- Open Access
Cellular phosphoinositides and the maturation of bluetongue virus, a non-enveloped capsid virus
© Bhattacharya and Roy; licensee BioMed Central Ltd. 2013
- Received: 11 December 2012
- Accepted: 1 March 2013
- Published: 5 March 2013
Bluetongue virus (BTV), a member of Orbivirus genus in the Reoviridae family is a double capsid virus enclosing a genome of 10 double-stranded RNA segments. A non-structural protein of BTV, NS3, which is associated with cellular membranes and interacts with outer capsid proteins, has been shown to be involved in virus morphogenesis in infected cells. In addition, studies have also shown that during the later stages of virus infection NS3 behaves similarly to HIV protein Gag, an enveloped viral protein. Since Gag protein is known to interact with membrane lipid phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2] and one of the known binding partners of NS3, cellular protein p11 also interacts with annexin a PI(4,5)P2 interacting protein, this study was designed to understand the role of this negatively charged membrane lipid in BTV assembly and maturation.
Over expression of cellular enzymes that either depleted cells of PI(4,5)P2 or altered the distribution of PI(4,5)P2, were used to analyze the effect of the lipid on BTV maturation at different times post-infection. The production of mature virus particles was monitored by plaque assay. Microscopic techniques such as confocal microscopy and electron microscopy (EM) were also undertaken to study localization of virus proteins and virus particles in cells, respectively.
Initially, confocal microscopic analysis demonstrated that PI(4,5)P2 not only co-localized with NS3, but it also co-localized with VP5, one of the outer capsid proteins of BTV. Subsequently, experiments involving depletion of cellular PI(4,5)P2 or its relocation demonstrated an inhibitory effect on normal BTV maturation and it also led to a redistribution of BTV proteins within the cell. The data was supported further by EM visualization showing that modulation of PI(4,5)P2 in cells indeed resulted in less particle production.
This study to our knowledge, is the first report demonstrating involvement of PI(4,5)P2 in a non-enveloped virus assembly and release. As BTV does not have lipid envelope, this finding is unique for this group of viruses and it suggests that the maturation of capsid and enveloped viruses may be more closely related than previously thought.
Bluetongue virus (BTV), a vector-borne animal pathogen has recently emerged in Europe causing high mortality in sheep. BTV is prototype of Orbivirus genus of the Reoviridae family. Like other family members, BTV is a non-enveloped icosahedral particle and is composed of seven structural proteins (VP1-VP7) organized in two concentric capsids . BTV enters the cells via receptor-mediated endocytosis and the two outer capsid proteins, VP2 and VP5 are involved in cell attachment and membrane penetration [2–6]. Although the membrane penetration protein VP5 is non-glycosylated, structurally it resembles the glycosylated fusion proteins of enveloped viruses, such as HIV, herpesviruses, vesicular stomatitis virus and influenza virus . The inner capsid or “core,” is comprised of the remaining five proteins, two major (VP7 and VP3), three minor enzymatic (VP1, VP4, VP6) and a genome of ten double-stranded RNA (dsRNA) segments. In addition, BTV also synthesizes four non-structural proteins (NS1, NS2, NS3/NS3A, NS4) in infected cells, of which the small NS3 protein is glycosylated. Upon infection, the core particles become active, synthesizing ten capped single-stranded RNA transcripts (ssRNAs) which extrude through the capsid pores into the cytoplasm. The newly synthesized core components are recruited by NS2, triggering the formation of virus-specific inclusion bodies (VIBs), the site of the core assembly [8, 9]. The addition of newly synthesized VP2 and VP5 onto the cores does not occur within VIBs [8, 10]. Instead these two proteins appear to be associated with NS3, the only protein of BTV that is glycosylated. NS3 has been localized to intracellular organelles (Golgi complex and Endoplasmic reticulum), cellular membranes and is associated with virus release [11–14]. It also interacts with Tsg101 [13, 14], a component of multivesicular bodies (MVBs) and with cellular protein p11 that forms a complex with annexin 2 [15, 16], a member of the cellular exocytotic pathway. Although it has been demonstrated that NS3 localizes to cellular membranes, the cellular components responsible for targeting NS3 to the cellular membrane have not yet been defined. Annexin-2, a binding partner of p11 has been demonstrated to interact with Phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2], a negatively charged lipid molecule in cellular membranes [17–22]. It is known that PI(4,5)P2 also interacts with members of the SNARE (soluble N-ethylmaleimide sensitive fusion protein receptors) superfamily . Interestingly, while NS3 binds p11, the outer capsid protein VP5 possesses a SNARE domain  indicating that BTV NS3 and VP5 may use these cellular components during virus morphogenesis.
The membrane lipid PI(4,5)P2 belongs to a family of lipid molecules that is collectively known as phosphoinositides . These lipid molecules are generally inter-converted by specific cellular lipid phosphatases and kinases. While the level of PI(4,5)P2 in cells is maintained by phosphatases such as polyphosphoinositide 5-phosphatase (5ptaseIV), a cellular kinase, namely phosphatidylinositol-4-phosphate 5-kinase generates the majority of PI(4,5)P2 in cells. More importantly, this cellular kinase itself is regulated by a number of factors including the small G protein ADP-ribosylation factor 6 (Arf6) . It is known that the expression of a constitutively active form of Arf6, defective for GTP hydrolysis (Arf6/Q67L), alters the localization of cellular PI(4,5)P2 by inducing the formation of PI(4,5)P2-enriched endosomal structures [27, 28]. Since annexin-2 and SNARE domains interact with PI(4,5)P2, and BTV has been shown to use similar egress machinery to HIV [13, 14], this current study was undertaken to investigate whether the membrane lipid PI(4,5)P2 plays any role in BTV maturation and assembly as it does in HIV.
For this purpose we used a combination of molecular, biochemical and microscopic techniques to investigate the effect of PI(4,5)P2 on BTV maturation. We found that when the level of PI(4,5)P2 was reduced by over expression of 5ptaseIV, the virus titres were also decreased significantly. Furthermore, BTV growth was also affected when PI(4,5)P2 distribution was altered to form cellular vesicles using a plasmid that expresses an Arf6 mutant (Arf6/Q67L). The results obtained strongly suggest that PI(4,5)P2 plays a key role in localizing BTV to cellular membranes and promotes efficient virus production. This observation is the first demonstration of the importance of membrane lipids in the morphogenesis of a non-enveloped virus.
BTV proteins associate with PI(4,5)P2 in infected cells
BTV particle production is affected when cellular PI(4,5)P2 level was perturbed
Subsequently, to investigate whether the depletion of PI(4,5)P2 hinders virus assembly, the total virus titres of the post-transfected cells infected with BTV were determined at 4 and 12 hrs. When viral titres at each time point were plotted either as the relative percentages of the titres in infected cells that were not transfected but infected (Figure 3B), or as total titres (Figure 3C), the virus titres in the cells over expressing 5ptaseIV were significantly reduced at 12 hrs post-infection in both HeLa (p = 0.008 and 0.003 in Figure 3B and 3C right, respectively) and BSR (p = 0.003 and 0.001 in Figure 3B and 3C left, respectively) cells. In contrast the reduction in virus titres of infected cells expressing Δ1 mutant was not significant either when the titres were plotted as a relative percentage (Figure 3B) of infected but not transfected cells (p = 0.07 in both HeLa and BSR cells) or as total titres (p = 0.1 and 0.2 in HeLa and BSR cells, respectively) (Figure 3C). Thus, depletion of PI(4,5)P2 inhibits virus titres but does not interfere with virus protein production in infected cells at 12 hrs. In order to negate the deleterious effect of the transfection reagent on virus replication, cells treated with only transfection reagent were also infected with BTV. There was no noticeable difference in virus titres between the cells that were transfected with plasmids or transfection reagent (data not shown) prior to infection.
Changing the normal distribution of PI(4,5)P2 decreases virus particle production
The effect of Arf6/Q67L on virus yield was further investigated by infecting the Arf6/Q67L expressing cells and analyzing the total titres by plaque assay as described in Materials and Methods (Figure 6B and C). When the relative titres were compared to control cells (Figure 6B, HeLa, right, and BSR, left) that were infected but not transfected, cells expressing the dominant negative plasmid, Arf6/Q67L showed significant reduction in viral tires at 12 hrs (p < 0.0001 in HeLa and p = 0.0007 in BSR) post-infection, but not at 4 hrs (p = 0.01 in HeLa and p = 0.2 in BSR). A similar trend was also observed for total viral tires (Figure 6C) where the reduction at 12 hrs was more significant (p = 0.003 and 0.001 for HeLa and BSR cells, respectively) than 4 hrs (p > 0.005 for HeLa and BSR) post infection in the both cell types. Since, no viral proteins were expressed at 4 hrs post-infection, this suggested that the viral particles counted at this early time post infection were the input virus particles and not newly assembled ones. Although the percentage of decrease in relative virus titre was more in HeLa cells (90%) than BSR (70%), similar trends in decrease of relative virus titre confirmed that perturbation of cellular PI(4,5)P2 inhibits virus production. Thus, the formation of PI(4,5)P2 enriched vesicles inhibits virus production but does not interfere with virus protein production.
Although BTV is a non-enveloped virus, the outer capsid protein VP5 possesses fusogenic property  as well as structural similarity with the fusion proteins of enveloped viruses . In addition, VP5 also possesses a SNARE domain  that is very similar to SNARE domains of cellular proteins that have been shown to interact with PI(4,5)P2[35, 36]. In addition a second BTV protein, NS3 has some functional similarities with HIV Gag [13, 14, 16] and it also interacts with cellular annexin2 , which in turn, interacts with PI(4,5)P2 present in membranes [17–22]. These compelling findings pointed at PI(4,5)P2 as the common denominator in the interactions between a non-enveloped virus (BTV) and host cells. In the case of enveloped viruses such as HIV, where the virus assembly occurs on the cellular membranes [37–40], the basic domain of HIV matrix protein (MA) has been suggested to contribute to the membrane binding of Gag by interacting with acidic phospholipids on the cytoplasmic leaflet of membranes [31, 41, 42]. Furthermore, a recent report has also shown that the subcellular localization of Gag in MLV infected cells is also determined by PI(4,5)P2.
This study therefore focused on the effect of PI(4,5)P2 during virus maturation. On the basis of earlier studies that have successively used PH-GFP as a marker for PI(4,5)P2 in cells , the same lipid marker was also utilized to study the role of the negatively charged lipid in BTV maturation. The experiments undertaken on particle production and protein synthesis were limited up to 12 hrs post-infection as the first replication cycle of BTV infection is completed by 16 hrs post-infection. Additionally, in order to negate whether the effects of lipid is not restricted to one particular cell type, two different cell types were analyzed for the affect of lipid on BTV morphogenesis. The sequestration of VP5 and NS3 to PI(4,5)P2-enriched endosomal vesicles by Arf6/Q67L expression and a decrease in relative virus titre in the presence of Arf6/Q67L indicated that disruption in the distribution pattern of PI(4,5)P2 hampered virus particle production. In addition since depletion of PI(4,5)P2 prior to BTV infection also decreased particle production, the results presented here strongly suggest that PI(4,5)P2 plays an important role in the BTV life cycle. As neither depleting the level of PI(4,5)P2 nor altering its distribution disrupted the level of viral proteins, this confirmed that although PI(4,5)P2 does play an active role in virus assembly, it does not have any role in viral protein production. Since the early time point (i.e., 4 hrs) showed some virus titres but no viral protein production, this indicated that the titres were due to the presence of the input virus. EM sectioning of BTV infected cells exhibited the attachment of viral particles to the outer surface of vesicle-like structures that were absent in PI(4,5)P2 depleted cells. Moreover, EM analyses of cells expressing Arf6/Q67L also showed a decrease in virus production. Some studies have reported a co-relation between expression of 5-phosphate IV and apoptosis related decrease in cell viability. However, since our experiments involving confocal microscopy (results not shown) of the BSR and HeLa cells over expressing 5-phosphate IV have not shown any of the gross cellular morphological changes that are usually visible in apoptotic cells, the decrease in virus titer due to reduction of PI(4,5)P2 can therefore be attributed to the effect of the absence of the lipid in cells and not due to apoptosis induced by over expression of 5-phosphate IV in transfected cells. In addition, previous research has also confirmed that BTV actively induces apoptosis in infected mammalian cells that does not have any negative impact on virus particle production .
It is well established that PI(4,5)P2 plays an important role in the generation and trafficking of intra-cytoplasmic vesicles via the cytoskeletal tracks [45, 46]. Studies in polarized epithelial cells have revealed that many newly synthesized proteins in the Golgi network, that are destined for the apical surface, are segregated into membrane components rich in sphingolipids and cholesterol . In comparison, proteins destined for the basolateral surface are sorted into vesicles that are predominantly composed of glycerophospholipid. Cells that are not overtly polarized, like the fibroblasts, also deploy these two distinct pathways [48, 49]. A study comprising of influenza virus hemagglutinin (HA) established that vesicles containing HA were delivered from the Golgi to the cell surface as the infection progressed in the cells. Based on this, it can be hypothesized that disruption of PI(4,5)P2 in cells either by its depletion or altered distribution hampers the generation of the intracytoplasmic vesicles that might act as hubs for BTV assembly in infected cells. This notion can be substantiated by the fact that NS3 interacts with the two outer capsid proteins of BTV, VP2 and VP5 [15, 24] and it also plays an essential role in virus egress [13, 14].
The combined data obtained from various experiments in this study provide conclusive evidence of the importance of PI(4,5)P2 in BTV infection which, to our knowledge, is the first report demonstrating involvement of PI(4,5)P2 in a non-enveloped virus assembly and release. It is also possible that the effect of PI(4,5)P2 on BTV maturation might also be due to changes in its cellular regulation caused by virus infection or that PI(4,5)P2 might affect BTV particle production due to its effect on NS3 and VP5 by perturbing the levels of either annexin-2 or SNARE proteins, the cellular binding partners of the two viral proteins. Further studies will be necessary to clarify this. Based on our current data it can be hypothesized that common elements may underlie the pathways of virus maturation used by both enveloped and non-enveloped viruses alike.
The principal findings of this research is that PI(4,5)P2 influences BTV maturation. In addition this is a unique demonstration of an essential role for negatively charged membrane lipid molecules in the morphogenesis of BTV. It also suggested that the egress pathways of capsid and enveloped viruses may be more closely related than commonly supposed.
Cells and viruses
HeLa (human cervical epithelial) and BSR (a derivative of Baby Hamster Kidney cell) were maintained as described previously . The BSR cells were used to propagate the BTV serotype (BTV-1 SA) and to determine the viral titre by plaque assay. For time course studies of viral infection, HeLa and BSR cell monolayers were washed with FCS-free growth medium and infected with BTV at an MOI of 1. Virus adsorptions were carried out for 30 minutes at 4°C, followed by incubation at 37°C in growth medium supplemented with 2% FCS for 4 and 12 hrs.
Reagents, buffers and antibodies
Reagents required for protein interaction and confocal microscopy studies were obtained as described previously . Except for VP2 , all the antibodies used against BTV proteins were generated in our laboratory. While various antibodies used in this study against the cellular proteins have been described previously , the mouse monoclonal anti-myc (9E10), rabbit polyclonal anti-HA and mouse monoclonal anti PI(4,5)P2 were obtained from Abcam (Cambridge,UK), Santa Cruz Biotechnology (SantaCruz, USA) and Molecular Probes (USA), respectively.
Plasmids expressing 5ptaseIV and Δ1 mutant lacking the phosphatase signature domain were donated by P. Majerus (Washington University School of Medicine, St. Louis) and Eric Freed (National Cancer Institute, NIH, Frederick, Maryland) respectively. The PHGFP expression plasmids and HA-tagged Arf6/Q67L were donated by T. Balla (National Institute of Child Health and Human Development, NIH) and J. Donaldson (National Heart, Lung, and Blood Institute, NIH) respectively.
HeLa cells were seeded in 12 well plates, transfected when 70% confluent with Lipofectaminine 2000 (Invitrogen) according to the manufacturer’s recommendations and incubated for 12 hrs at 37°C. Subsequently they were infected with the virus, incubated for the various time points and then processed for titration assays, western blot or confocal microscopy assays as described below.
Cell extracts from BTV-infected and either transfected or treated with tranfecting reagent were collected, freeze thawed three times and virus titres were determined by plaque assays using BSR cells as described previously . The total viral titer was determined and normalized to the titer obtained for infected but untransfected cells. The mean and standard error of the reduction mediated by the inhibitor were calculated (Sigma Plot 2000; Systat Software Inc.).
SDS-PAGE separated proteins were transferred onto a Hybond enhanced chemiluminescence nitrocellulose membrane (GE Healthcare, Uppsala Sweden) and probed with appropriate antibodies. Subsequently, the blots were incubated with alkaline phosphatase conjugated secondary antibodies and developed with BCIP-NBT substrate (Sigma-Aldrich). The western blots were repeated 2 times on three independent experiments.
Mammalian cells were seeded in 24 well plates on 13-mm-diameter coverslips, transfected with the expression plasmids and infected with BTV. Subsequently the cells were processed for confocal microscopy as described previously . After analyzing with a Zeiss LSM 510 confocal microscope, the images were obtained using LSM 510 image browser software and processed using Photoshop Elements 2.0 software (Adobe).
Electron microscopy (EM)
HeLa cells were transfected with the expression plasmids followed by infection with virus and incubated for 12 hrs at 37°C. The cells were then processed for EM as described previously  and examined by a Hitachi H7000 electron microscope. The experiment was repeated twice and 3 different sections per independent experiment were analyzed for the distribution of virus particles. The statistical analysis of the virus particles were undertaken by Sigma Plot 2000 (Systat Software Inc.) and Excel (Microsoft).
We thank J. Donaldson (National Heart, Lung, and Blood Institute, NIH) and P. Majerus (Washington University School of Medicine, St. Louis), T. Balla (National Institute of Child Health and Human Development, NIH) and E. Freed (NIH, Frederick, Maryland) for providing plasmids. We also thank Maria McCrossan (LSHTM) for technical help with electron microscopy experiments and Theresa Ward (LSHTM) for valuable comments in the preparation of the manuscript. This work was funded by NIH, USA.
- Roy P, Noad R: Bluetongue virus assembly and morphogenesis. Curr Top Microbiol Immunol 2006, 309: 87-116. 10.1007/3-540-30773-7_4PubMedGoogle Scholar
- Eaton BT, Crameri GS: The site of bluetongue virus attachment to glycophorins from a number of animal erythrocytes. J Gen Virol 1989, 70: 3347-3353. 10.1099/0022-1317-70-12-3347PubMedView ArticleGoogle Scholar
- Hassan SH, Roy P: Expression and functional characterization of bluetongue virus VP2 protein: Role in cell entry. J Virol 1999, 73: 9832-9842.PubMedPubMed CentralGoogle Scholar
- Hassan SH, Wirblich C, Forzan M, Roy P: Expression and functional characterization of bluetongue virus VP5 protein: role in cellular permeabilization. J Virol 2001, 75: 8356-8367. 10.1128/JVI.75.18.8356-8367.2001PubMedPubMed CentralView ArticleGoogle Scholar
- Forzan M, Wirblich C, Roy P: A capsid protein of nonenveloped Bluetongue virus exhibits membrane fusion activity. PNAS 2004, 101: 2100-2105. 10.1073/pnas.0306448101PubMedPubMed CentralView ArticleGoogle Scholar
- Forzan M, Marsh M, Roy P: Bluetongue virus entry into cells. J Virol 2007, 81: 4819-4827. 10.1128/JVI.02284-06PubMedPubMed CentralView ArticleGoogle Scholar
- Zhang X, Boyce M, Bhattacharya B, Zhang X, Scheina S, Roy P, Zhou ZH: Bluetongue virus coat protein VP2 contains a sialic acid-binding domain and VP5 has similarities to enveloped virus fusion proteins. PNAS 2010, 107: 6292-6297. 10.1073/pnas.0913403107PubMedPubMed CentralView ArticleGoogle Scholar
- Modrof J, Lymperopoulos K, Roy P: Phosphorylation of Bluetongue Virus Nonstructural Protein 2 Is Essential for Formation of Viral Inclusion Bodies. J Virol 2005, 79: 10023-10031. 10.1128/JVI.79.15.10023-10031.2005PubMedPubMed CentralView ArticleGoogle Scholar
- Kar AK, Ghosh M, Roy P: Mapping the assembly of Bluetongue virus scaffolding protein VP3. Virology 2004, 324: 387-399. 10.1016/j.virol.2004.04.018PubMedView ArticleGoogle Scholar
- Kar AK, Bhattacharya B, Roy P: Bluetongue virus RNA binding protein NS2 is a modulator of viral replication and assembly. BMC Mol Biol 2007, 8: 4. 10.1186/1471-2199-8-4PubMedPubMed CentralView ArticleGoogle Scholar
- Wu X, Chen SY, Iwata H, Compans RW, Roy P: Multiple glycoproteins synthesized by the smallest RNA segment (S10) of bluetongue virus. J Virol 1992, 66: 7104-7112.PubMedPubMed CentralGoogle Scholar
- Hyatt AD, Zhao Y, Roy P: Release of bluetongue virus-like particles from insect cells is mediated by BTV nonstructural protein NS3/NS3A. Virology 1993, 193: 592-603. 10.1006/viro.1993.1167PubMedView ArticleGoogle Scholar
- Wirblich C, Bhattacharya B, Roy P: Nonstructural protein 3 of bluetongue virus assists virus release by recruiting ESCRT-I protein Tsg101. J Virol 2006, 80: 460-473. 10.1128/JVI.80.1.460-473.2006PubMedPubMed CentralView ArticleGoogle Scholar
- Celma CC, Roy P: A viral nonstructural protein regulates bluetongue virus trafficking and release. J Virol 2009, 83: 6806-6816. 10.1128/JVI.00263-09PubMedPubMed CentralView ArticleGoogle Scholar
- Beaton AR, Rodriguez J, Reddy YK, Roy P: The membrane trafficking protein calpactin forms a complex with bluetongue virus protein NS3 and mediates virus release. Proc Natl Acad Sci USA 2002, 99: 13154-13159. 10.1073/pnas.192432299PubMedPubMed CentralView ArticleGoogle Scholar
- Celma CC, Roy P: Interaction of calpactin light chain (S100A10/p11) and a viral NS protein is essential for intracellular trafficking of nonenveloped bluetongue virus. J Virol 2011, 85: 4783-4791. 10.1128/JVI.02352-10PubMedPubMed CentralView ArticleGoogle Scholar
- Ayala-Sanmartin J, Henry JP, Pradel LA: Cholesterol regulates membrane binding and aggregation by annexin 2 at submicromolar Ca(2+) concentration. Biochim Biophys Acta 2001, 1510: 18-28. 10.1016/S0005-2736(00)00262-5PubMedView ArticleGoogle Scholar
- Hayes MJ, Merrifield CJ, Shao D, Ayala-Sanmartin J, Schorey CD, Levine TP, Proust J, Curran J, Bailly M, Moss SE: Annexin A2 binding to phosphatidylinositol 4,5-bisphosphate on endocytic vesicles is regulated by the stress response pathway. J Biol Chem 2004, 279: 14157-14164. 10.1074/jbc.M313025200PubMedPubMed CentralView ArticleGoogle Scholar
- Rescher U, Ruhe D, Ludwig C, Zobiack N, Gerke V: Annexin 2 is a phosphatidylinositol (4,5)-bisphosphate binding protein recruited to actin assembly sites at cellular membranes. J Cell Sci 2004, 117: 3473-3480. 10.1242/jcs.01208PubMedView ArticleGoogle Scholar
- Chasserot-Golaz S, Vitale N, Umbrecht-Jenck E, Knight D, Gerke V, Bader MF: Annexin 2 promotes the formation of lipid microdomains required for calcium-regulated exocytosis of dense-core vesicles. Mol Biol Cell 2005, 16: 1108-1119. 10.1091/mbc.E04-07-0627PubMedPubMed CentralView ArticleGoogle Scholar
- Gokhale NA, Abraham A, Digman MA, Gratton E, Cho W: Phosphoinositide specificity of and mechanism of lipid domain formation by annexin A2-p11 heterotetramer. J Biol Chem 2005, 280: 42831-42840. 10.1074/jbc.M508129200PubMedView ArticleGoogle Scholar
- Volker G, Creutze CE, Moss SE: Annexins: linking Ca2+ signalling to membrane dynamics. Nat Rev Mol Cell Biol 2005, 6: 449-461. 10.1038/nrm1661Google Scholar
- Somanath S, Barg S, Marshall C, Silwood CJ, Turner MD: High extracellular glucose inhibits exocytosis through disruption of syntaxin 1A-containing lipid rafts. Biochem Biophys Res Commun 2009, 389: 241-246. 10.1016/j.bbrc.2009.08.126PubMedView ArticleGoogle Scholar
- Bhattacharya B, Roy P: Bluetongue virus outer capsid protein VP5 interacts with membrane lipid rafts via a SNARE domain. J Virol 2008, 27: 27.Google Scholar
- Simonsen A, Wurmser A, Emr SD, Stenmark H: The role of phosphoinositides in membrane transport. Curr Opin Cell Biol 2001, 13: 485-492. 10.1016/S0955-0674(00)00240-4PubMedView ArticleGoogle Scholar
- Donaldson JG: Multiple roles for Arf6: sorting, structuring, and signaling at the plasma membrane. J Biol Chem 2003, 278: 41573-41576. 10.1074/jbc.R300026200PubMedView ArticleGoogle Scholar
- Brown FD, Rozelle AL, Yin HL, Balla T, Donaldson JG: Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic. J Cell Biol 2001, 154: 1007-1017. 10.1083/jcb.200103107PubMedPubMed CentralView ArticleGoogle Scholar
- Aikawa Y, Martin TF: ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5)bisphosphate required for regulated exocytosis. J Cell Biol 2003, 162: 647-659. 10.1083/jcb.200212142PubMedPubMed CentralView ArticleGoogle Scholar
- Várnai P, Bala T: Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools. J Cell Biol 1998, 143: 501-510. 10.1083/jcb.143.2.501PubMedPubMed CentralView ArticleGoogle Scholar
- Kisseleva MV, Wilson MP, Majerus PW: The isolation and characterization of a cDNA encoding phospholipid-specific inositol polyphosphate 5-phosphatase. J Biol Chem 2000, 275: 20110-20116. 10.1074/jbc.M910119199PubMedView ArticleGoogle Scholar
- Ono A, Ablan SD, Lockett SJ, Nagashima K, Freed EO: Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane. PNAS 2004, 101: 14889-14894. 10.1073/pnas.0405596101PubMedPubMed CentralView ArticleGoogle Scholar
- Kar AK, Iwatani N, Roy P: Assembly and intracellular localization of the bluetongue virus core protein VP3. J Virol 2005, 79: 11487-11495. 10.1128/JVI.79.17.11487-11495.2005PubMedPubMed CentralView ArticleGoogle Scholar
- Kisseleva MV, Cao L, Majerus PW: Phosphoinositide-specific inositol polyphosphate 5-phosphatase IV inhibits Akt/Protein Kinase B phosphorylation and leads to apoptotic cell death. J Biol Chem 2002, 277: 6266-6272. 10.1074/jbc.M105969200PubMedView ArticleGoogle Scholar
- Honda A, Nogami M, Yokozeki T, Yamazaki M, Nakamura H, Watanabe H, Kawamoto K, Nakayama K, Morris AJ, Frohman MA, Kanaho Y: Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation. Cell Biol 1999, 99: 521-532.Google Scholar
- Holz RW, Hlubek M, Sorensen SD, Fisher SK, Balla T, Ozaki S, Prestwich GD, Stuenkel EL, Bittner MA: A pleckstrin homology domain specific for phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) and fused to green fluorescent protein identifies plasma membrane PtdIns-4,5-P2 as being important in exocytosis. J Biol Chem 2000, 275: 17878-17885. 10.1074/jbc.M000925200PubMedView ArticleGoogle Scholar
- Bai J, Tucker WC, Chapman ER: PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane. Nat Struct Mol Biol 2004, 11: 36-44. 10.1038/nsmb709PubMedView ArticleGoogle Scholar
- Raposo G, Moore M, Innes D, Leijendekker R, Leigh-Brown A, Benaroch P, Geuze H: Human macrophages accumulate HIV-1 particles in MHC II compartments. Traffic 2002, 3: 718-729. 10.1034/j.1600-0854.2002.31004.xPubMedView ArticleGoogle Scholar
- Pelchen-Matthews A, Kramer B, Marsh M: Infectious HIV-1 assembles in late endosomes in primary macrophages. J Cell Biol 2003, 162: 443-455. 10.1083/jcb.200304008PubMedPubMed CentralView ArticleGoogle Scholar
- Nydegger S, Foti M, Derdowski A, Spearman P, Thali M: HIV-1 egress is gated through late endosomal membranes. Traffic 2003, 4: 902-910. 10.1046/j.1600-0854.2003.00145.xPubMedView ArticleGoogle Scholar
- Sherer NM, Lehmann MJ, Jimenez-Soto LF, Ingmundson A, Horner SM, Cicchetti G, Allen PG, Pypaert M, Cunningham JM, Mothes W: Visualization of retroviral replication in living cells reveals budding into multivesicular bodies. Traffic 2003, 4: 785-801. 10.1034/j.1600-0854.2003.00135.xPubMedView ArticleGoogle Scholar
- Zhou W, Parent LJ, Wills JW, Resh MD: Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids. J Virol 1994, 68: 2556-2569.PubMedPubMed CentralGoogle Scholar
- Chukkapalli V, Hogue IB, Boyko V, Hu WS, Ono A: Interaction between the human immunodeficiency virus type 1 gag matrix domain and phosphatidylinositol-(4,5)-bisphosphate is essential for efficient gag membrane binding. J Virol 2008, 82: 2405-2417. 10.1128/JVI.01614-07PubMedPubMed CentralView ArticleGoogle Scholar
- Hamard-Peron E, Juillard F, Saad JS, Roy C, Roingeard P, Summers MF, Darlix JL, Picart C, Muriaux D: Targeting of murine leukemia virus gag to the plasma membrane is mediated by PI(4,5)P2/PS and a polybasic region in the matrix. J Virol 2010, 84: 503-515. 10.1128/JVI.01134-09PubMedPubMed CentralView ArticleGoogle Scholar
- Stewart ME, Roy P: Role of cellular caspases, nuclear factor-kappa B and interferon regulatory factors in Bluetongue virus infection and cell fate. Virol J 2010, 7: 362. 10.1186/1743-422X-7-362PubMedPubMed CentralView ArticleGoogle Scholar
- Kanzaki M, Furukawa M, Raab W, Pessin JE: Phosphatidylinositol 4,5-bisphosphate regulates adipocyte actin dynamics and GLUT4 vesicle recycling. J Biol Chem 2004, 279: 30622-30633. 10.1074/jbc.M401443200PubMedView ArticleGoogle Scholar
- Cremona O, Di Paolo G, Wenk MR, Lüthi A, Kim WT, Takei K, Daniell L, Nemoto Y, Shears SB, Flavell RA, McCormick DA, De Camilli P: Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell 1999, 99: 179-188. 10.1016/S0092-8674(00)81649-9PubMedView ArticleGoogle Scholar
- Harder TS, Simons K: Caveolae, DIGs and the dynamics of sphingolipid-cholesterol microdomains. Curr Opin Cell Biol 1997, 9: 534-542. 10.1016/S0955-0674(97)80030-0PubMedView ArticleGoogle Scholar
- Yoshimori T, Keller P, Roth MG, Simons K: Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells. J Cell Biol 1996, 133: 247-256. 10.1083/jcb.133.2.247PubMedView ArticleGoogle Scholar
- Harder T, Scheiffele P, Verkade P, Simons K: Lipid domain structure of the plasma membrane revealed by patching of membrane components. J Cell Biol 1998, 141: 929-942. 10.1083/jcb.141.4.929PubMedPubMed CentralView ArticleGoogle Scholar
- DeMaula CD, Heidner HW, Rossitto PV, Pierce CM, MacLachlan NJ: Neutralization determinants of United States bluetongue virus serotype 10. Virology 1993, 195: 292-296. 10.1006/viro.1993.1377PubMedView ArticleGoogle Scholar
- Bhattacharya B, Roy P: Role of lipids on entry and exit of bluetongue virus, a complex non-enveloped virus. Viruses 2010, 2: 1218-1235. 10.3390/v2051218PubMedPubMed CentralView ArticleGoogle Scholar
- Bhattacharya B, Noad RJ, Roy P: Interaction between Bluetongue virus outer capsid protein VP2 and vimentin is necessary for virus egress. Virol J 2007, 4: 7. 10.1186/1743-422X-4-7PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.