Background:  Our lab has recently demonstrated that M-R5 virus treatment alters gene expression profiles in macrophages, including factors involved in signal transduction, within 48 hours. We hypothesize that the cellular receptor complex serves not only in recognition and viral entry, but also initiates signal transduction events required for efficient viral replication. We predict a difference in gene expression profiles of whole primary macrophage cultures treated with envelopes that mediate macrophage entry vs an envelope restricted at entry.

Methods:  Affymetrix HG-U133A GeneChip was used to identify altered sentinel genetic networks altered in primary macrophages from 3 donors treated with 3 different envelopes that differed in cell-tropism and co-receptors. Hierarchical clustering and visualization of results were performed using Cluster and TreeView. Class comparison and leave-one-out cross-validation statistical analyses were performed using BRB ArrayTools software platform. Results were confirmed by quantitative polymerase chain reaction (QT-PCR), Western blot, and cytokine secretion assays.

Results:  M-R5 envelope and D-X4 envelope treatment effect on gene expression was detected within 15 to 24 hours. Class comparison identified 250 genes statistically altered by M-R5 at 15 hours, in contrast to 106 by T-X4 (p = 0.01). Leave-one-out cross-validation experiments used artificial intelligence modeling to correctly predict the 15 hour M-R5 treatment across all donor pairs, indicating these genes are specific to treatment and independent of background. M-R5 primarily altered genes involved in signal transduction pathways. Several signal transduction genes altered by M-R5 function in pathways altered by whole virus. TaqMan assays confirm results observed in microarray analysis. We identified the induction of several genes, including NPY and NFATC4, by M-R5 envelope that may be important for virus to establish infection in host cell.

Conclusions:  In contrast to T-X4 envelope, M-R5 envelopes induce distinct gene expression profiles in macrophages. D-X4 envelope signals in macrophages, but with delayed kinetics compared to M-R5. Results suggest that envelope signaling serves as a “priming factor” to permit viral entry and replication. Differences in transcriptomes of primary macrophages as a consequence of envelope signaling through CCR5 as opposed to CXCR4 represent novel therapeutic targets to control viral replication.