Hijacked highways: plant virus modulation of vector proteins from entry to exit.

Systemic and Specific Delivery of Small Interfering RNAs to the Liver Mediated by Apolipoprotein A-I

The current model for cell-to-cell movement of plant viruses holds that transport requires virus-encoded movement proteins that intimately associate with endoplasmic reticulum membranes. We have examined the early stages of the integration into endoplasmic reticulum membranes of a double-spanning viral movement protein using photocross-linking. We have discovered that this process is cotranslational and proceeds in a signal recognition particle-dependent manner. In addition, nascent chain photocross-linking to Sec61alpha and translocating chain-associated membrane protein reveal that viral membrane protein insertion takes place via the translocon, as with most eukaryotic membrane proteins, but that the two transmembrane segments of the viral protein leave the translocon and enter the lipid bilayer together.

Diverse animal and plant viruses are able to translocate their virions between neighboring cells via intercellular connections. In this work, we analyze the virion assembly and cell-to-cell movement of a plant closterovirus and reveal a strong correlation between these two processes. The filamentous virions of a closterovirus possess a long body formed by the major capsid protein (CP) and a short tail formed by the minor capsid protein (CPm). Genetic and biochemical analyses show that the functions of these virion components are distinct. A virion body is required primarily for genome protection, whereas a tail represents a specialized device for cell-to-cell movement. Furthermore, tail assembly is mediated by the viral Hsp70 homolog (Hsp70h) that becomes an integral part of the virion. Inactivation of the ATPase domain of Hsp70h results in assembly of tailless virions that are incapable of translocation. A dual role for the viral molecular chaperone Hsp70h in virion assembly and transport, combined with the previous finding of this protein in intercellular channels, allowed us to propose a model of closteroviral movement from cell to cell.

The 2b proteins encoded by cucumovirus act as post-transcriptional gene silencing suppressors to counter host defence during infection. Here we report the crystal structure of Tomato aspermy virus 2b (TAV2b) protein bound to a 19 bp small interfering RNA (siRNA) duplex. TAV2b adopts an all alpha-helix structure and forms a homodimer to measure siRNA duplex in a length-preference mode. TAV2b has a pair of hook-like structures to recognize simultaneously two alpha-helical turns of A-form RNA duplex by fitting its alpha-helix backbone into two adjacent major grooves of siRNA duplex. The conserved pi-stackings between tryptophan and the 5'-terminal base of siRNA duplex from both ends enhance the recognition. TAV2b further oligomerizes to form a dimer of dimers through the conserved leucine-zipper-like motif at its amino-terminal alpha-helix. Biochemical experiments suggest that TAV2b might interfere with the post-transcriptional gene silencing pathway by directly binding to siRNA duplex.

How important is the clathrin-dependent endocytic pathway for entry of viruses into host cells? While it is widely accepted that Semliki Forest virus (SFV), an enveloped virus, requires this pathway there are conflicting data concerning the closely related Sindbis virus, as well as varying results with picornaviruses such as human rhinovirus 14 (HRV 14) and poliovirus. We have examined the entry mode of SFV, Sindbis virus, HRV 14 and poliovirus using a method that identifies single infected cells. This assay takes advantage of the observation that the clathrin-dependent endocytic pathway is specifically and potently arrested by overexpression of dynamin mutants that prevent clathrin-coated pit budding. Using HeLa cells and conditions of low multiplicity of infection to favor use of the most avid pathway of cell entry, it was found that SFV, Sindbis virus and HRV 14 require an active clathrin-dependent endocytic pathway for successful infection. In marked contrast, infection of HeLa cells by poliovirus did not appear to require the clathrin pathway.

RNA interference inhibitors were initially discovered in plant viruses, representing a unique mechanism employed by these viruses to counteract host RNA interference. This mechanism has found extensive applications in plant disease resistance breeding and other fields; however, the impact of such interference inhibitors on insect cell RNA interference remains largely unknown. In this study, we screened three distinct interference inhibitors from plant and mammal viruses that act through different mechanisms and systematically investigated their effects on the insect cell cycle and baculovirus infection period at various time intervals. Our findings demonstrated that the viral suppressors of RNA silencing (VSRs) derived from plant and mammal viruses significantly attenuated the RNA interference effect in insect cells, as evidenced by reduced apoptosis rates, altered gene regulation patterns in cells, enhanced expression of exogenous proteins, and improved production efficiency of recombinant virus progeny. Further investigations revealed that the early expression of VSRs yielded superior results compared with late expression during RNA interference processes. Additionally, our results indicated that dsRNA-binding inhibition exhibited more pronounced effects than other modes of action employed by these interference inhibitors. The outcomes presented herein provide novel insights into enhancing defense mechanisms within insect cells using plant and mammal single-stranded RNA virus-derived interference inhibitors and have potential implications for expanding the scope of transformation within insect cell expression systems.

TAR RNA-binding protein, TRBP, was recently discovered to be an essential partner for Dicer and a crucial component of the RNA-induced silencing complex (RISC), a critical element of the RNA interference (RNAi) of the cell apparatus. Human TRBP was originally characterized and cloned 15 years ago based on its high affinity for binding the HIV-1 encoded leader RNA, TAR. RNAi is used, in part, by cells to defend against infection by viruses. Here, we report that transfected TAR RNA can attenuate the RNAi machinery in human cells. Our data suggest that TAR RNA sequesters TRBP rendering it unavailable for downstream Dicer-RISC complexes. TAR-induced inhibition of Dicer-RISC activity in transfected cells was partially relieved by exogenous expression of TRBP.

BackgroundRNA interference (RNAi) in animals and post-transcriptional gene silencing (PTGS) in plants are related phenomena whose functions include the developmental regulation of gene expression and protection from transposable elements and viruses. Plant viruses respond by expressing suppressor proteins that interfere with the PTGS system.ResultsHere we demonstrate that both transient and constitutive expression of the Tobacco etch virus HC-Pro silencing suppressor protein, which inhibits the maintenance of PTGS in plants, prevents dsRNA-induced RNAi of a lacZ gene in cultured Drosophila cells. Northern blot analysis of the RNA present in Drosophila cells showed that HC-Pro prevented degradation of lacZ RNA during RNAi but that there was accumulation of the short (23nt) RNA species associated with RNAi. A mutant HC-Pro that does not suppress PTGS in plants also does not affect RNAi in Drosophila. Similarly, the Cucumber mosaic virus 2b protein, which inhibits the systemic spread of PTGS in plants, does not suppress RNAi in Drosophila cells. In addition, we have used the Drosophila system to demonstrate that the 16K cysteine-rich protein of Tobacco rattle virus, which previously had no known function, is a silencing suppressor protein.ConclusionThese results indicate that at least part of the process of RNAi in Drosophila and PTGS in plants is conserved, and that plant virus silencing suppressor proteins may be useful tools to investigate the mechanism of RNAi.

Effect of fish oil on agonist-induced receptor internalization of the PG F2α receptor and cell signaling in bovine luteal cells in vitro

Hepatocytes, the major epithelial cells in the liver, are highly polarized. Their plasma membranes are separated by tight junctions into sinusoidal–basolateral and canalicular–apical domains, which contain distinct sets of proteins and lipids. A normal membrane polarity is vital for hepatocytes to perform many diverse functions, such as canalicular bile secretion and simultaneous sinusoidal secretion of large quantities of serum proteins into blood. Hepatocyte polarity is lost in many diseases like cholestasis. A complete understanding of the molecular mechanisms involved in hepatocyte polarity therefore is of considerable significance to both liver cell biology and the pathogenesis of liver diseases. To maintain the distinct distribution of proteins and lipids in the canalicular and sinusoidal membranes, hepatocytes have developed complex polarized trafficking and retention mechanisms. The establishment of membrane polarity in hepatocytes begins during liver embryogenesis. During hepatocyte differentiation, specific routes and mechanisms are defined for the delivery of plasma membrane proteins. How this polarity is maintained in adult hepatocytes and is generated during development are two major unanswered questions in this field. In this review, we first give an overview of the current knowledge about how membrane polarity is maintained in hepatocytes with the focus on recent developments in polarized protein trafficking. We then present a hypothetical model about how membrane polarity may be generated during liver development, and finally, we present an example of how membrane polarity is altered in cholestasis. Readers are directed to recent reviews for discussion of lipid trafficking in hepatocytes.1, 2 TGN, trans-Golgi network; DPP IV, dipeptidyl peptidase IV; pIgA-R, polymeric immunoglobulin A receptor; GPI, glycosyl-phosphatidylinositol; 5′NT, 5′-nucleotidase; MDCK, Madin-Darby canine kidney cells; ATP, adenosine triphosphate; Mdr1, multidrug resistance protein 1; Bsep, bile salt export pump; GFP, green fluorescent protein; Mrp2, multidrug resistant associated protein; LDL-R, low density lipoprotein receptor; AP, adaptor protein; SNARE, the soluble NSF attachment protein receptor; ERM, ezrin-radixin-moesin; TLCA, taurolithocholic acid. Three major intracellular trafficking pathways are involved in the polarized expression of plasma membrane proteins in hepatocytes. Through biogenesis, a plasma membrane protein is first synthesized in the endoplasmic reticulum, modified in Golgi, and finally sorted at the trans-Golgi network (TGN). From the TGN, they traffic along the post-Golgi biosynthetic pathway to the canalicular or sinusoidal membrane domain. After a protein reaches the plasma membrane, it can be either retained or internalized (endocytosed). After endocytosis, some proteins are transported to the opposite membrane domain of hepatocytes, a process known as transcytosis. During the past two decades, a key quest in liver cell biology has been to understand the trafficking routes and molecular mechanisms that are used by various canalicular and sinusoidal proteins as they journey along these pathways. Previous studies have demonstrated that proteins destined for the sinusoidal membrane in hepatocytes traffic from the Golgi complex to their destination directly (Fig. 1A).3 However, most canalicular proteins traffic by an indirect route, first to the sinusoidal membrane, followed by transcytosis through basolateral early endosomes and a subapical compartment, before final targeting to the canalicular domain (Fig. 1A). These canalicular proteins include single transmembrane proteins (such as dipeptidyl peptidase IV [DPP IV], aminopeptidase N, and polymeric immunoglobulin A receptor [pIgA-R]) and the glycosyl-phosphatidylinositol (GPI)-anchored proteins like 5′-nucleotidase (5′NT). Until recently, the indirect transcytotic pathway was thought to be the only route by which canalicular proteins reach their destination in hepatocytes. In contrast, most apical proteins traffic directly from TGN to the apical membrane in Madin-Darby canine kidney (MDCK) cells, a polarized kidney cell line, whereas both direct and indirect pathways operate in the retinal pigment and intestinal epithelial cells.4, 5 The post-Golgi biosynthetic trafficking and endocytic routes in hepatocytes. A hepatocyte couplet is shown with the basolateral–sinusoidal and apical–canalicular membranes separated by tight junctions (TJ). (A) Sinusoidal membrane proteins (blue circles) take a direct route (blue arrows) from the TGN to the basolateral membrane. Most canalicular proteins (green patches) are targeted initially to the basolateral membrane and then undergo transcytosis (green arrows) through the basolateral early endosome (BEE) and a subapical compartment (SAC) before reaching the canalicular membrane. (B) In contrast, Mdr1 (solid red patch) traffics directly (red arrow) from the TGN to the canalicular membrane. Bsep (hatched red patch) may traffic first to a subapical compartment (also termed as SAC) and then to the canalicular membrane. It remains unclear if Bsep and the transcytosed proteins traffic through the same SAC. (C) Some sinusoidal proteins recycle between the basolateral membrane, BEE, and SAC (dashed blue lines). The SAC functions as a common sorting station for both transcytosed and endocytosed proteins.65 Some sinusoidal proteins are targeted to late endosome (LE) and lysosomes (L) for degradation after endocytosis. At the canalicular membrane, most canalicular proteins are very slowly endocytosed with little recycling (dashed thin green lines). When an apical phosphoinositide 3 kinase is inhibited, some canalicular proteins (green patches) accumulate in lysosomes.46 (D) Mdr1 and Bsep both may recycle (dashed red lines) between the canalicular membranes and subapical compartment(s). The exact endocytic routes for Mdr1 and Bsep remain to be determined (indicated by ?). Possible intracellular compartments en route may include the apical early endosome (AEE), SAC, the apical recycling endosome (ARE), and lysosome for degradation. BC, bile canaliculi, N, nucleus. Recently, two canalicular adenosine triphosphate (ATP)–binding cassette transporters, the multidrug resistance protein 1 (Mdr1) and the bile salt export pump (Bsep), were shown to traffic directly from the Golgi to the canalicular domain (Fig. 1B). These findings were based on in vivo pulse-chase labeling in rats6 and expression of green fluorescent protein (GFP) fusion constructs in WIF-B cells, a polarized hepatic hybrid cell line.7 In addition to Mdr1 and Bsep, newly synthesized sphingolipids also reach the canalicular membrane directly from the TGN in hepatocytes.8 However, it remains unclear whether another major canalicular ATP-binding cassette transporter, the multidrug resistant-associated protein (Mrp2), which contains PDZ domains (a motif originally identified in postsynaptic protein PSD-95, Drosophila tumor suppressor Dlg and tight junction protein ZO-1; and used extensively for forming molecular scaffold as described below), also traffic directly from the Golgi to canalicular membrane. Indeed, a recent study demonstrated that Mrp2 resides in the same intracellular vesicles as pIgA-R, a canalicular protein that undergoes transcytosis.9 Much of our knowledge of the molecular mechanisms involved in polarized trafficking comes from work on epithelial cells other than hepatocytes.10 Direct basolateral targeting often involves cytoplasmic sorting signals, which can be either dileucine or tyrosine-based motifs.11 Two sinusoidal membrane proteins, the low-density lipoprotein receptor (LDL-R) and the sodium-taurocholate cotransporter, contain two tyrosine-based sorting signals.12, 13 Mutation of a critical glycine adjacent to the high-capacity tyrosine signal in LDL-R results in the mistargeting of LDL-R to the canalicular membrane in the WIF-B cells.14 Interestingly, β-turn structures, which are common to tyrosine-based basolateral sorting and endocytosis signals, are not present in the two tyrosine signals of sodium-taurocholate cotransporters.13 Basolateral sorting signals are believed to be recognized at the TGN by adaptor protein (AP) complexes, which are tetrameric protein complexes involved in both biosynthetic and endocytic sorting.15 Hepatocytes do not express μ1B, one important subunit of the AP-1B complex.16 μ1B is indispensable in the sorting of many basolateral proteins in kidney epithelial cells.17 It is not clear if hepatocytes use an unknown isoform of μ1 or a distinct AP complex to recognize the sinusoidal protein cargos. After they are bound to the AP complex, the sinusoidal proteins may be targeted and fused to the sinusoidal membrane, a process mediated in some epithelial cells by the soluble NSF (N-ethyl Maleimide-sensitive fusion protein) attachment protein receptor (SNARE) and an octomeric protein complex, which in yeast is called the exocyst-sec6/8 complex.18 The exact composition of the basolateral SNARE and exocyst complexes in hepatocytes and their roles in sinusoidal targeting have not been established. The canalicular transcytosing protein pIgA-R also takes a transcytotic route in MDCK and intestinal epithelial cells. Transcytosis of pIgA-R is stimulated by binding of immunoglobulin A and regulated by phosphorylation.19 pIgA-R contains a series of targeting signals within a cytoplasmic domain of 103 amino acids, which include a basolateral sorting signal and two tyrosine-based endocytosis signals.19 Notably, the basolateral sorting signal of pIgA-R is not dileucine or tyrosine based, and hence is different from the direct sinusoidal sorting signals discussed above. Different mechanisms other than the AP complex may determine the basolateral sorting of pIgA-R. The sorting signals in other single transmembrane proteins that undergo transcytosis are less well defined.20 Compared with direct basolateral sorting, the signals and machinery responsible for apical sorting are less well understood. Thus far, three types of apical sorting signals have been described: (1) GPI anchors, (2) transmembrane domains, and (3) N-glycans.10 In addition, the sphingolipid- and cholesterol-enriched domains, also known as lipid rafts, function as sorting platforms for apical cargos in the TGN.21 Lipid rafts are characterized by insolubility to detergent extraction at cold temperature. The enrichment of cholesterol and sphingolipid in canalicular membrane of hepatocytes has long been noted.22 Two recent independent studies demonstrate that lipid rafts mediate both transcytosis and direct apical trafficking of canalicular proteins in hepatocytes.23, 24 Slimane et al.23 showed that a GPI-anchored model protein, GPI-GFP, reaches the apical membrane of hepatic HepG2 cells by transcytosis and largely is present in lipid rafts, which are insoluble in both Triton X-100 and Lubrol WX at 4°C (Triton rafts). In contrast, two direct trafficking apical proteins, Mdr1-GFP and ATP7B (a P-type copper transporting ATPase), are contained in a distinct type of raft (Lubrol rafts), which are insoluble in Lubrol WX but are soluble in the stronger detergent, Triton X-100. Pulse-chase labeling indicated that both Triton and Lubrol rafts are formed at the TGN in HepG2 cells. Cholesterol depletion moderately increased the solubility of GPI-GFP to Triton X-100 without affecting its apical sorting. However, the same treatment led to missorting of Mdr1-GFP to the basolateral domain in HepG2 cells. Triton and Lubrol rafts also have been described in MDCK cells and segregate the GPI-anchored proteins from multispanning transmembrane proteins in the apical membrane.25 Nyasae et al.24 examined the solubility of a number of WIF-B cell apical proteins to Triton X-100 at 4°C. Two GPI-anchored proteins, 5′NT and CD59, are virtually insoluble to Triton X-100. In contrast, the single transmembrane proteins DDP IV and pIgA-R are completely soluble. Thus, not all of these canalicular proteins are contained in rafts, although they all use the indirect transcytotic pathway. Other mechanisms in addition to rafts must determine their indirect apical sorting. Moreover, the detergent-insoluble apical proteins seem to enter lipid rafts at different stages in the transcytotic pathway and with different kinetics.24 In this study, cholesterol depletion after 60 minutes severely impaired transcytosis of both raft-associated (CD59) and nonassociated proteins (DDP IV), presumably by blocking transcytotic flux from basolateral endosomes.24 Interestingly, cholesterol depletion did not affect the apical trafficking of GPI-GFP after 180 minutes in HepG2 cells in the study by Slimane et al.23 It is conceivable that the model protein GPI-GFP may not reflect faithfully the apical sorting of endogenous GPI-anchored canalicular proteins like CD59 and 5′NT. Despite this discrepancy, these two studies23, 24 together show that lipid rafts mediate the apical trafficking of some canalicular proteins in hepatocytes and that cholesterol is probably required for both Triton and Lubrol rafts. Furthermore, these two studies emphasize the complexity of membrane microdomains in the plasma membrane of hepatocytes. Judged by the sole criteria of detergent insolubility, the canalicular membrane consists of at least two types of rafts and a pool of single transmembrane proteins, some of which may be partly raft associated.23, 24 Lipid rafts also exist in the sinusoidal membrane of hepatocytes because some transcytosing proteins may have been incorporated into rafts before their transit to the sinusoidal membrane. In addition, caveolae, a special type of raft, have been detected at microvilli beneath the sinusoidal membranes of hepatocytes26 and contain raft-resident signaling proteins such as Ras and protein kinase C.27 Taken together, these studies suggest that both the canalicular and sinusoidal membranes can be viewed as a mosaic of raft-associated and nonassociated microdomains. A comprehensive analysis of detergent solubility of all canalicular and sinusoidal proteins will be required to appreciate fully the diversity of microdomains in the plasma membrane of hepatocytes and their functional implication. It also would be interesting to examine directly rafts and other microdomains in live hepatic cells using high-resolution fluorescence microscopy, which has been used successfully to examine raft-resident proteins in several cell types.28 Although it is not yet certain how raft mechanisms mediate the apical sorting of canalicular proteins, the work by Slimane et al.23 suggests that Lubrol rafts may segregate multispanning transmembrane proteins and may influence their direct apical sorting in HepG2 cells. It remains to be determined if other ATP-binding cassette proteins that traffic directly to the canalicular domain, such as Bsep, reside in the same type of lipid rafts. As discussed above, most GPI-anchored and single transmembrane proteins traffic directly from the TGN to the apical membrane in MDCK cells but indirectly via transcytosis to the canalicular membrane in hepatocytes. Why is there such a difference? Presumably, the plasma membrane and sorting mechanisms are organized differently between these two cell types. For example, secretory forms of DPP IV and pIgA-R (lacking the transmembrane regions) are secreted across the apical membrane in MDCK cells,29, 30 but across the basolateral domain in hepatic WIF-B cells.31 In hepatocytes, most proteins are secreted primarily across the sinusoidal domain. Canalicular proteins that undergo transcytosis seem to be sorted similarly at the TGN as the sinusoidal secreted proteins, that is, they are initially targeted to the sinusoidal domain. Basolateral endosomes also have been proposed to function as a sorting station for proteins that undergo transcytosis to the apical membrane in hepatocytes.31 In contrast, Mdr1 and Bsep must be sorted differently because they traffic directly to the canalicular domain. How these proteins exactly are sorted through the TGN remains to be determined. Transcytosis is not a phenomenon unique to hepatocytes because a number of apical proteins are delivered partly by transcytosis and partly by direct targeting to the apical membrane in intestinal epithelial cells.32 Transcytosis also seems to be regulated during epithelial differentiation. In Fisher rat thyroid cells, transcytosis functions as a major apical sorting mechanism for DPP IV in early epithelial differentiation, but is replaced by direct apical targeting in more differentiated epithelial cells.33 It is worth noting that few mammalian cells have robust protein secretion across the basolateral membrane like hepatocytes. Therefore, sinusoidal protein secretion, which is established early in hepatocyte differentiation and persists in adult liver, may be the reason why transcytosis is a major apical sorting mechanism for most canalicular proteins in adult hepatocytes. Interestingly, Bsep, a direct trafficking canalicular protein, is detected in rat liver only after birth,34 consistent with the notion that direct apical targeting may be established in hepatocytes at a later stage of differentiation. In contrast, Mrp2 is detected at the bile canaliculi before birth,34 consistent with the possibility that Mrp2 may be sorted via the transcytotic rather than the direct pathway. To understand fully the mechanistic difference in polarized trafficking between hepatocytes and other epithelial cells, a comprehensive comparison of the molecules and complexes involved in protein sorting will be required. For example, soluble NSF (N-ethyl maleimide-sensitive fusion protein) attachment protein-23, a key component of the SNARE complex, is present at both the apical and basolateral membranes of many epithelial cells but is absent from the canalicular membrane of hepatocytes.3 The apical SNARE complex mediates the docking and fusion of Triton rafts (containing GPI-anchored proteins) to the apical membrane in MDCK cells.35 It remains to be determined if the absence of soluble NSF attachment protein-23 in the canalicular membrane contributes to the preference of transcytosis as a major apical delivery mechanism in hepatocytes. Several sinusoidal membrane proteins, such as LDL-R, the transferrin receptor, and the asialoglycoprotein receptor, have a dynamic residence at the plasma membrane and undergo endocytosis (Fig. 1C).36 What about canalicular proteins? Tuma et al.37 recently demonstrated that several indirect pathway proteins (DPP IV, aminopeptidase N, and 5′NT) as well as MRP2 largely are retained at the apical membrane in differentiated hepatic WIF-B cells (Fig. 1C). However, these proteins recycle between plasma membrane and a novel intracellular compartment in less differentiated Fao and Clone 9 cells, which contain only would-be apical proteins. These data suggest that apical retention mechanisms are established during hepatocyte differentiation. In contrast, Mdr1-GFP and Bsep-GFP (which presumably reflect Mdr1 and Bsep) undergo dynamic endocytosis and recycle between the canalicular membrane and a subapical region when expressed in WIF-B cells (Fig. 1D).7, 38 Why is there such a difference in the dynamics of plasma membrane proteins in hepatocytes? First, the actin-based cytoskeleton is thought to be responsible for retention of domain-specific proteins.39, 40 Proteins like ezrin-radixin-moesin (ERM) bind to both actin and single transmembrane proteins directly41 and interact with multispanning proteins like Mrp2 via their PDZ domains.42 Therefore, ERM proteins mediate the formation of a molecular scaffold between apical membrane and actin cytoskeleton, which leads to the retention of these canalicular proteins. The degree of retention for single transmembrane proteins and Mrp2 may be different because disruption of actin filaments affected their localization differently in WIF-B cells.37 In addition, knockout of radixin in mice led to a significant decrease in the canalicular membrane expression of Mrp2 but not DDP IV, Mdr1, or Bsep.43 At present, it is not known how ERM or other retention mechanisms operate underneath the sinusoidal membrane. Second, dynamic membrane proteins like the transferrin receptor and raft-resident proteins are endocytosed by the clathrin- and caveolae-mediated pathways, respectively.44, 45 At present, it is not known which pathways mediate the fast apical endocytosis of Mdr1 and Bsep and the slow apical endocytosis of indirect pathway proteins and Mrp2. Several mechanisms may explain why Mdr1 and Bsep recycle rather than being retained in the canalicular membrane like Mrp2: (1) Mdr1 and possibly Bsep reside in distinct lipid rafts,23 which may confer different lateral diffusion properties, and (2) Mdr1 and Bsep do not posses PDZ motifs and presumably do not bind to tethering ERM proteins. Despite a tight apical retention mechanism, slow endocytosis and subsequent targeting to lysosomes may still function as a means to remove and degrade the indirect pathway proteins like aminopeptidase N (2%–3% degraded per hour37) and Mrp2 from the canalicular membrane. Inhibition of Vps34p, an apical phosphoinositide 3 kinase by wortmannin or injection of inhibitory Vps34p antibodies, led to accumulation of apical proteins like 5′-NT and MRP2 in lysosomal vacuoles in WIF-B cells.46, 47 Phosphoinositide 3 kinase generates secondary lipid messengers from inositol, which in turn regulates many steps of both biosynthetic and endocytic sorting. Thus far, we have considered how hepatic membrane polarity is maintained. How is such a complex polarity generated in hepatocytes during liver development and in particular how are the transcytotic delivery and apical retention mechanisms established? A number of electron microscopic studies have documented the ultrastructural changes in hepatocyte progenitor cells and hepatocytes at different stages of liver development.48-50 In chick embryos, the earliest liver parenchymal cells are not polarized.48 As the liver bud invades the perihepatic mesoderm, the parenchymal cells become polarized at day 5 when canaliculi-specific antigens are first detected. By day 7, the liver rudiment shows ultrastructural characteristics of bile canaliculi, and a canalicular network can be seen.48 From this point on, hepatocytes are polarized, but the bile canaliculi do not attain a fully several after Interestingly, in hepatocytes, intracellular bile canaliculi have been that may canalicular domains and may be to the intracellular apical compartment in Fao cells described by Tuma et al.37 The development of membrane polarity is to the process of hepatocyte differentiation. To understand fully the molecular involved in the of hepatocyte a analysis of the trafficking cytoskeleton, and signaling molecules at different stages of hepatic differentiation are required. on recent work from other epithelial we that the of hepatic polarity involves the in In hepatocyte progenitor cells, would-be sinusoidal and canalicular membrane proteins are sorted at the TGN into distinct vesicles by basolateral sorting signals and raft mechanisms (Fig. cells the these proteins may be into the but these microdomains are with other in the plasma membrane. Some intracellular compartments may be formed and may contain the would-be canalicular proteins, an stage before formation of the canalicular membrane. As the liver bud invades the perihepatic mesoderm, the of hepatocytes is by or (Fig. or the a process mediated by cell molecules like may be to the of and may to the of domain-specific or both a membrane or a that in turn may a number of (Fig. As by et and these include the establishment of apical junction complexes junction and the of signaling and polarized attachment of actin and which then to the of the cytoskeleton in the would-be canalicular and sinusoidal proteins distinct on the plasma membrane, followed by the of apical and basolateral the canalicular and basolateral domains are formed in hepatocytes (Fig. and actin cytoskeleton also are established in a polarized shown in A hypothetical model for the of membrane polarity during hepatocyte differentiation. Several stages of during hepatocyte differentiation. (A) hepatocyte progenitor cells are not polarized. The would-be sinusoidal (blue circles) and would-be canalicular proteins (green and red for indirect and direct pathway proteins, are sorted at the TGN and are in the plasma membrane. Some intracellular apical compartments may be formed that contain canalicular proteins. (B) As the liver bud invades the perihepatic mesoderm, of hepatocytes is by exocyst complexes may be to the to the subsequent of domain-specific (C) a membrane which the formation of apical The actin network to the complexes, to the and of the cytoskeleton in hepatocytes. canalicular and sinusoidal proteins traffic to the plasma membrane (indicated by arrows) and the canalicular and sinusoidal (D) membrane polarity is fully established in hepatocytes. The and of are The subapical actin cytoskeleton network to tight junctions and the canalicular membrane, forming as a the canalicular domain. BC, bile tight During this specific that liver differentiation may influence the expression and distribution of specific membrane targeting molecules soluble NSF attachment retention molecules the ERM and the of cytoskeleton the subapical actin these the mechanisms for polarized protein For example, hepatocyte and the formation of tight junctions during and may function similarly in liver differentiation. of to of Fao cells these cells to bile canaliculi and to functional of cell Thus bile which are secreted only at the later stage of liver development, also may affect hepatocyte membrane polarity and the of bile canaliculi, possibly by signaling through To these at the molecular will to in of the expression and signaling between the and Fao at different during development of cell polarity may interesting about when and how transcytosis and direct apical trafficking is established in to hepatocyte of hepatocyte polarity is a of liver by or injection of leads to a of canalicular proteins such as pIgA-R and DPP IV to the domain and accumulation in subapical (Fig. and treatment also the of transcytotic with an in both the number and of transcytotic vesicles within the subapical intracellular in which may from fusion of transcytotic vesicles and may bile canaliculi both and These intracellular seem to the apical compartments in less differentiated hepatic cells and liver, as discussed above. In liver, the tight junctions are also the between bile and Taken together, these suggest that transcytosis of canalicular proteins and leads to a or of membrane The mechanisms responsible for altered cell polarity in are understood. However, the of to the trafficking of Bsep to the canalicular domain is to be in hepatocytes in and can both transcytosis and the expression of Mrp2 in the canalicular The these inhibitory of and the of and to the canalicular membrane, which may in turn affect the trafficking of Mrp2. It is conceivable that bile also may directly or indirectly affect the trafficking of other plasma membrane proteins in hepatocytes. Much work is in membrane polarity and polarized trafficking in hepatocytes. leads to the of canalicular proteins (green patches) to the sinusoidal and lateral domains, of transcytotic vesicles red and the of intracellular junctions also are a between the and taurolithocholic the targeting and of transcytosed proteins and Mrp2 to the canalicular membrane, with the of to the canalicular membrane. the inhibitory of and of to the canalicular membrane. studies described in this have significant into how sinusoidal and canalicular proteins traffic to and reside in the plasma membrane. However, the molecular mechanisms by which hepatocytes and maintain membrane polarity still remain understood. more of the polarized sorting machinery is required. by the of hepatocyte differentiation, we may into how hepatocyte polarity is generated and how the preference of hepatocytes to proteins to the apical canalicular membrane by transcytosis is established. studies of polarized trafficking in hepatocytes have been largely because of developments such as described in this the a understanding of this complex yet vital of hepatocytes to and maintain their polarity in and The and Tuma for during the of this and and for We to are not because of of

Plant viruses are obligate organisms that require host components for movement within and between cells. A mechanistic understanding of virus movement will allow the identification of new methods to control virus systemic spread and serve as a model system for understanding host macromolecule intra- and intercellular transport. Recent studies have moved beyond the identification of virus proteins involved in virus movement and their effect on plasmodesmal size exclusion limits to the analysis of their interactions with host components to allow movement within and between cells. It is clear that individual virus proteins and replication complexes associate with and, in some cases, traffic along the host cytoskeleton and membranes. Here, we review these recent findings, highlighting the diverse associations observed between these components and their trafficking capacity. Plant viruses operate individually, sometimes within virus species, to utilize unique interactions between their proteins or complexes and individual host cytoskeletal or membrane elements over time or space for their movement. However, there is not sufficient information for any plant virus to create a complete model of its intracellular movement; thus, more research is needed to achieve that goal.

5-Hydroxytryptamine 2A (5-HT2A) receptors, a major site of action of clozapine and other atypical antipsychotic medications, are, paradoxically, internalized in vitro and in vivo by antagonists and agonists. The mechanisms responsible for this paradoxical regulation of 5-HT2A receptors are unknown. In this study, the arrestin and dynamin dependences of agonist- and antagonist-mediated internalization were investigated in live cells using green fluorescent protein (GFP)-tagged 5-HT2A receptors (SR2-GFP). Preliminary experiments indicated that GFP tagging of 5-HT2A receptors had no effect on either the binding affinities of several ligands or agonist efficacy. Likewise, both the native receptor and SR2-GFP were internalized via endosomes in vitro. Experiments with a dynamin dominant-negative mutant (dynamin K44A) demonstrated that both agonist- and antagonist-induced internalization were dynamin-dependent. By contrast, both the agonist- and antagonist-induced internalization of SR2-GFP were insensitive to three different arrestin (Arr) dominant-negative mutants (Arr-2 V53D, Arr-2-(319-418), and Arr-3-(284-409)). Interestingly, 5-HT2A receptor activation by agonists, but not antagonists, induced greater Arr-3 than Arr-2 translocation to the plasma membrane. Importantly, the agonist-induced internalization of 5-HT2A receptors was accompanied by differential sorting of Arr-2, Arr-3, and 5-HT2A receptors into distinct plasma membrane and intracellular compartments. The agonist-induced redistribution of Arr-2 and Arr-3 into intracellular vesicles and plasma membrane compartments distinct from those involved in 5-HT2A receptor internalization implies novel roles for Arr-2 and Arr-3 independent of 5-HT2A receptor internalization and desensitization.

Intracellular movement is an important step for the initial spread of virus in plants during infection. This process requires virus-encoded movement proteins (MPs) and their interaction with host factors. Despite the large number of known host factors involved in the movement of different viruses, little is known about host proteins that interact with one of the MPs encoded by potexviruses, the triple-gene-block protein 3 (TGBp3). The main obstacle lies in the relatively low expression level of potexviral TGBp3 in hosts and the weak or transient nature of interactions. Here, we used TurboID-based proximity labeling to identify the network of proteins directly or indirectly interacting with the TGBp3 of a potexvirus, Bamboo mosaic virus (BaMV). Endoplasmic reticulum (ER) luminal-binding protein 4 and calreticulin 3 of Nicotiana benthamiana (NbBiP4 and NbCRT3, respectively) associated with the functional TGBp3-containing BaMV movement complexes, but not the movement-defective mutant, TGBp3M. Fluorescent microscopy revealed that TGBp3 colocalizes with NbBiP4 or NbCRT3 and the complexes move together along ER networks to cell periphery in N. benthamiana. Loss- and gain-of-function experiments revealed that NbBiP4 or NbCRT3 is required for the efficient spread and accumulation of BaMV in infected leaves. In addition, overexpression of NbBiP4 or NbCRT3 enhanced the targeting of BaMV TGBp1 to plasmodesmata (PD), indicating that NbBiP4 and NbCRT3 interact with TGBp3 to promote the intracellular transport of virion cargo to PD that facilitates virus cell-to-cell movement. Our findings revealed additional roles for NbBiP4 and NbCRT3 in BaMV intracellular movement through ER networks or ER-derived vesicles to PD, which enhances the spread of BaMV in N. benthamiana.

The genomes of many plant viruses have a coding capacity limited to <10 proteins, yet it is becoming increasingly clear that individual plant virus proteins may interact with several targets in the host for establishment of infection. As new functions are uncovered for individual viral proteins, virologists have realized that the apparent simplicity of the virus genome is an illusion that belies the true impact that plant viruses have on host physiology. In this review, we discuss our evolving understanding of the function of the P6 protein of Cauliflower mosaic virus (CaMV), a process that was initiated nearly 35 years ago when the CaMV P6 protein was first described as the 'major inclusion body protein' (IB) present in infected plants. P6 is now referred to in most articles as the transactivator (TAV)/viroplasmin protein, because the first viral function to be characterized for the Caulimovirus P6 protein beyond its role as an inclusion body protein (the viroplasmin) was its role in translational transactivation (the TAV function). This review will discuss the currently accepted functions for P6 and then present the evidence for an entirely new function for P6 in intracellular movement.

Plant viruses are significant pathogens that pose a major threat to global agriculture, causing extensive crop losses and economic damage. Understanding the molecular biology of plant viruses is crucial for developing effective control strategies. Plant viruses are primarily composed of nucleic acids (RNA or DNA) encased in a protein coat, and they rely on host cellular machinery for replication and movement within the plant. Key molecular mechanisms include virus entry, replication, movement, and host-virus interactions, such as the manipulation of host gene expression and suppression of plant defense responses. Molecular techniques have greatly advanced our understanding of these processes. Techniques such as Reverse Transcription Polymerase Chain Reaction (RT-PCR), Next-Generation Sequencing (NGS), and CRISPR-Cas systems enable precise detection, identification, and characterization of plant viruses at the genetic level. These tools also facilitate the study of viral evolution and the development of resistance in plants. Control strategies for plant viruses are multifaceted. Breeding for virus-resistant cultivars is an approach which is often achieved through conventional breeding or genetic engineering. Transgenic plants expressing viral coat protein genes or RNA interference (RNAi) constructs have shown promise in conferring resistance. Additionally, cross-protection, where a mild strain of the virus is used to protect against a more virulent strain, is another effective method. Cultural practices, such as crop rotation, sanitation, and vector control, play essential roles in managing virus spread. Integrated Pest Management (IPM) strategies that combine biological, chemical, and cultural practices are vital for sustainable control.