Chikungunya virus (CHIKV) is a mosquito-borne alphavirus known to cause epidemics resulting in predominantly symptomatic infections, which in rare cases cause long term debilitating arthritis and arthralgia. Significant progress has been made in understanding the roles of canonical RNA sensing pathways in the host recognition of CHIKV; however, less is known regarding antagonism of CHIKV by cytosolic DNA sensing pathways like that of cyclic GMP-AMP synthase (cGAS) and Stimulator of Interferon Genes (STING). With the use of cGAS or STING null cells we demonstrate that the pathway restricts CHIKV replication in fibroblasts and immune cells. We show that DNA accumulates in the cytoplasm of infected cells and that CHIKV blocks DNA dependent IFN-β transcription. This antagonism of DNA sensing is via an early autophagy-mediated degradation of cGAS and expression of the CHIKV capsid protein is sufficient to induce cGAS degradation. Furthermore, we identify an interaction of CHIKV nsP1 with STING and map the interaction to 23 residues in the cytosolic loop of the adaptor protein. This interaction stabilizes the viral protein and increases the level of palmitoylated nsP1 in cells. Together, this work supports previous publications highlighting the relevance of the cGAS-STING pathway in the early detection of (+)ssRNA viruses and provides direct evidence that CHIKV interacts with and antagonizes cGAS-STING signaling.
Injury to the specialized epithelial cells of the glomerulus (podocytes) underlies the pathogenesis of all forms of proteinuric kidney disease; however, the specific genetic changes that mediate podocyte dysfunction after injury are not fully understood. Here, we performed a large-scale insertional mutagenic screen of injury-resistant podocytes isolated from mice and found that increased expression of the gene Rap1gap, encoding a RAP1 activation inhibitor, ameliorated podocyte injury resistance. Furthermore, injured podocytes in murine models of disease and kidney biopsies from glomerulosclerosis patients exhibited increased RAP1GAP, resulting in diminished glomerular RAP1 activation. In mouse models, podocyte-specific inactivation of Rap1a and Rap1b induced massive glomerulosclerosis and premature death. Podocyte-specific Rap1a and Rap1b haploinsufficiency also resulted in severe podocyte damage, including features of podocyte detachment. Over-expression of RAP1GAP in cultured podocytes induced loss of activated β1 integrin, which was similarly observed in kidney biopsies from patients. Furthermore, preventing elevation of RAP1GAP levels in injured podocytes maintained β1 integrin-mediated adhesion and prevented cellular detachment. Taken together, our findings suggest that increased podocyte expression of RAP1GAP contributes directly to podocyte dysfunction by a mechanism that involves loss of RAP1-mediated activation of β1 integrin. IntroductionPodocytes, the terminally differentiated visceral epithelial cells of the glomerulus, are responsible for forming and regulating the kidney filtration barrier. These cells have a remarkably complex cellular morphology, extending numerous interdigitating foot processes that surround the glomerular capillary walls and form unique specialized intercellular junctions known as slit diaphragms. The importance of slit diaphragms is enforced by the abundance of glomerular disorders that are caused by mutations in genes that encode components of this complex. A podocyte's intricate shape is maintained by a well-organized and dynamic actin cytoskeleton that is tightly regulated. In all forms of human proteinuric kidney disease, the podocyte undergoes cytoskeletal remodeling that results in foot process effacement and loss of normal filtration barrier selectivity, a process that is common to nearly all forms of podocyte injury, regardless of the underlying cause (1). The molecular mechanisms driving foot process effacement versus recovery are only beginning to be understood and are paramount to the identification of novel therapeutic strategies for proteinuria.The original goals of our studies were not only to identify pathways that are dysregulated in podocytes in response to injury, but also to select for those pathways that have the largest functional impact when dysregulated. To accomplish this, we designed and
HIV integrates into the host genome to create a persistent viral reservoir. Stimulation of CD4+ memory T lymphocytes with common γc-chain cytokines renders these cells more susceptible to HIV infection, making them a key component of the reservoir itself. IL-15 is up-regulated during primary HIV infection, a time when the HIV reservoir established. Therefore, we investigated the molecular and cellular impact of IL-15 on CD4+ T-cell infection. We found that IL-15 stimulation induces SAM domain and HD domain-containing protein 1 (SAMHD1) phosphorylation due to cell cycle entry, relieving an early block to infection. Perturbation of the pathways downstream of IL-15 receptor (IL-15R) indicated that SAMHD1 phosphorylation after IL-15 stimulation is JAK dependent. Treating CD4+ T cells with Ruxolitinib, an inhibitor of JAK1 and JAK2, effectively blocked IL-15–induced SAMHD1 phosphorylation and protected CD4+ T cells from HIV infection. Using high-resolution single-cell immune profiling using mass cytometry by TOF (CyTOF), we found that IL-15 stimulation altered the composition of CD4+ T-cell memory populations by increasing proliferation of memory CD4+ T cells, including CD4+ T memory stem cells (TSCM). IL-15–stimulated CD4+ TSCM, harboring phosphorylated SAMHD1, were preferentially infected. We propose that IL-15 plays a pivotal role in creating a self-renewing, persistent HIV reservoir by facilitating infection of CD4+ T cells with stem cell-like properties. Time-limited interventions with JAK1 inhibitors, such as Ruxolitinib, should prevent the inactivation of the endogenous restriction factor SAMHD1 and protect this long-lived CD4+ T-memory cell population from HIV infection.
Focal segmental glomerulosclerosis (FSGS) is a leading cause of nephrotic syndrome and end-stage renal disease worldwide.Although the mechanisms underlying this important disease are poorly understood, the glomerular podocyte clearly plays a central role in disease pathogenesis. In the current work, we demonstrate that the homophilic adhesion molecule sidekick-1 (sdk-1) is up-regulated in podocytes in FSGS both in rodent models and in human kidney biopsy samples. Transgenic mice that have podocyte-specific overexpression of sdk-1 develop gradually progressive heavy proteinuria and severe FSGS. We also show that sdk-1 associates with the slit diaphragm linker protein MAGI-1, which is already known to interact with several critical podocyte proteins including synaptopodin, ␣-actinin-4, nephrin, JAM4, and -catenin. This interaction is mediated through a direct interaction between the carboxyl terminus of sdk-1 and specific PDZ domains of MAGI-1. In vitro expression of sdk-1 enables a dramatic recruitment of MAGI-1 to the cell membrane. Furthermore, a truncated version of sdk-1 that is unable to bind to MAGI-1 does not induce podocyte dysfunction when overexpressed. We conclude that the up-regulation of sdk-1 in podocytes is an important pathogenic factor in FSGS and that the mechanism involves disruption of the actin cytoskeleton possibly via alterations in MAGI-1 function. Focal segmental glomerulosclerosis (FSGS)2 is an important cause of end-stage renal disease worldwide, accounting for ϳ20% of all dialysis patients (1). In fact, the frequency of this disease has dramatically increased over the last 20 years, making it the most common cause of primary nephrotic syndrome in adults (1). Multiple cohort studies show progression to end-stage renal disease in 50 -70% of cases at 10 years, giving FSGS one of the worst prognoses among primary glomerular diseases (2, 3).The diagnosis of FSGS is based on the clinical findings of proteinuria and specific histopathological changes that include glomerular sclerosis, glomerular tuft collapse, and synechia formation. In the early stages, these changes are both focal, affecting a subset of glomeruli, and segmental, involving a portion of the glomerular tuft. Although the idiopathic form of FSGS is the most common, secondary FSGS occurs in association with other underlying conditions including HIV-associated nephropathy (HIVAN), among others.Although the pathogenic mechanisms underlying this disease are poorly understood, the podocyte, the visceral epithelial cell of the glomerulus, plays a central role. Multiple genetic studies using both human and murine models demonstrate that the development of FSGS is initiated by podocyte dysfunction (4). In humans, mutations in the podocyte-specific genes nephrin (5), podocin (6), ␣-actinin-4 (7), TRPC6 (8, 9), and others all disrupt podocyte function, leading to inherited forms of FSGS. Two recent landmark studies showed a strong association of non-coding variants in the podocyte-expressed gene myh9 with susceptibility to HIVAN and FSG...
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