4?7]. Importantly, the concentration of both neuropeptides that effectively inhibited HIV-1 production (10 nM) also possesses a variety of immunomodulatory roles [38?1], suggesting that the ability of these peptides to influence HIV-1 replication in macrophages is associated with their immunoregulatory activities on these cells. The ability of VIP and PACAP to down-regulate HIV-1 production became even more evident in light of the experiment testing their additive and synergistic activity on infected macrophages. Both neuropeptides presented an additive effect at 5 nM concentration, and showed potent synergy at 1 nM concentration, and their combined effects at these concentrations were the same as those observed following treatment with each molecule individually at 10 nM concentration (Fig. 2). Furthermore, the addition of sub-optimal concentrations of the receptor agonists to HIV-1-infected macrophages restricted the viral growth in a similar manner to treatment with equivalent doses of VIP and PACAP (Fig. 5). Thus, even taking into account that the activation of the VPAC1 receptor may favor HIV-1 replication, we believe that the concomitant engagement of the three receptors by the native neuropeptides, as presumably occurs in lymphoid tissues, will indeed enhance the macrophage resistance to HIV-1 growth. VIP promoted HIV-1 inhibition through stimulation of the receptors VPAC1 and VPAC2 but not through stimulation of PAC1. This effect likely occurred due to the high affinity of VIP for VPAC1 and VPAC2 and its low affinity for PAC1. The ability of PACAP to diminish HIV-1 replication, on the other hand, resulted from its ligation of all three receptors because its effect was only abrogated when all three receptors were blocked. Because the PACAP affinity for PAC1 is higher than its affinity for VPAC1 or VPAC2 [8], it is conceivable that it inhibits HIV-1 upon preferential binding to PAC1, but can also exert an inhibitory effect following ligation to VPAC1 and VPAC2 in the absence or hindrance of PAC1, or in a situation of excess PACAP concentration. We defined the receptors preference for the HIV-1 inhibitory activity of VIP and PACAP using specific receptor agonists. The combination of these molecules not only mimicked the effects of the natural neuropeptides but also established the receptors VP.
