Abstract
BackgroundHerpesviruses have evolved chemokines and chemokine receptors, which modulate the recruitment of human leukocytes during the inflammatory response to infection. Early post-infection, human herpesvirus 6A (HHV-6A) infected cells express the chemokine receptor U51A and chemokine U83A which have complementary effects in subverting the CC-chemokine family thereby controlling anti-viral leukocyte recruitment. Here we show that, to potentiate this activity, the viral chemokine can also avoid clearance by scavenger chemokine receptors, DARC and D6, which normally regulate an inflammatory response. Conversely, U83A delays internalisation of its signalling target receptor CCR5 with diversion to caveolin rich membrane domains. This mechanism can redirect displaced human chemokines to DARC and D6 for clearance of the anti-viral inflammatory response, leaving the viral chemokine unchecked.MethodsCell models for competitive binding assays were established using radiolabeled human chemokines and cold U83A on CCR5, DARC or D6 expressing cells. Flow cytometry was used to assess specific chemotaxis of CCR5 bearing cells to U83A, and internalisation of CCR5 specific chemokine CCL4 after stimulation with U83A. Internalisation analyses were supported by confocal microscopy of internalisation and co-localisation of CCR5 with caveosome marker caveolin-1, after virus or human chemokine stimulation.ResultsU83A displaced efficiently human chemokines from CCR5, with a high affinity of 0.01nM, but not from DARC or D6. Signalling via CCR5 resulted in specific chemoattraction of primary human leukocytes bearing CCR5. However, U83A effective binding and signalling to CCR5 resulted in delayed internalisation and recycling up to 2 hours in the absence of continual re-stimulation. This resulted in diversion to a delayed caveolin-linked pathway rather than the rapid clathrin mediated endocytosis previously shown with human chemokines CCL3 or CCL4.ConclusionU83A diverts human chemokines from signalling, but not regulatory or scavenger, receptors facilitating their clearance, while occupying signalling receptors at the cell surface. This can enhance virus specific inflammation, facilitating dissemination to replication sensitive leukocytes while evading clearance; this has implications for linked neuro-inflammatory pathologies.
Highlights
Herpesviruses have evolved chemokines and chemokine receptors, which modulate the recruitment of human leukocytes during the inflammatory response to infection
U83A efficiently displaces human chemokines from CCR5 signalling chemokine receptor in model system to compare to regulatory receptors In order to investigate U83A activities on regulatory receptors and to compare them to those on signalling receptors, U83A binding competition was screened against chemokines relevant for regulatory receptor specificity
CCL3 and CCL5 were selected for the comparisons as their binding can be competed by U83A on signalling receptors CCR1 and CCR5 as shown in previous tests on CCR5 bearing astrocytic U373, monocytic U937, peripheral blood mononuclear cells (PBMC) as well as model CCR1 or CCR5 transfected COS-7 cells [2,8]
Summary
Herpesviruses have evolved chemokines and chemokine receptors, which modulate the recruitment of human leukocytes during the inflammatory response to infection. U83A delays internalisation of its signalling target receptor CCR5 with diversion to caveolin rich membrane domains This mechanism can redirect displaced human chemokines to DARC and D6 for clearance of the antiviral inflammatory response, leaving the viral chemokine unchecked. To overcome the chemokine defence mechanism and redirect it towards enhanced virus persistence, HHV-6 encodes two chemokine receptors (U12 and U51) and one chemokine (U83) [2,3,4,5] These are potential virulence factors in linked inflammatory pathologies. U83A from HHV-6A is a high affinity broad-range yet selective agonist for CC-chemokine receptors CCR1, CCR4, CCR5, CCR6 and CCR8, while HHV-6B U83B is a low affinity CCR2 ligand [2,4] This disparity suggests U83 plays a key role in tropism and pathology differences between variant strains. It is important to establish effects of U83A in an inflammatory response
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have