The Role of CCR5 in Vaccinia virus Pathogenesis

Viral appropriation of chemokine receptors is an effective way to prevent a host immune response against the invading virus. Many viruses, including poxviruses, subvert the host immune response by encoding several chemokine receptor homologues, capable of binding to and thereby precluding chemokines from activating their cognate cell surface receptors. All poxviruses employ strategies to modulate chemokine activity, including virus-encoded chemokine-binding proteins, receptor homologues and ligand mimics. The potential for the involvement of certain chemokine receptors in poxviral infection was suggested in studies utilizing the rabbit poxvirus, myxoma. Specifically, CCR5 was implicated in mediating cell target susceptibility to infection. Our data suggest virus-CCR5 interactions may lead to the selective activation of distinct signaling pathways that are advantageous for the virus. VACV, a member of the poxvirus family, produces two structurally distinct forms of virions, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV), for which the immediate events following cell entry are ill-defined. Using confocal microscopy, we provided evidence that IMV and EEV enter both permissive and non-permissive cells, and that introduction of CCR5 into non-permissive cells – mouse fibroblasts and human PM1 T cells - renders them permissive for VACV replication. We showed that virus activation of CCR5 leads to the selective activation of distinct signaling pathways that are advantageous for the virus. We demonstrated that VACV infection in permissive cells is inhibited by siRNA knockdown of cell surface CCR5 expression and by the CCR5 antagonist, TAK-779. The importance of tyrosine phosphorylation of CCR5 was suggested by the observation that introduction of a CCR5 mutant, in which all the intracellular tyrosines are replaced by phenylalanines, effectively reduces VACV infection in permissive cells. Moreover, tyrosine-339 was implicated in CCR5 as the critical residue for mediating viral infection, since cells expressing CCR5.Y339F do not support viral replication. The cascade of events that leads to permissive phenotype of these cells includes phosphorylation activation of multiple signaling effectors: Jak-2, IRS-2, ERK1/ 2 and Grb2. These data were supported by findings that viral replication in permissive CCR5 expressing cells is blocked by Herbimycin A, and the Jak2 inhibitor, tyrophostin AG490, but not pertussis toxin. Viewed altogether, a critical role of post-entry events, specifically intracellular tyrosine phosphorylation events, was established in determining permissiveness of cells to VACV replication. Furthermore, evidence was provided that introduction of CCR5 in primary human T cells renders them permissive to VACV replication. Since permissive infection of T cells might represent a mechanism for VACV dissemination throughout the lymphatic system, we hypothesized that the absence of CCR5 may be protective against VACV infection in vivo. To test this hypothesis, wild-type and CCR5 null mice were challenged with VACV by intranasal inoculation. In time course studies we identified aggressive viral replication in the lungs and spleens of CCR5+/+ mice, with no evidence of infection in the CCR5-/- mice. Moreover, associated with VACV infection, we provided evidence for CD4+ and CD8+ T as well as CD11c+ and F4/80+ cell infiltration into the lungs of CCR5+/+ but not CCR5-/- mice, and showed that CCR5-expressing T cells harbor replicating virus. We showed that this CCR5-dependence is VACV-specific, since CCR5-/- mice were as susceptible to intranasal influenza (A/WSN/33) infection as CCR5+/+ mice. In a final series of experiments we provided evidence that adoptive transfer of CCR5+/+ bone marrow into CCR5-/- mice restored VACV permissiveness, with evidence of lung and spleen infection. Taken together, our data showed a critical and novel role for CCR5 in VACV infection and dissemination in vivo. Moreover, our confocal studies suggested a possible physical interaction between cellular proteins and the VACV in cytosole. Using mass spectrometry-based proteomics, glomulin was identified as a host cell protein that interacts with VACV. Knockdown of glomulin expression in human PM1.CCR5 T cells reduced VACV infection. We demonstrate that treatment of PM1.CCR5 T cells with a c-Met phosphorylation inhibitor led to a significant reduction in VACV infectivity. The data indicated that inhibition of c-Met phosphorylation, reduces the cytosolic availability of activated glomulin, thus leading to a decrease in VACV infectivity. These data identify glomulin as a permissivity factor for VACV infection, and as a potential therapeutic target for VACV.

Keywords: Chemokine receptors
Vaccinia virus
Advisor: Fish, Eleanor N.