Viperin triggers ribosome collision-dependent translation inhibition to restrict viral replication.

Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation. We found that the radical SAM activity of viperin is required for translation inhibition and that this is mediated by viperin's enzymatic product, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Viperin triggers ribosome collisions and activates the MAPKKK ZAK pathway that in turn activates the GCN2 arm of the integrated stress response pathway to inhibit translation. The study illustrates the importance of translational repression in the antiviral response and identifies viperin as a translation regulator in innate immunity.

Role for lysosomal phospholipase A2 in iNKT cell-mediated CD1d recognition.

Invariant natural killer T (iNKT) cells recognize self lipid antigens presented by CD1d molecules. The nature of the self-antigens involved in the development and maturation of iNKT cells is poorly defined. Lysophospholipids are self-antigens presented by CD1d that are generated through the action of phospholipases A1 and A2. Lysosomal phospholipase A2 (LPLA2, group XV phospholipase A2) resides in the endocytic system, the main site where CD1d antigen acquisition occurs, suggesting that it could be particularly important in CD1d function. We find that Lpla2(-/-) mice show a decrease in iNKT cell numbers that is neither the result of a general effect on the development of lymphocyte populations nor of effects on CD1d expression. However, endogenous lipid antigen presentation by CD1d is reduced in the absence of LPLA2. Our data suggest that LPLA2 plays a role in the generation of CD1d complexes with thymic lipids required for the normal selection and maturation of iNKT cells.

Endoplasmic reticulum glycoprotein quality control regulates CD1d assembly and CD1d-mediated antigen presentation.

The non-classical major histocompatibility complex (MHC) homologue CD1d presents lipid antigens to innate-like lymphocytes called natural-killer T (NKT) cells. These cells, by virtue of their broad cytokine repertoire, shape innate and adaptive immune responses. Here, we have assessed the role of endoplasmic reticulum glycoprotein quality control in CD1d assembly and function, specifically the role of a key component of the quality control machinery, the enzyme UDP glucose glycoprotein glucosyltransferase (UGT1). We observe that in UGT1-deficient cells, CD1d associates prematurely with ß2-microglobulin (ß2m) and is able to rapidly exit the endoplasmic reticulum. At least some of these CD1d-ß2m heterodimers are shorter-lived and can be rescued by provision of a defined exogenous antigen, α-galactosylceramide. Importantly, we show that in UGT1-deficient cells the CD1d-ß2m heterodimers have altered antigenicity despite the fact that their cell surface levels are unchanged. We propose that UGT1 serves as a quality control checkpoint during CD1d assembly and further suggest that UGT1-mediated quality control can shape the lipid repertoire of newly synthesized CD1d. The quality control process may play a role in ensuring stability of exported CD1d-ß2m complexes, in facilitating presentation of low abundance high affinity antigens, or in preventing deleterious responses to self lipids.

Fatty acid auxotrophy in Drosophila larvae lacking SREBP.

SREBPs are membrane bound transcription factors that are crucial for normal lipid synthesis in animal cells. Here, we show that Drosophila lacking dSREBP die before the third larval instar. Mutant larvae exhibit pronounced growth defects prior to lethality, along with substantial deficits in the transcription of genes required for fatty acid synthesis. Compared to wild-type larvae, mutants contain markedly less fatty acid, although its composition is unaltered. Dietary supplementation with fatty acids rescues mutants to adulthood. The most effective fatty acid, oleate, rescues 80% of homozygotes. Rescue by dSREBP requires expression only in fat body and gut. Larvae expressing dSREBP prior to pupariation complete development and are viable as adults even when dSREBP expression is subsequently extinguished. The role, if any, of dSREBP in adults is not yet apparent. These data indicate that dSREBP deficiency renders Drosophila larvae auxotrophic for fatty acids.

Alternative processing of sterol regulatory element binding protein during larval development in Drosophila melanogaster.

Sterol regulatory element binding protein (SREBP) is a major transcriptional regulator of lipid metabolism. Nuclear Drosophila SREBP (dSREBP) is essential for larval development in Drosophila melanogaster but dispensable in adults. dSREBP(-) larvae die at second instar owing to loss of dSREBP-mediated transcription but survive to adulthood when fed fatty acids. Activation of SREBP requires two separate cleavages. Site-1 protease (S1P) cleaves in the luminal loop of the membrane-bound SREBP precursor, cutting it in two. The NH(2)- and COOH-terminal domains remain membrane bound owing to their single membrane-spanning helices. The NH(2)-terminal cleavage product is the substrate for site-2 protease (S2P), which cleaves within its membrane-spanning helix to release the transcription factor. In mice, loss of S1P is lethal but the consequences of loss of S2P in animals remain undefined. All known functions of SREBP require its cleavage by S2P. We isolated Drosophila mutants that eliminate all dS2P function (dS2P(-)). Unexpectedly, larvae lacking dS2P are viable. They are deficient in transcription of some dSREBP target genes but less so than larvae lacking dSREBP. Despite loss of dS2P, dSREBP is processed in mutant larvae. Therefore, larvae have an alternative cleavage mechanism for producing transcriptionally active dSREBP, and this permits survival of dS2P mutants.

COPI activity coupled with fatty acid biosynthesis is required for viral replication.

During infection by diverse viral families, RNA replication occurs on the surface of virally induced cytoplasmic membranes of cellular origin. How this process is regulated, and which cellular factors are required, has been unclear. Moreover, the host-pathogen interactions that facilitate the formation of this new compartment might represent critical determinants of viral pathogenesis, and their elucidation may lead to novel insights into the coordination of vesicular trafficking events during infection. Here we show that in Drosophila cells, Drosophila C virus remodels the Golgi apparatus and forms a novel vesicular compartment, on the surface of which viral RNA replication takes place. Using genome-wide RNA interference screening, we found that this step in the viral lifecycle requires at least two host encoded pathways: the coat protein complex I (COPI) coatamer and fatty acid biosynthesis. Our results integrate, clarify, and extend numerous observations concerning the cell biology of viral replication, allowing us to conclude that the coupling of new cellular membrane formation with the budding of these vesicles from the Golgi apparatus allows for the regulated generation of this new virogenic organelle, which is essential for viral replication. Additionally, because these pathways are also limiting in flies and in human cells infected with the related RNA virus poliovirus, they may represent novel targets for antiviral therapies.

Spontaneous jamming and unjamming in a hopper with multiple exit orifices.

We show that the flow of granular material inside a two-dimensional flat bottomed hopper is altered significantly by having more than one exit orifice. For hoppers with small orifice widths, intermittent flow through one orifice enables the resumption of flow through the adjacent jammed orifice, thus displaying a sequence of jamming and unjamming events. Using discrete element simulations, we show that the total amount of granular material (i.e., avalanche size) emanating from all the orifices combined can be enhanced by about an order of magnitude difference by simply adjusting the interorifice distance. The unjamming is driven primarily by fluctuations alone when the interorifice distance is large, but when the orifices are brought close enough, the fluctuations along with the mean flow cause the flow to unjam.

Flow of granular matter in a silo with multiple exit orifices: jamming to mixing.

We investigate the mixing characteristics of dry granular material while draining down a silo with multiple exit orifices. The mixing in the silo, which otherwise consists of noninteracting stagnant and flowing regions, is observed to improve significantly when the flow through specific orifices is stopped intermittently. This momentary stoppage of flow through the orifice is either controlled manually or is chosen by the system itself when the orifice width is small enough to cause spontaneous jamming and unjamming. We observe that the overall mixing behavior shows a systematic dependence on the frequency of closing and opening of specific orifices. In particular, the silo configuration employing random jamming and unjamming of any of the orifices shows early evidence of chaotic mixing. When operated in a multipass mode, the system exhibits a practical and efficient way of mixing particles.