e Interferon-inducible transmembrane (IFITM) protein family members IFITM1, -2, and -3 restrict the infection of multiple enveloped viruses. Significant enrichment of a minor IFITM3 allele was recently reported for patients who were hospitalized for seasonal and 2009 H1N1 pandemic flu. This IFITM3 allele lacks the region corresponding to the first amino-terminal 21 amino acids and is unable to inhibit influenza A virus. In this study, we found that deleting this 21-amino-acid region relocates IFITM3 from the endosomal compartments to the cell periphery. This finding likely underlies the lost inhibition of influenza A virus that completes its entry exclusively within endosomes at low pH. Yet, wild-type IFITM3 and the mutant with the 21-amino-acid deletion inhibit HIV-1 replication equally well. Given the pH-independent nature of HIV-1 entry, our results suggest that IFITM3 can inhibit viruses that enter cells via different routes and that its N-terminal region is specifically required for controlling pHdependent viruses.

Previous studies suggested that tyrosine kinase activation is an important signal transduction event in the IL-1 response of chondrocytes. The present study identifies the mitogenactivated protein (MAP) kinases extracellular signal-regulated kinase (ERK)-1 and ERK-2 as major tyrosine phosphorylated proteins in IL-1 stimulated chondrocytes. Kinase assays on immunoprecipitates with myelin basic protein as substrate showed that ERK-1 and ERK-2 activation was detectable within 5 min after IL-1 stimulation and decreased to baseline within 60 min. Analysis of other members of the MAP kinase family showed that chondrocytes also express c-Jun NH 2 terminal kinase (JNK)-1, JNK-2, and p38 proteins. These kinases were time-dependently activated by IL-1. Among other chondrocyte activators tested, only TNF activated all three of the MAP kinase subgroups. JNK and p38 were not activated by any of the other cytokines and growth factors tested. However, ERK was also activated by PDGF, IGF-1, and IL-6. Phorbol 12-myristate 13-acetate, calcium ionophore, and cAMP analogues only increased ERK activity but had no significant effects on JNK or p38.These results suggest differential activation of MAP kinase subgroups by extracellular stimuli. ERK is activated in response to qualitatively diverse extracellular stimuli and various second messenger agonists. In contrast, JNK and p38 are only activated by IL-1 or TNF, suggesting that these kinases participate in the induction of the catabolic program in cartilage. ( J. Clin. Invest. 1996. 98:2425-2430.)

Members of the interferon-induced transmembrane (IFITM) protein family inhibit the entry of a wide range of viruses. Viruses often exploit the endocytosis pathways to invade host cells and escape from the endocytic vesicles often in response to low pH. Localization to these endocytic vesicles is essential for IFITM3 to interfere with the cytosolic entry of pH-dependent viruses. However, the nature of the sorting signal that targets IFITM3 to these vesicles is poorly defined. In this study, we report that IFITM3 possesses a YxxΦ sorting motif, i.e., 20-YEML-23, that enables IFITM3 to undergo endocytosis through binding to the μ2 subunit of the AP-2 complex. IFITM3 accumulates at the plasma membrane as a result of either mutating 20-YEML-23, depleting the μ2 subunit, or overexpressing μ2 mutants. Importantly, blocking endocytosis of IFITM3 abrogates its ability to inhibit pH-dependent viruses. We have therefore identified a critical sorting signal, namely 20-YEML-23, that controls both the endocytic trafficking and the antiviral action of IFITM3. This finding also reveals that as an endocytic protein, IFITM3 first arrives at the plasma membrane before it is endocytosed and further traffics to the late endosomes where it acts to impede virus entry.

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Activation of monocytes by bacterial lipopolysaccharides (LPSs) is a central component in the pathogenesis of septic shock syndrome. Interleukin 10 (IL-10) is a potent monocyte-deactivating factor and transcriptionally inhibits LPS-induced expression of proinflammatory mediators. The intracellular snali pathways of LPS have been only partially characterized and me ms of IL-10 signaling remain unknown. We show that LPS activates the protein tyrosine kinase (PTK) p56od> and that this is associated with tyrosine phosphorylation of the protooncogene product Vav.

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The ART (Activator of Replication and Transcription) protein of Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus-8 (HHV-8), is encoded by the ORF50 gene. It is expressed as an immediate-early gene and plays a crucial role in the transition between latency and productive infection. HHV-8 ART is a transcriptional transactivator which can up-regulate viral gene expression. Transient expression assays showed that ART strongly activated ORF57 and K8 promoter-directed gene expression in both CV-1 and BJAB cells. The ART target site was mapped to a 40-bp region compassing nt 81904 to 81943 on the ORF57 promoter. When linked upstream to a heterologous SV40 promoter, this region by itself was able to confer ART responsiveness. This 40-bp segment contains a 16-bp consensus sequence which is also found in the K8 promoter region located between nt 74769 to 74784. Deletion of the fragment including this 16-bp consensus abrogated the ART responsiveness of the K8 promoter. The role of this 16-bp consensus in ART transactivation was further supported by site-directed mutagenesis. Mutations of the conserved nucleotides within the 16-bp consensus in the ORF57 promoter dramatically impaired its responsiveness to ART. Fusion protein analysis with chimeric proteins containing the DNA binding domain of yeast transactivator Gal4 (residues 1 to 147) and different ART segments defined an acidic C-terminal region (amino acids [aa] 527 to 634) as a potent activator. Deletions of this activation domain in the ART protein resulted in a decrease or loss of its ability to activate ORF57 and K8 promoters containing the ART responsive element in transfected cells. How the ART activation domain activates ORF57 and K8 gene expression through the 16-bp consensus sequence remains to be determined.