Background information. Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization.Results. Among the 105 proteins identified in the CLD fraction by LC-MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte-specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl-CoA synthetases) and for TAG hydrolysis. In differentiated Caco-2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3-β-hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA-IV (apolipoprotein A-IV), which is specifically expressed by enterocytes and has been proposed to play many functions in vivo, including the formation of lipoproteins and the control of their size. The association of ApoA-IV with CLD was confirmed by confocal and immunoelectron microscopy and validated in vivo in the jejunum of mice fed with a high-fat diet.Conclusions. We report for the first time the protein endowment of Caco-2/TC7 enterocyte CLDs. Our results suggest that their formation and mobilization may participate in the control of enterocyte TRL secretion in a cell-specific manner.

bHerpes simplex virus 1 (HSV-1) is a neurotropic virus that travels long distances through cells using the microtubule network. Its 125-nm-diameter capsid is a large cargo which efficiently recruits molecular motors for movement. Upon entry, capsids reach the centrosome by minus-end-directed transport. From there, they are believed to reach the nucleus by plus-end-directed transport. Plus-end-directed transport is also important during egress, when capsids leave the nucleus to reach the site of envelopment in the cytoplasm. Although capsid interactions with dynein and kinesins have been described in vitro, the actual composition of the cellular machinery recruited by herpesviruses for capsid transport in infected cells remains unknown. Here, we identify the spectraplakin protein, dystonin/BPAG1, an important cytoskeleton cross-linker involved in microtubule-based transport, as a binding partner of the HSV-1 protein pUL37, which has been implicated in capsid transport. Viral replication is delayed in dystonin-depleted cells, and, using video microscopy of living infected cells, we show that dystonin depletion strongly inhibits capsid movement in the cytoplasm during egress. This study provides new insights into the cellular requirements for HSV-1 capsid transport and identifies dystonin as a nonmotor protein part of the transport machinery.

During the post-prandial phase, intestinal triglyceride-rich lipoproteins (TRL) i.e. chylomicrons are the main contributors to the serum lipid level, which is linked to coronary artery diseases. Hypertriglyceridemia can originate from decreased clearance or increased production of TRL. During lipid absorption, enterocytes produce and secrete chylomicrons and transiently store lipid droplets (LDs) in the cytosol. The dynamic fluctuation of triglycerides in cytosolic LDs suggests that they contribute to TRL production and may thus control the length and amplitude of the post-prandial hypertriglyceridemia. In this review, we will describe the recent advances in the characterization of enterocytic LDs. The role of LDs in chylomicron production and secretion as well as potential previously unsuspected functions in the metabolism of vitamins, steroids and prostaglandins and in viral infection will also be discussed.

Cytosolic lipid droplets (LDs) are observed in enterocytes of jejunum during lipid absorption. One important function of the intestine is to secrete chylomicrons, which provide dietary lipids throughout the body, from digested lipids in meals. The current hypothesis is that cytosolic LDs in enterocytes constitute a transient pool of stored lipids that provides lipids during interprandial period while lowering chylomicron production during the post-prandial phase. This smoothens the magnitude of peaks of hypertriglyceridemia. Here, we review the composition and functions of lipids and associated proteins of enterocyte LDs, the known physiological functions of LDs as well as the role of LDs in pathological processes in the context of the intestine.

In enterocytes, the dynamic accumulation and depletion of triacylglycerol (TAG) in lipid droplets (LD) during fat absorption suggests that cytosolic LD-associated TAG contribute to TAG-rich lipoprotein (TRL) production. To get insight into the mechanisms controlling the storage/secretion balance of TAG, we used as a tool hepatitis C virus core protein, which localizes onto LDs, and thus may modify their protein coat and decrease TRL secretion. We compared the proteome of LD fractions isolated from Caco-2/TC7 enterocytes expressing or not hepatitis C virus core protein by a differential proteomic approach (isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with liquid chromatography and tandem mass spectrometry). We identified 42 proteins, 21 being involved in lipid metabolism. Perilipin-2/ADRP, which is suggested to stabilize long term-stored TAG, was enriched in LD fractions isolated from Caco-2/TC7 expressing core protein while perilipin-3/TIP47, which is involved in LD synthesis from newly synthesized TAG, was decreased. Endoplasmic reticulum-associated proteins were strongly decreased, suggesting reduced interactions between LD and endoplasmic reticulum, where TRL assembly occurs. For the first time, we show that 17β-hydroxysteroid dehydrogenase 2 (DHB2), which catalyzes the conversion of 17-keto to 17 β-hydroxysteroids and which was the most highly enriched protein in core expressing cells, is localized to LD and interferes with TAG secretion, probably through its capacity to inactivate testosterone. Overall, we identified potential new players of lipid droplet dynamics, which may be involved in the balance between lipid storage and secretion, and may be altered in enterocytes in pathological conditions such as insulin resistance, type II diabetes and obesity.

Secondary envelopment of herpes simplex virus type 1 has been demonstrated as taking place at the trans-Golgi network (TGN). The inner tegument proteins pUL36 and pUL37 and the envelope glycoproteins gD and gE are known to be important for secondary envelopment. We compared the cellular localizations of capsids from a virus mutant lacking the UL37 gene with those of a virus mutant lacking the genes encoding gD and gE. Although wild-type capsids accumulated at the TGN, capsids of the pUL37− mutant were distributed throughout the cytoplasm and showed no association with TGN-derived vesicles. This was in contrast to capsids from a gD−gE− mutant, which accumulated in the vicinity of TGN vesicles, but did not colocalize with them, suggesting that they were transported to the TGN but were unable to undergo envelopment. We conclude that the inner tegument protein pUL37 is required for directing capsids to the TGN, where secondary envelopment occurs.

The UL33 protein of herpes simplex virus type 1 (HSV-1) is thought to be a component of the terminase complex that mediates the cleavage and packaging of viral DNA. In this study we describe the generation and characterization of a series of 15 UL33 mutants containing insertions of five amino acids located randomly throughout the 130-residue protein. Of these mutants, seven were unable to complement the growth of the UL33-null virus dlUL33 in transient assays and also failed to support the cleavage and packaging of replicated amplicon DNA into capsids. The insertions in these mutants were clustered between residues 51 and 74 and between 104 and 116, within the most highly conserved regions of the protein. The ability of the mutants to interact with the UL28 component of the terminase was assessed in immunoprecipitation and immunofluorescence assays. All four mutants with insertions between amino acids 51 and 74 were impaired in this interaction, whereas two of the three mutants in the second region (with insertions at positions 111 and 116) were not affected. These data indicate that the ability of UL33 to interact with UL28 is probably necessary, but not sufficient, to support viral growth and DNA packaging.During the packaging of the double-stranded DNA genome of herpes simplex virus type 1 (HSV-1), the cleavage of replicated concatemeric viral DNA into single-genome lengths is tightly coupled to its insertion into preassembled spherical procapsids. Upon genome insertion, the internal scaffold protein of the procapsid is lost, and the capsid shell angularizes. Genetic analysis has revealed that successful packaging requires a cis-acting DNA sequence (the a sequence) together with seven proteins, encoded by the UL6, UL15, UL17, UL25, UL28, UL32, and UL33 genes (6, 10). By analogy with doublestranded bacteriophage, the encapsidation of HSV-1 DNA is thought to be mediated by a heteromultimeric terminase enzyme. It is envisaged that the terminase is involved in the recognition of packaging signals present in the concatemers and the association with procapsids via an interaction with the capsid portal protein. Terminase initiates packaging by cleaving at an a sequence present between adjacent genomes within concatemers and subsequently provides energy for genome insertion through the hydrolysis of ATP. Packaging is terminated by a second cleavage event at the next similarly orientated a sequence, resulting in the encapsidation of a unitlength genome.An accumulating body of evidence suggests that the HSV-1 terminase is comprised of the UL15, UL28, and UL33 gene products. Viruses lacking a functional version of any of these three proteins are unable to initiate DNA packaging, and uncleaved concatemers and abortive B-capsids (angularized forms containing scaffold but no DNA) accumulate in the nuclei of infected cells (2,4,5,11,25,27,30,36,38). Protein sequence comparisons revealed a distant relationship between UL15 and the large subunit of bacteriophage T4 terminase, gp17, including the presence of Walker A and B box...

bDuring infection by herpes simplex virus 1 (HSV-1), the viral capsid is transported around the cytoplasm along the microtubule (MT) network. Although molecular motors have been implicated in this process, the composition of the molecular machinery required for efficient directional transport is unknown. We previously showed that dystonin (BPAG1) is recruited to HSV-1 capsids by the capsid-bound tegument protein pUL37 to promote efficient cytoplasmic transport of capsids during egress. Dystonin is a cytoskeleton cross-linker which localizes at MT plus ends and has roles in retrograde and anterograde transport in neurons. In this study, we investigated the role of dystonin during the entry stages of HSV-1 infection. Because of the way in which the MT network is organized, capsids are required to change their direction of motion along the MTs as they travel from the point of entry to the nucleus, where replication takes place. Thus, capsids first travel to the centrosome (the principal microtubule organizing center) by minus-end-directed transport and then switch polarity and travel to the nucleus by plus-end-directed transport. We observed that transport of capsids toward the centrosome was slowed, but not blocked, by dystonin depletion. However, transport of capsids away from the centrosome was significantly impaired, causing them to accumulate in the vicinity of the centrosome and reducing the numbers reaching the nucleus. We conclude that, during entry of HSV-1, dystonin has a specific role in plus-ended transport of capsids from the centrosome to the nucleus.A successful outcome of infection demands precise control of particle movement around the cell. The cell has a number of transport mechanisms available, but the most important for herpesviruses is the microtubule (MT) network (1, 2), which is the main route of movement between the cell surface, where virus entry and exit take place, and the nucleus, which is the site of virus transcription, DNA replication, and capsid assembly. The MT network is typically organized around one or more microtubuleorganizing centers (MTOCs), with the MT minus ends anchored at the MTOC and the plus ends radiating outwards (3). Because of this arrangement, a herpesvirus capsid has to switch polarity in order to travel from the plasma membrane to the nucleus. Thus, the capsids travel from the plasma membrane to the centrosome (the principal MTOC in most cell types) by minus-end-directed transport but must then transfer to another MT to complete its journey by plus-end-directed transport. The direction of transport along MTs is determined by the molecular motors that transport the cargo. These are of two basic types, kinesins and dynein, which carry out plus-end-and minus-end-directed transport, respectively. Association of herpes simplex virus 1 (HSV-1) capsids with molecular motors, such as dynein or kinesins, has been reported in vitro (4), and kinesin 3 interaction with the viral membrane protein pUs9 was shown to be important for anterograde transport of pseudorabies virus ...