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As a possible route for invasion of the CNS, circulating poliovirus (PV) in the blood is believed to traverse the blood-brain barrier (BBB), resulting in paralytic poliomyelitis. However, the underlying mechanism is poorly understood. In this study, we demonstrated that mouse transferrin receptor 1 (mTfR1) is responsible for PV attachment to the cell surface, allowing invasion into the CNS via the BBB. PV interacts with the apical domain of mTfR1 on mouse brain capillary endothelial cells (MBEC4) in a dose-dependent manner via its capsid protein (VP1). We found that F-G, G-H, and H-I loops in VP1 are important for this binding. However, C-D, D-E, and E-F loops in VP1-fused Venus proteins efficiently penetrate MBEC4 cells. These results imply that the VP1 functional domain responsible for cell attachment is different from that involved in viral permeation of the brain capillary endothelium. We observed that co-treatment of MBEC4 cells with excess PV particles but not dextran resulted in blockage of transferrin transport into cells. Using the Transwell in vitro BBB model, transferrin co-treatment inhibited permeation of PV into MBEC4 cells and delayed further viral permeation via mTfR1 knockdown. With mTfR1 as a positive mediator of PV-host cell attachment and PV permeation of MBEC4 cells, our results indicate a novel role of TfR1 as a cellular receptor for human PV receptor/CD155-independent PV invasion of the CNS., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Werner syndrome helicase (WRN) plays a role in maintaining genomic stability. The lack of WRN results in Werner syndrome, a rare autosomal recessive genetic disorder, which causes premature aging accompanied by many complications such as rare forms of cancer and type 2 diabetes. However, the underlying mechanisms of these complications, arising due to the loss of WRN, are poorly understood. In this study, we demonstrated the function of WRN in transcriptional regulation of NF-κB targets. WRN physically interacts via its RecQ C-terminal (RQC) domain with the Rel homology domain of both the RelA (p65) and the p50 subunits of NF-κB. In the steady state, WRN is recruited to HIV-1 long terminal repeat (LTR), a typical NF-κB-responsive promoter, as well as the p50/p50 homodimer, in an NF-κB site-dependent manner. The amount of WRN on LTR increased along with the transactivating RelA/p50 heterodimer in response to TNF-α stimulation. Further, a knockdown of WRN reduced the transactivation of LTR in exogenous RelA/p50-introduced or TNF-α-stimulated cells. Additionally, knockdown of WRN reduced TNF-α stimulation-induced activation of the endogenous promoter of IL-8, an NF-κB-responsive gene, and WRN increased its association with the IL-8 promoter region together with RelA/p50 after TNF-α stimulation. In conjunction with studies that have shown NF-κB to be a key regulator of aging and inflammation, our results indicate a novel role of WRN in transcriptional regulation. Along with NF-κB, the loss of WRN is expected to result in incorrect regulation of downstream targets and leads to immune abnormalities and homeostatic disruption., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

We have previously shown that DPF2 (requiem/REQ) functions as a linker protein between the SWI/SNF complex and RelB/p52 NF-κB heterodimer and plays important roles in NF-κB transactivation via its noncanonical pathway. Using sensitive 293FT reporter cell clones that had integrated a SWI/SNF-dependent NF-κB reporter gene, we find in this study that the overexpression of DPF1, DPF2, DPF3a, DPF3b, and PHF10 significantly potentiates the transactivating activity of typical NF-κB dimers. Knockdown analysis using 293FT reporter cells that endogenously express these five proteins at low levels clearly showed that DPF3a and DPF3b, which are produced from the DPF3 gene by alternative splicing, are the most critical for the RelA/p50 NF-κB heterodimer transactivation induced by TNF-α stimulation. Our data further show that this transactivation requires the SWI/SNF complex. DPF3a and DPF3b are additionally shown to interact directly with RelA, p50, and several subunits of the SWI/SNF complex in vitro and to be co-immunoprecipitated with RelA/p50 and the SWI/SNF complex from the nuclear fractions of cells treated with TNF-α. In ChIP experiments, we further found that endogenous DPF3a/b and the SWI/SNF complex are continuously present on HIV-1 LTR, whereas the kinetics of RelA/p50 recruitment after TNF-α treatment correlate well with the viral transcriptional activation levels. Additionally, re-ChIP experiments showed DPF3a/b and the SWI/SNF complex associate with RelA on the endogenous IL-6 promoter after TNF-α treatment. In conclusion, our present data indicate that by linking RelA/p50 to the SWI/SNF complex, DPF3a/b induces the transactivation of NF-κB target gene promoters in relatively inactive chromatin contexts.

The SWI/SNF chromatin remodeling complex plays pivotal roles in mammalian transcriptional regulation. In this study, we identify the human requiem protein (REQ/DPF2) as an adaptor molecule that links the NF-kappaB and SWI/SNF chromatin remodeling factor. Through in vitro binding experiments, REQ was found to bind to several SWI/SNF complex subunits and also to the p52 NF-kappaB subunit through its nuclear localization signal containing the N-terminal region. REQ, together with Brm, a catalytic subunit of the SWI/SNF complex, enhances the NF-kappaB-dependent transcriptional activation that principally involves the RelB/p52 dimer. Both REQ and Brm were further found to be required for the induction of the endogenous BLC (CXCL13) gene in response to lymphotoxin stimulation, an inducer of the noncanonical NF-kappaB pathway. Upon lymphotoxin treatment, REQ and Brm form a larger complex with RelB/p52 and are recruited to the BLC promoter in a ligand-dependent manner. Moreover, a REQ knockdown efficiently suppresses anchorage-independent growth in several cell lines in which the noncanonical NF-kappaB pathway was constitutively activated. From these results, we conclude that REQ functions as an efficient adaptor protein between the SWI/SNF complex and RelB/p52 and plays important roles in noncanonical NF-kappaB transcriptional activation and its associated oncogenic activity.