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Impaired lymphoid chemokine-mediated migration due to a block on the chemokine receptor switch in human cytomegalovirus-infected dendritic cells.

ABSTRACT: Dendritic cell (DC) migration from the site of infection to the site of T-cell priming is a crucial event in the generation of antiviral T-cell responses. Here we present to our knowledge the first functional evidence that human cytomegalovirus (HCMV) blocks the migration of infected monocyte-derived DCs toward lymphoid chemokines CCL19 and CCL21. DC migration is blocked by viral impairment of the chemokine receptor switch at the level of the expression of CCR7 molecules. The inhibition occurs with immediate-early-early kinetics, and viral interference with NF-kappaB signaling is likely to be at least partially responsible for the lack of CCR7 expression. DCs which migrate from the infected cultures are HCMV antigen negative, and consequently they do not stimulate HCMV-specific CD8(+) T cells, while CD4(+)-T-cell activation is not impaired. Although CD8(+) T cells can also be activated by alternative antigen presentation mechanisms, the spatial segregation of naive T cells and infected DCs seems a potent mechanism of delaying the generation of primary CD8(+)-T-cell responses and aiding early viral spread.

SUBMITTER: Moutaftsi M

PROVIDER: S-EPMC353728 | biostudies-other | 2004 Mar

REPOSITORIES: biostudies-other

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Project description:An open reading frame (ORF), US28, with homology to mammalian chemokine receptors has been identified in the genome of human cytomegalovirus (HCMV). Its protein product, pUS28, has been shown to bind several human CC chemokines, including RANTES, MCP-1, and MIP-1 alpha, and the CX(3)C chemokine fractalkine with high affinity. Addition of CC chemokines to cells expressing pUS28 was reported to cause a pertussis toxin-sensitive increase in the concentration of cytosolic free Ca(2+). Recently, pUS28 was shown to mediate constitutive, ligand-independent, and pertussis toxin-insensitive activation of phospholipase C via G(q/11)-dependent signaling pathways in transiently transfected COS-7 cells. Since these findings are not easily reconciled with the former observations, we analyzed the role of pUS28 in mediating CC chemokine activation of pertussis toxin-sensitive G proteins in cell membranes and phospholipase C in intact cells. The transmembrane signaling functions of pUS28 were studied in HCMV-infected cells rather than in cDNA-transfected cells. Since DNA sequence analysis of ORF US28 of different laboratory and clinical strains had revealed amino acid sequence differences in the amino-terminal portion of pUS28, we compared two laboratory HCMV strains, AD169 and Toledo, and one clinical strain, TB40/E. The results showed that infection of human fibroblasts with all three HCMV strains led to a vigorous, constitutively enhanced formation of inositol phosphates which was insensitive to pertussis toxin. This effect was critically dependent on the presence of the US28 ORF in the HCMV genome but was independent of the amino acid sequence divergence of the three HCMV strains investigated. The constitutive activity of pUS28 is not explained by expression of pUS28 at high density in HCMV-infected cells. The pUS28 ligands RANTES and MCP-1 failed to stimulate binding of guanosine 5'-O-(3-[(35)S]thiotriphosphate to membranes of HCMV-infected cells and did not enhance constitutive activation of phospholipase C in intact HCMV-infected cells. These findings raise the possibility that the effects of CC chemokines and pertussis toxin on G protein-mediated transmembrane signaling previously observed in HCMV-infected cells are either independent of or not directly mediated by the protein product of ORF US28.

Project description:Patients with Fragile X syndrome, the leading monogenetic cause of autism, suffer from impairments related to the prefrontal cortex, including working memory and attention. Synaptic inputs to the distal dendrites of layer 5 pyramidal neurons in the prefrontal cortex have a weak influence on the somatic membrane potential. To overcome this filtering, distal inputs are transformed into local dendritic Na+ spikes, which propagate to the soma and trigger action potential output. Layer 5 extratelencephalic (ET) prefrontal cortex (PFC) neurons project to the brainstem and various thalamic nuclei and are therefore well positioned to integrate task-relevant sensory signals and guide motor actions. We used current clamp and outside-out patch clamp recording to investigate dendritic spike generation in ET neurons from male wild-type and Fmr1 knockout (FX) mice. The threshold for dendritic spikes was more depolarized in FX neurons compared to wild-type. Analysis of voltage responses to simulated in vivo 'noisy' current injections showed that a larger dendritic input stimulus was required to elicit dendritic spikes in FX ET dendrites compared to wild-type. Patch clamp recordings revealed that the dendritic Na+ conductance was significantly smaller in FX ET dendrites. Taken together, our results suggest that the generation of Na+ -dependent dendritic spikes is impaired in ET neurons of the PFC in FX mice. Considering our prior findings that somatic D-type K+ and dendritic hyperpolarization-activated cyclic nucleotide-gated-channel function is reduced in ET neurons, we suggest that dendritic integration by PFC circuits is fundamentally altered in Fragile X syndrome. KEY POINTS: Dendritic spike threshold is depolarized in layer 5 prefrontal cortex neurons in Fmr1 knockout (FX) mice. Simultaneous somatic and dendritic recording with white noise current injections revealed that larger dendritic stimuli were required to elicit dendritic spikes in FX extratelencephalic (ET) neurons. Outside-out patch clamp recording revealed that dendritic sodium conductance density was lower in FX ET neurons.

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Impaired lymphoid chemokine-mediated migration due to a block on the chemokine receptor switch in human cytomegalovirus-infected dendritic cells.

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