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.
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