[PMC free article] [PubMed] [CrossRef] [Google Scholar] 69. EMCV replication post-entry. These findings establish that ADAM9 is required for the early stage of EMCV infection, likely for virus entry or viral genome delivery to the cytosol. genus of the family known to cause myocarditis, diabetes, and neurologic and reproductive disorders in rodents and nonhuman primates (1). The virus was first isolated in 1944 from a gibbon that died suddenly from pulmonary edema and myocarditis (2) and later isolated from diseased pigs (3). Since its discovery, EMCV has been isolated globally in an extensive range of animal species (4,C7). Rodents, specifically rats, are believed to be the natural reservoir hosts of EMCV, while infection of other animal species may result from occasional cross-species transmission by ingestion of contaminated food, water, or infected carcasses (8,C11). EMCV has also emerged as a pathogen capable of causing Methasulfocarb large zoonotic pandemics and decimating domestic animal populations, making it an important veterinary pathogen. While human infections are rare, EMCV can cause symptomatic disease in humans, manifesting as a mild, nonspecific febrile illness (12,C15). Infection is more prevalent among humans with occupational exposure to animals, particularly hunters (16,C18), suggesting a strong zoonotic potential for EMCV. While serious human EMCV infections are generally rare, EMCV rapidly kills human cells such as HeLa cells as well as primary human cells in culture (19, 20). EMCV is a well-accepted and widely used model for studying mechanisms of virus-mediated immune suppression, viral myocarditis, and insulin-dependent diabetes (21,C25). However, little is known about the receptor requirements of EMCV. The virus receptor on host cells is often a key factor in influencing viral tropism for particular tissues, which subsequently results in various disease manifestations of infection. Thus, understanding viral pathogenesis often hinges on identifying the cellular molecules that the virus binds to facilitate cell entry and subsequent infection. Here, we employed a functional genomics approach to identify genes responsible for EMCV-induced lytic infection in both human and murine cells. Using a genome-wide CRISPR-Cas9 screen, we identified ADAM9 as a major EMCV dependency factor Methasulfocarb (EDF). ADAMs (a disintegrin and metalloproteinase domain) are a family of transmembrane metalloproteinases that play important roles in growth factor and cytokine signaling as well as cell-cell signaling, adhesion, and extracellular matrix remodeling (26,C35). In animals, including humans, ADAM9 is ubiquitously expressed in cells of the developing heart, brain, retina, lung, fibroblasts, neutrophils, and platelets (27, 30, 34,C50). Approximately half of the ADAM family members, including ADAM9, have proteolytic capabilities that modulate the activity of cytokines, chemokines, and growth factors; their associated receptors; and cell adhesion molecules (27, 35, 37, 45). Rabbit polyclonal to ARHGAP21 ADAMs have been implicated in a range of human cancers, inflammatory diseases, wound healing, and microbial infections; however, very little is known about the role of ADAMs in viral infection. This study demonstrates that ADAM9 functions as a major EDF involved in the early infection of both human and murine cells. RESULTS Methasulfocarb CRISPR-Cas9 screening identifies EMCV dependency factors (EDFs). EMCV infection is rapidly lytic in human and murine cells (51,C54). We took advantage of the high lytic potential of EMCV and the power of CRISPR genetic screening (53, 55) to discover virus-host interaction genes that mediated virus infection and, thus, rendered the cells susceptible to EMCV-induced cell death. HeLa cells stably expressing Cas9 were used for screening (53, 55). In initial optimization experiments, we determined that HeLa cells were killed by EMCV within 24?h of infection at a multiplicity of infection (MOI) of 0.1. The rapid lysis of HeLa cells with EMCV infection allowed us to screen for EDFs using pooled single-guide RNAs (sgRNAs) since we could identify such mutant cells by their resistance to EMCV-induced cell death, i.e., these mutants would no longer be susceptible to EMCV infection and would survive EMCV challenge. We screened for EDFs using a CRISPR-Cas9 pooled human gene Methasulfocarb screen (Fig.?1). H1-HeLa cells (stably expressing a human-codon optimized pyogenesCas9) were transduced with the GeCKOv2 sgRNA library (53, 54), a complex lentiviral library that targets 19,052 human genes. The GeCKOv2 library contains six unique sgRNAs per gene in two half-libraries (A and B). Methasulfocarb Libraries A and B each contain three unique sgRNAs per gene, and the lentiviral libraries were transduced in parallel into H1-HeLa cells. Stable sgRNA-expressing cells were selected with puromycin for 48?h to allow sgRNA-guided CRISPR-Cas9-mediated gene deletion within each.
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- Each adjustable was stratified the following: 0: absent, or zero alterations; +: mild; ++: moderate; +++: intense
- Finish mounting quickly within 30 s?1 min
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