SummarySortilin, also known as neurotensin receptor 3 (NTR3), is a transmembrane protein with a dual function. It acts as a receptor for neuromediators and growth factors at the plasma membrane, but it has also been implicated in binding and transport of some lysosomal proteins. However, the role of sortilin during phagosome maturation has not been investigated before. Here, we show that in macrophages, sortilin is mainly localized in the Golgi and transported to latex-bead phagosomes (LBPs). Using live-cell imaging and electron microscopy, we found that sortilin is delivered to LBPs in a manner that depends on its cytoplasmic tail. We also show that sortilin participates in the direct delivery of acid sphingomyelinase (ASM) and prosaposin (PS) to the phagosome, bypassing fusion with lysosomal compartments. Further analysis confirmed that ASM and PS are targeted to the phagosome by sortilin in a Brefeldin-A-sensitive pathway. Analysis of primary macrophages isolated from Sort1 -/-mice indicated that the delivery of ASM and PS, but not pro-cathepsin D, to LBPs was severely impaired. We propose a pathway mediated by sortilin by which selected lysosomal proteins are transported to the phagosome along a Golgi-dependent route during the maturation of phagosomes.

Phagosome maturation is an essential part of the innate and adaptive immune response. Although it is well established that several Ras-related proteins in brain (Rab) proteins become associated to phagosomes, little is known about how these phagosomal Rab proteins influence phagosome maturation. Here, we show a specific role for Rab34 and mammalian uncoordinated 13-2 (Munc13-2) in phagolysosome biogenesis and cargo delivery. Rab34 knockdown impaired the fusion of phagosomes with late endosomes/lysosomes and high levels of active Rab34 promoted this process. We demonstrate that Rab34 enhances phagosome maturation independently of Rab7 and coordinates phagolysosome biogenesis through sizeselective transfer of late endosomal/lysosomal cargo into phagosomes. More importantly, we show that Rab34 mediates phagosome maturation through the recruitment of the protein Munc13-2. Finally, we report that the alternative maturation pathway controlled by Rab34 is critical for mycobacterial killing because Rab34 silencing resulted in mycobacterial survival, and Rab34 expression led to mycobacterial killing. Altogether, our studies uncover Rab34/ Munc13-2 as a critical part of an alternative Rab7-independent phagosome maturation machinery and lysosome-mediated killing of mycobacteria.

Viral immune evasion is currently understood to focus on deflecting CD8 T cell recognition of infected cells by disrupting antigen presentation pathways. We evaluated viral interference with the ultimate step in cytotoxic T cell function, the death of infected cells. The viral inhibitor of caspase-8 activation (vICA) conserved in human cytomegalovirus (HCMV) and murine CMV (MCMV) prevents the activation of caspase-8 and proapoptotic signaling. We demonstrate the key role of vICA from either virus, in deflecting antigen-specific CD8 T cell-killing of infected cells. vICA-deficient mutants, lacking either UL36 or M36, exhibit greater susceptibility to CD8 T cell control than mutants lacking the set of immunoevasins known to disrupt antigen presentation via MHC class I. This difference is evident during infection in the natural mouse host infected with MCMV, in settings where virus-specific CD8 T cells are adoptively transferred. Finally, we identify the molecular mechanism through which vICA acts, demonstrating the central contribution of caspase-8 signaling at a point of convergence of death receptor-induced apoptosis and perforin/granzyme-dependent cytotoxicity.

Cytomegalovirus (CMV) is a betaherpesvirus that latently infects most adult humans worldwide and is a major cause of morbidity and mortality in immunocompromised hosts. Latent human CMV (HCMV) is believed to reside in precursors of myeloid-lineage leukocytes and monocytes, which give rise to macrophages and dendritic cells (DC). We report here that human monocyte-derived DC (mo-DC) suppress HCMV infection in coculture with infected fibroblast target cells in a manner dependent on the effector-to-target ratio. Intriguingly, optimal activation of mo-DC was achieved under coculture conditions and not by direct infection with HCMV, implying that mo-DC may recognize unique molecular patterns on, or within, infected fibroblasts. We show that HCMV is controlled by secreted factors that act by priming defenses in target cells rather than by direct viral neutralization, but we excluded a role for interferons (IFNs) in this control. The expression of lytic viral genes in infected cells and the progression of infection were significantly slowed, but this effect was reversible, indicating that the control of infection depended on the transient induction of antiviral effector molecules in target cells. Using immediate early or late-phase reporter HCMVs, we show that soluble factors secreted in the cocultures suppress HCMV replication at both stages of the infection and that their antiviral effects are robust and comparable in numerous batches of mo-DC as well as in primary fibroblasts and stromal cells. Human cytomegalovirus is a widespread opportunistic pathogen that can cause severe disease and complications in vulnerable individuals. This includes newborn children, HIV AIDS patients, and transplant recipients. Although the majority of healthy humans carry this virus throughout their lives without symptoms, it is not exactly clear which tissues in the body are the main reservoirs of latent virus infection or how the delicate balance between the virus and the immune system is maintained over an individual's lifetime. Here, for the first time, we provide evidence for a novel mechanism of direct virus control by a subset of human innate immune cells called dendritic cells, which are regarded as a major site of virus latency and reactivation. Our findings may have important implications in HCMV disease prevention as well as in development of novel therapeutic approaches.