Dendritic cells (DCs) are key instigators of adaptive immune responses. Using an alphaviral expression cloning technology, we have identified the chemokine CCL19 as a potent inducer of T cell proliferation in a DC-T cell coculture system. Subsequent studies showed that CCL19 enhanced T cell proliferation by inducing maturation of DCs, resulting in upregulation of costimulatory molecules and the production of proinflammatory cytokines. Moreover, CCL19 programmed DCs for the induction of T helper type (Th) 1 rather than Th2 responses. Importantly, only activated DCs that migrated from the periphery to draining lymph nodes, but not resting steady-state DCs residing within lymph nodes, expressed high levels of CCR7 in vivo and responded to CCL19 with the production of proinflammatory cytokines. Migrating DCs isolated from mice genetically deficient in CCL19 and CCL21 (plt/plt) presented an only partially mature phenotype, highlighting the importance of these chemokines for full DC maturation in vivo. Our findings indicate that CCL19 and CCL21 are potent natural adjuvants for terminal activation of DCs and suggest that chemokines not only orchestrate DC migration but also regulate their immunogenic potential for the induction of T cell responses.

Familial prion diseases are linked to point and insertional mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP to the infectious isoform. In this report, we have investigated the subcellular localization of PrP molecules carrying pathogenic mutations using immunofluorescence staining, immunogold labeling, and PrPgreen fluorescent protein chimeras. To facilitate visualization of the mutant proteins, we have utilized a novel Sindbis viral replicon engineered to produce high protein levels without cytopathology. We demonstrate that several different pathogenic mutations have a common effect on the trafficking of PrP, impairing delivery of the molecules to the cell surface and causing a portion of them to accumulate in the endoplasmic reticulum. These observations suggest that protein quality control in the endoplasmic reticulum may play an important role in prion diseases, as it does in some other inherited human disorders. Our experiments also show that chimeric PrP molecules with the sequence of green fluorescent protein inserted adjacent to the glycolipidation site are post-translationally modified and localized normally, thus documenting the utility of these constructs in cell biological studies of PrP.

The assembly and budding of Sindbis virus, a prototypic member of the alphavirus subgroup in the family Togaviridae, requires a specific interaction between the nucleocapsid core and the membrane-embedded glycoproteins El and E2. These glycoproteins are modified posttranslationally by the addition of palmitic acid, and inhibitors of acylation interfere with this budding process (M. J. Schlesinger and C. Malfer, J. Biol. Chem. 257:9887-9890, 1982). This report describes the use of site-directed mutagenesis to identify two of the acylation sites in the E2 glycoprotein as the cysteines near the carboxyl terminus of the protein which is oriented to the cytoplasmic domain of this type 1 transmembrane protein. Additional mutations were made at two prolines within a hydrophobic sequence of E2 that is highly conserved among several alphaviruses, and the mutant viruses were aberrant in assembly and particle formation. These data support earlier studies indicating that the native structure of the cytoplasmic domain of E2 is essential for proper assembly of this enveloped virus.

The two glycoproteins that form the external spikes of the alphaviruses are type 1 membrane proteins whose transmembrane domains of hydrophobic amino acids are close to the carboxyl termini of the polypeptides and anchor the proteins in the lipid bilayer. Most of the members of the alphavirus genus contain within this transmembrane sequence one or more highly conserved cysteines, which are positioned close to the cytoplasmic face of the lipid bilayer. Cysteines in the cytoplasmic domains of the alphavirus glycoproteins and other enveloped viruses have been shown to be modified by palmitylation. To determine whether the transmembranal cysteines in Sindbis virus also were palmitylated, we used site-directed mutation to change the single transmembranal cysteine in the E1 glycoprotein and two of the transmembranal cysteines in the E2 glycoprotein to alanines. Transfection of RNA transcribed from the differently mutated Sindbis virus cDNAs led to production of infectious virus. Cells infected with the mutant virions and labeled with [3H]-palmitic acid showed that the E1 mutant no longer contained fatty acid in the E1 glycoprotein and that the extent of palmitylation was reduced about twofold in the E2 glycoprotein of virions containing the E2 mutations. At early times postinfection, the mutants grew slightly slower than the wild type in cultures of chicken embryo fibroblasts and secreted about half the amount of virus particles as wild type, but little difference was found at later time points. A triple mutant containing both the E1 and E2 mutations formed virions deficient in palmitylation of both glycoproteins, and this mutant had growth properties that were similar to those of the independent E1 and E2 mutants. Virions with the mutated glycoproteins that were deficient in fatty acid were more susceptible than the wild-type virions to inactivation by the detergent Triton X-100.

Libraries of cloned cDNA were prepared from complete genomic RNA and isolated S RNA of the Bulgarian L3 isolate of tomato spotted wilt virus (TSWV-L3). Northern blotting of TSWV genomic RNA detected clones specific for the L, M and S RNAs in the library from complete RNA. S RNA-specific clones selected from both libraries covered approximately 2-8 kb (about 95 %) of the S RNA. Sequencing of these clones showed TSWV-L3 S RNA to be ambisense. It contains two open reading frames (ORFs); one of 1401 nucleotides located on the viral RNA encodes an Mr 52400 (52K) protein, and the other of 774 nucleotides on the complementary strand encodes an Mr 28 900 (29K) protein. Expression of the 29K ORF in bacteria and immunological analysis of the fusion protein synthesized confirmed that the 29K protein is the N protein of TSWV-L3. Comparison with the published sequence for the S RNA of a Brazilian TSWV isolate, CNPH1, revealed almost complete identity in the amino acid sequences for the 29K protein, but several clustered amino acid exchanges in the putative 52K protein. In addition, the separating non-translated intergenic region of the S RNA of the Bulgarian isolate is 81 nucleotides longer than that of CNPH1.