Differential coreceptor expression allows for independent evolution of non-syncytium-inducing and syncytium-inducing HIV-1.

We demonstrated previously that CD45RA(+) CD4(+) T cells are infected primarily by syncytium-inducing (SI) HIV-1 variants, whereas CD45RO(+) CD4(+) T cells harbor both non-SI (NSI) and SI HIV-1 variants. Here, we studied evolution of tropism for CD45RA(+) and CD45RO(+) CD4(+) cells, coreceptor usage, and molecular phylogeny of coexisting NSI and SI HIV-1 clones that were isolated from four patients in the period spanning SI conversion. NSI variants were CCR5-restricted and could be isolated throughout infection from CD45RO(+) CD4(+) cells. SI variants seemed to evolve in CD45RO(+) CD4(+) cells, but, in time, SI HIV-1 infection of CD45RA(+) CD4(+) cells equaled infection of CD45RO(+) CD4(+) cells. In parallel with this shift, SI HIV-1 variants first used both coreceptors CCR5 and CXCR4, but eventually lost the ability to use CCR5. Phylogenetically, NSI and SI HIV-1 populations diverged over time. We observed a differential expression of HIV-1 coreceptors within CD45RA(+) and CD45RO(+) cells, which allowed us to isolate virus from purified CCR5(+) CXCR4(-) and CCR5(-) CXCR4(+) CD4(+) cells. The CCR5(+) subset was exclusively infected by CCR5-dependent HIV-1 clones, whereas SI clones were preferentially isolated from the CXCR4(+) subset. The differential expression of HIV-1 coreceptors provides distinct cellular niches for NSI and SI HIV-1, contributing to their coexistence and independent evolutionary pathways.

Susceptibility of in vitro stimulated PBMC to infection with NSI HIV-1 is associated with levels of CCR5 expression and beta-chemokine production.

Susceptibility of PHA/rIL-2-stimulated PBMC from 14 healthy blood donors for NSI HIV-1 infection was analyzed in relation to CCR5 expression and beta-chemokine production. After 1 week of culture in the presence of rIL-2, but not at the moment of inoculation, CCR5 surface expression was positively and beta-chemokine production was inversely associated with susceptibility to NSI HIV-1 infection. Surprisingly, no association was observed between CCR5 genotype and in vitro NSI HIV-1 susceptibility, which was in agreement with similar levels of CCR5 surface expression and beta-chemokine production in CCR5Delta32/+ and CCR5 +/+ PBMC after PHA/rIL-2 stimulation. In contrast to what was observed in vitro, CCR5 genotype did associate with CCR5 surface expression levels in vivo in resting as well as in activated CD4(+) T cell populations that were identified by the expression of CD45RO, CD27, HLA-DR, and CD69. The association between CCR5 expression and susceptibility to infection by NSI HIV-1 observed in vitro might offer an explanation for the in vivo observed protective effect of CCR5 polymorphisms that influence CCR5 expression on disease progression.

Vascular-endothelial-cadherin modulates endothelial monolayer permeability.

Vascular endothelial (VE)-cadherin is the endothelium-specific member of the cadherin family of homotypic cell adhesion molecules. VE-cadherin, but not the cell adhesion molecule platelet/endothelial cell adhesion molecule (PECAM-1), markedly colocalizes with actin stress fibers at cell-cell junctions between human umbilical vein endothelial cells. Inhibition of VE-cadherin-mediated, but not PECAM-1-mediated, adhesion induced reorganization of the actin cytoskeleton, loss of junctional VE-cadherin staining and loss of cell-cell adhesion. In functional assays, inhibition of VE-cadherin caused increased monolayer permeability and enhanced neutrophil transendothelial migration. In a complementary set of experiments, modulation of the actin cytoskeleton was found to strongly affect VE-cadherin distribution. Brief stimulation of the beta2-adrenergic receptor with isoproterenol induced a loss of actin stress fibers resulting in a linear, rather than 'jagged', VE-cadherin distribution. The concomitant, isoproterenol-induced, reduction in monolayer permeability was alleviated by a VE-cadherin-blocking antibody. Finally, cytoskeletal reorganization resulting from the inactivation of p21Rho caused a diffuse localization of VE-cadherin, which was accompanied by reduced cell-cell adhesion. Together, these data show that monolayer permeability and neutrophil transendothelial migration are modulated by VE-cadherin-mediated cell-cell adhesion, which is in turn controlled by the dynamics of the actin cytoskeleton.