The snake venom rhodocytin has been reported to bind to integrin alpha2beta1 and glycoprotein (GP) Ibalpha on platelets, but it is also able to induce activation independent of the 2 receptors and of GPVI. Using rhodocytin affinity chromatography, we have identified a novel C-type lectin receptor, CLEC-2, in platelets that confers signaling responses to rhodocytin when expressed in a cell line. CLEC-2 has a single tyrosine residue in a YXXL motif in its cytosolic tail, which undergoes tyrosine phosphorylation upon platelet activation by rhodocytin or an antibody to CLEC-2, but not to collagen, thrombin receptor agonist peptide (TRAP), or convulxin. Tyrosine phosphorylation of CLEC-2 and other signaling proteins by rhodocytin is inhibited by the Src family kinase inhibitor PP2. Further, activation of murine platelets by rhodocytin is abolished in the absence of Syk and PLCgamma2, and partially reduced in the absence of LAT, SLP-76, and Vav1/Vav3. These findings define a novel signaling pathway in platelets whereby activation of CLEC-2 by rhodocytin leads to tyrosine phosphorylation of its cytosolic tail, binding of Syk and initiation of downstream tyrosine phosphorylation events, and activation of PLCgamma2. CLEC-2 is the first C-type lectin receptor to be found on platelets which signals through this novel pathway.

CD8 + T lymphocytes play a key role in host defense, in particular against important persistent viruses, although the critical functional properties of such cells in tissue are not fully defined. We have previously observed that CD8 + T cells specific for tissue-localized viruses such as hepatitis C virus express high levels of the C-type lectin CD161. To explore the significance of this, we examined CD8 + CD161 + T cells in healthy donors and those with hepatitis C virus and defined a population of CD8 + T cells with distinct homing and functional properties. These cells express high levels of CD161 and a pattern of molecules consistent with type 17 differentiation, including cytokines (e.g., IL-17, IL-22), transcription factors (e.g., retinoic acid-related orphan receptor γ-t, P = 6 × 10 −9 ; RUNX2, P = 0.004), cytokine receptors (e.g., IL-23R, P = 2 × 10 −7 ; IL-18 receptor, P = 4 × 10 −6 ), and chemokine receptors (e.g., CCR6, P = 3 × 10 −8 ; CXCR6, P = 3 × 10 −7 ; CCR2, P = 4 × 10 −7 ). CD161 + CD8 + T cells were markedly enriched in tissue samples and coexpressed IL-17 with high levels of IFN-γ and/or IL-22. The levels of polyfunctional cells in tissue was most marked in those with mild disease ( P = 0.0006). These data define a T cell lineage that is present already in cord blood and represents as many as one in six circulating CD8 + T cells in normal humans and a substantial fraction of tissue-infiltrating CD8 + T cells in chronic inflammation. Such cells play a role in the pathogenesis of chronic hepatitis and arthritis and potentially in other infectious and inflammatory diseases of man.

SummaryThe HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan-processing enzymes, so that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664) maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens.

Increased understanding of the role of protein- and lipid-linked carbohydrates in a wide range of biological processes has led to interest in drugs that target the enzymes involved in glycosylation. But given the importance of carbohydrates in fundamental cellular processes such as protein folding, therapeutic strategies that modulate, rather than ablate, the activity of enzymes involved in glycosylation are likely to be a necessity. Two such approaches that use imino sugars to affect glycosylation enzymes now show considerable promise in the treatment of viral infections, such as hepatitis B, and glucosphingolipid storage disorders, such as Gaucher disease.

We show that hepatitis C virus (HCV) p7 protein forms ion channels in black lipid membranes. HCV p7 ion channels are inhibited by long-alkyl-chain iminosugar derivatives, which have antiviral activity against the HCV surrogate bovine viral diarrhea virus. HCV p7 presents a potential target for antiviral therapy.H epatitis C virus (HCV) is the major cause of chronic hepatitis with a significant risk of end-stage liver cirrhosis and hepatocellular carcinoma (1). HCV belongs to the family Flaviviridae, which consists of three genera: flaviviruses, pestiviruses, and hepaciviruses. In the absence of both a suitable small animal model and a reliable in vitro infectivity assay for HCV, potential antiviral drugs initially have been tested by using a related pestivirus, bovine viral diarrhea virus (BVDV) (2). BVDV in vitro infectivity assays were used to demonstrate that long-alkyl-chain iminosugar derivatives containing either the glucose analogue deoxynojirimycin (DNJ) or the galactose analogue deoxygalactonojirimycin (DGJ) are potent antiviral inhibitors (3).DNJ derivatives inhibit endoplasmic reticulum (ER) ␣-glucosidases I and II (4, 5), and this inhibition leads to the misfolding of many host-and virus-encoded glycoproteins, including the envelope glycoproteins of BVDV (6) and HCV (7). Previous experiments have shown that the antiviral effect of the longalkyl-chain derivative N-nonyl-DNJ (NN-DNJ) is more pronounced than that of the short-alkyl-chain derivative N-butyl-DNJ (NB-DNJ), although the latter achieves a more effective ER ␣-glucosidase inhibition in cellulo. In addition, long-alkylchain DGJ derivatives that are not recognized by and do not inhibit ER ␣-glucosidases also show potent antiviral activity (3). Therefore, ER ␣-glucosidase inhibition does not correlate directly with the observed antiviral effect and is ruled out as the sole antiviral mechanism.The additional mechanism of action apparently is associated with the length of the alkyl side chain, because the short-chain N-butyl-DGJ (NB-DGJ) shows no antiviral activity, whereas the long-alkyl-chain derivative NN-DGJ is a potent inhibitor (3).The predominant antiviral mechanism is proposed to be mediated directly or indirectly by an effect of the long-alkyl side chains on the membrane and͞or membrane proteins, because treatment with long-alkyl-chain iminosugars affects the dimerization of viral membrane glycoproteins and alters the membrane glycoprotein composition of secreted BVDV virions but does not influence either viral RNA replication or protein synthesis (3).We decided to investigate the small membrane-spanning protein p7 as a potential target of long-alkyl-chain iminosugar derivatives, because flaviviruses such as dengue virus and Japanese encephalitis virus (8), which do not contain p7, are not inhibited by long-alkyl-chain DGJ derivatives, whereas pestiviruses are (3). Pesti-and hepaciviruses both contain the p7 protein.Most functional data about p7 are derived from the pestivirus p7, a 70-aa protein very similar to HCV p7. Functional data hav...