Rotavirus Replication: the Role of Lipid Droplets


Chapter 2.5

Rotavirus Replication: the Role of Lipid Droplets



W. Cheung

E. Gaunt

A. Lever

U. Desselberger    Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, United Kingdom


Abstract


As is the case for all viruses, their replication depends on the interaction of viral components with cellular organelles and proteins. Here the role of the cellular organelles lipid droplets for rotavirus replication is reviewed. Newly formed rotavirus viroplasms interact with lipid droplets during the replication cycle, as shown by confocal microscopy, fluorescence resonance energy transfer, equilibrium ultracentrifugation of rotavirus-infected cell extracts, and lipid analyses. Disturbance of the cellular lipid droplet homoeostasis with chemical compounds inducing lipolysis or blockage of fatty acid biosynthesis was shown to reduce the number and size of viroplasms, the amount of newly synthesized rotavirus dsRNA and the infectivity of viral progeny. Thus, rotavirus has joined the growing list of viruses interacting with lipid droplets during their replication, opening a new area for search of antivirals.



Keywords


rotavirus replication

viroplasm

lipid droplets

lipid metabolism

cellular proteins


1. Introduction


Rotaviruses (RVs), a genus of the Reoviridae family, are a major cause of acute gastroenteritis (AGE) in infants and young children and in the young of a large variety of other mammalian and avian species. The genome of Rotavirus consists of 11 segments of double-stranded (ds) RNA, which encode 6 structural (VP) and 5–6 nonstructural (NSP) proteins. Upon entry into gut epithelial cells, the infectious, triple-layered RV particles (TLPs) are processed to become double-layered particles (DLPs), which actively transcribe single-stranded (ss) RNAs of (+) polarity from all 11 genomic segments to be released into the cytoplasm. These RNAs act either as mRNAs and are translated into viral proteins, or they become templates for progeny genomes and are replicated into dsRNAs to be packaged into newly formed virion particles. Early virion morphogenesis and viral RNA replication occur in cytoplasmic inclusion bodies termed “viroplasms”. DLPs released from viroplasms “mature” to TLPs in the endoplasmic reticulum (ER) before they are released from cells, either by lysis or a budding process (for details see Estes and Greenberg, 2013). The RV nonstructural proteins NSP2 and NSP5 are essential components of viroplasms. The transfection of NSP2- and NSP5-expressing plasmids into uninfected cells leads to the formation of viroplasm-like structures (VLS) (Fabbretti et al., 1999). Disruption of NSP2 or NSP5 synthesis in RV-infected cells by specific siRNAs, intrabodies or the use of NSP2- or NSP5-specific temperature sensitive mutants at the nonpermissive temperature abolishes viroplasm formation and the production of infectious viral progeny (Silvestri et al., 2004; Vascotto et al., 2004; Campagna et al., 2005).

Throughout its replication cycle, RV depends on and interacts with various components of the host cell. Here the interaction of the RV viroplasms with the cellular organelles lipid droplets is described.

2. Viroplasms


RV-encoded NSP2 and NSP5 are essential components of viroplasms, which also contain several other viral proteins including VP1, VP2, VP3, VP6, and NSP4. NSP2 forms octamers (Taraporewala et al., 2002); grooves in the NSP2 octamer are binding sites for which NSP5 dimers and ssRNAs compete, possibly regulating the balance between RV RNA replication and translation (Jiang et al., 2006). In addition NSP2 interacts with tubulin (Martin et al., 2010; Criglar et al., 2014) and induces microtubule depolymerization and stabilization by acetylation (Eichwald et al., 2012). Cellular components of the autophagy pathway are also involved in viroplasm formation and RV replication (Berkova et al., 2006; Berkova et al., 2007; Contin et al., 2011; López et al., 2011; Crawford et al., 2012; Arnoldi et al., 2014).

3. Lipid droplets


Lipid droplets (LDs) are the principal cellular storage sites of triacylglycerols and sterol esters (Herker and Ott, 2012) and are surrounded by a phospholipid monolayer into which numerous proteins including perilipin A and adipose differentiation-related protein (ADRP) are inserted (Martin and Parton, 2006; Walther and Farese, 2012; Brasaemle and Wolins, 2012). LDs are dynamic organelles with functions in the cell other than energy storage including protein trafficking and interaction with the other cellular components such as mitochondria, lysosomes, and the plasma membrane (Heaton and Randall, 2011; Saka and Valdivia, 2012). Examination of cellular components associated with viroplasms revealed proteins characteristic of cellular LDs.

4. Interaction of viroplasms with lipid droplets


Viroplasms interact with and possibly trigger the formation of LDs, inducing and recruiting them during the replication cycle (Cheung et al., 2010; Saxena et al., 2015). The initial discovery of the interaction of RV viroplasms with LDs was based on the observation, in RV-infected MA104 and Caco-2 cells, of colocalization of viroplasm-associated viral proteins (eg, NSP2 and NSP5) with cellular proteins typically found inserted into the LD-surface phospholipid monolayer (perilipin A and ADRP) (Cheung et al., 2010; Cheung, 2010; Fig. 2.5.1). LDs were also found to colocalize with VLS (Fabbretti et al., 1999) in uninfected cells (Cheung et al., 2010), indicating that complexes of coexpressed NSP2 and NSP5 are sufficient to interact with LDs. In addition, MA104 cells constitutively expressing the fusion protein NSP5-EGFP and infected with RV showed—as expected—concentration of NSP5-EGFP in viroplasms with close spatial proximity to LD-associated proteins as evidenced by fluorescence resonance energy transfer (FRET) (Cheung et al., 2010).

image

Figure 2.5.1 NSP2 colocalizes with lipid droplet-associated proteins perilipin A and ADRP in viroplasms of rotavirus-infected Caco-2 cells.
Confocal images of rotavirus-infected Caco-2 cells at 8 h p.i. Viroplasms were detected with anti-NSP2 antibodies followed by visualization with Alexa Fluor 488 (green)-labeled species-specific secondary antibody, while LD-associated proteins were detected with anti-perilipin A and anti-ADRP antibodies followed by reaction with Alexa Fluor 633 (red)-labeled species-specific secondary antibody. Scale bar: 10 μm. (Source: From Cheung W, PhD Thesis, University of Cambridge, Cambridge, 2010.)

LD recruitment (measured by perilipin A localization) was found to depend on viroplasm formation in rotavirus-infected cells. MA104 cells pretreated with siRNAs targeting NSP5 mRNAs [of either the porcine OSU (siOSU) or the simian SA11 (siSA11) strains] (Campagna et al., 2005) were infected with the OSU rotavirus strain for 6 h, and the localization of NSP5 and perilipin A was investigated using immunofluorescence and confocal microscopy. As shown in Fig. 2.5.2, siOSU largely blocked both, viroplasm formation and perilipin A recruitment, in comparison to cells treated with an irrelevant siRNA (siSA11) (Cheung, 2010). The yield of infectious progeny from the siOSU treated cells was 100 times lower than that of the siSA11-treated or the untreated cells (data not shown) (Cheung, 2010).

image

Figure 2.5.2 Inhibition of viroplasm formation by NSP5-specific siRNA in RV-infected cells affects recruitment of perilipin A and by implication of LDs.
MA104 cells pretreated with siRNAs directed against NSP5 of RV strains OSU (panel A) or SA11 (panel B) were infected with the OSU RV strain at a m.o.i. of 5 and fixed at 6 h p.i., followed by staining with NSP5- (green) and perilipin A- (red) specific antibodies (as indicated in Legend of Figure 2.5.1). siRNA directed against the OSU strain inhibits viroplasm formation in most cells (panel A), while siRNA directly against the SA11 strain has not effect (panel B). Untreated, infected (panel C) and uninfected (panel D) cells served as controls. Scale bar: 20 μm. (Source: From Cheung W, PhD Thesis, University of Cambridge, Cambridge, 2010)

RV-infected cells contain significantly higher concentrations of lipids than do uninfected cells (Kim and Chang, 2011; Gaunt et al., 2013b). The link between viroplasms and LDs was further strengthened by equilibrium ultracentrifugation through iodixanol gradients of RV-infected cell extracts (detergent-free): viral dsRNA sedimented in the same low-density fractions (1.11–1.15 g/mL) as NSP5 and perilipin A, markers of viroplasms and LDs, respectively. By contrast, purified RV DLPs spiked into uninfected cell extracts passed through the gradient without localization in the low density fractions (Cheung et al., 2010). Lipid components preferentially found in LDs (triacylglycerol, ceramide, sphingomyelin, phosphatidylinositol, phosphatidic acid) were also concentrated in the low-density fractions, further supporting the physical interaction between viroplasms and LDs (Gaunt et al., 2013b).

5. Lipid droplet homoeostasis and rotavirus replication


The association between RV “factories” and the cellular energy reservoirs prompted exploration of whether disturbance of the homoeostasis of cellular lipid metabolism might also affect RV replication. Treatment of cells with a combination of isoproterenol, a beta-adrenergic agonist, and isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, raises the level of intracellular cAMP, leading to phosphorylation of hormone-dependent lipase, which catalyses fragmentation of LDs (lipolysis) (Gross et al., 2006; Marcinkiewicz et al., 2006). LD formation can also be inhibited by antagonists of various enzymes involved in fatty acid synthesis. Triacsin C [N-(((2E,4E,7E)-undeca-2,4,7-trienylidene) amino) nitrous amide] is a specific inhibitor of long chain acyl coenzyme A synthetases (Igal et al., 1997; Namatame et al., 1999) and prevents LD formation in Huh7 cells (Zou et al., 2010); C75 (tetrahydro-4-methylene-2R-octyl-5-oxo-3S-furancarboxylic acid) is an inhibitor of the fatty acid synthase (FAS) complex and significantly reduces LD accumulation (Schmid et al., 2005); TOFA [5-(tetradecyloxy)-2-furoic acid] inhibits the enzyme acetyl-CoA carboxylase 1 (ACC1), acting early in the fatty acid biosynthesis pathway (Parker et al., 1977; Halvorson and McCune, 1984; Fukuda and Ontko, 1984).

Pretreatment of cells with nontoxic concentrations of all of these: a combination of [isoproterenol + IBMX], or triacsin C, or TOFA alone reduced the number and size of viroplasms generated by a subsequent RV infection (data not shown). The amounts of newly synthesized RV dsRNA declined by 3.8- to 5.9-fold, and the infectivity of viral progeny by 20- to 50-fold (Table 2.5.1) (Cheung et al., 2010; Gaunt et al., 2013a). Interestingly, treatment appeared to protect cells from RV-induced cytopathicity since a higher percentage of infected, drug-treated cells was viable at 16 h p.i. compared to those infected but not treated (Cheung et al., 2010). TOFA still had an inhibitory effect when added to RV-infected cells as late as at 4 h after infection. Specific siRNA knockdown of ACC1 (verified by Western blot) produced a qualitatively similar effect to chemical ACC1 inhibition (Gaunt et al., 2013a). Triacsin C and analogs were also found to inhibit RV replication by Kim et al. (2012). Inhibition of RV replication by C75 was only marginal, most likely due to the low chemotherapeutic index (Gaunt et al., 2013a). More recently it was shown that inhibitors of lipolysis and of transport of fatty acids to mitochondria decreased RV replication by 94–97% (Crawford et al., 2013), but the mechanism of action remains to be fully understood.


Table 2.5.1


Comparison of Inhibitory Effects of Different Compounds Affecting Lipid Droplet Homoeostasis on Rotavirus Replication



















































Treatment of cells Viral dsRNA Infectivity of progeny


Relative valuesa Diffb log TCID50/mLc Diffb
Isoproterenol 1.00
8.2
+ IBMXd + 0.25 4.0-fold 6.5 50-fold
Triacsin Cd 1.00
7.5

+ 0.26 3.8-fold 6.2 20-fold
TOFAe 1.00
8.4

+ 0.17 5.9-fold 6.7 50-fold



a Calculated from densitometric values of RNA gels (Cheung, 2010)


b Underlining indicates statistical difference.


c S.E. values not shown


d From: Cheung et al., J. Virol. 2010; 84: 6782–6798.


e From: Gaunt et al., J. Gen. Virol. 2013a: 94: 1310–1317.


Interestingly, the decrease of RV RNA production in TOFA-treated cells was disproportionately smaller than the decrease in infectivity (Gaunt et al., 2013a). This effect was analysed further by CsCl gradient ultracentrifugation of RVs (Arnoldi et al., 2007) synthesized after infection of untreated or TOFA-treated cells. TOFA treatment led to a 2-fold reduction in double-layered particles (DLPs) but a 20-fold reduction in triple-layered particles (TLPs, the infectious virions), compared to untreated cells (Table 2.5.2). These data were confirmed by electrophoresis of DLPs and TLPs on nondenaturing agarose gels and subsequent densitometry; the infectivity of RV progeny from the TOFA-treated cells was also decreased by 10-fold (data not shown). The results suggest that TOFA treatment, apart from interfering with DLP synthesis in viroplasms, may also affect the lipid composition of the endoplasmic reticulum (ER) where RV particle maturation from DLPs to TLPs occurs.


Table 2.5.2


Effect of TOFA on the Production of Rotavirus DLPs and TLPs ug/mL Protein Ratio TOFA/Untr

































ug/mL protein Ratio TOFA/Untr
TOFA Untr
DLP TLP DLP TLP DLP TLP
7.6 1.2 21.9 18.2 0.48 0.05
±2.6a ±1.1 ±13.1 ±10.4 ± 0.19 ±0.03



TOFA, [5-(Tetradecyloxy)-2-furoic acid]; DLP, double-layered particles; TLP, triple-layered RV particles


a Arithmetic mean ± standard error (N = 2)


Farnesoid X receptor (FXR) and its natural ligands bile acids [such as chenodeoxycholic acid (CDCA)] play major roles in cholesterol and lipid homeostasis (Makishima et al., 1999; Parks et al., 1999; Trauner and Boyer, 2003; Watanabe et al., 2004). Treatment of MA104 cells with CDCA, deoxycholic acid (DCA), and other FXR agonists led to a reduction of cellular triglyceride content (while RV infection, as mentioned, increased it); after RV infection, this treatment was correlated with a significant reduction of viral replication in a dose-dependent manner, with downregulation of cellular lipids as a possible contributing factor. In a mouse model of RV infection, oral administration of CDCA significantly reduced fecal RV shedding (Kim and Chang, 2011), suggesting that FXR agonists could become a treatment option against RV disease. However, more extensive experiments with animal models of RV infection are required to pursue this idea.

Infection of HT29 cells with RV in the presence of nontoxic concentrations of stilbenoids, cannabinoid receptor antagonists, led to a 10- to 20-fold decrease of the infectivity of viral progeny; one of the suggested mechanisms of action was interference with lipid homoeostasis, since stilbenoids are very lipophilic (Ball et al., 2015). However, more work is needed to explore this interesting observation in more detail.

6. Lipid droplets, lipid homoeostasis and replication of viruses and other microbes


Rotaviruses have joined the growing list of viruses and microbes that interact with LDs during their replication and depend on cellular lipid homoeostasis, including hepatitis C virus (Miyanari et al., 2007; Boulant et al., 2007; Shavinskaya et al., 2007; Salloum et al., 2013; Paul et al., 2014; Liefhebber et al., 2014; Shahidi et al., 2014; Filipe et al., 2015), dengue virus (Samsa et al., 2009; Jain et al., 2014; Soto-Acosta et al., 2014), GB virus B (Hope et al., 2002), bunyavirus (Wu et al., 2014) and the intracellular parasites Chlamydia (Kumar et al., 2006), Mycobacterium tuberculosis (Daniel et al., 2011) and Mycobacterium leprae (Mattos et al., 2011). On a wider scale, FA biosynthesis has been recently recognized as being essential for the replication of a larger number of viruses such as enteroviruses, West Nile virus, human cytomegalovirus, Kaposi sarcoma-associated herpes virus and Epstein Barr virus (Chukkapalli et al., 2012).

7. Future work on lipid droplets and rotavirus replication


Future work should be aimed at identifying which genes/proteins involved in LD homoeostasis (Guo et al., 2008) are important for the interaction of LDs with viroplasms and what is the basis for the observed inhibition of TLP compared to DLP production in TOFA-treated cells. Most importantly, the in vivo effects of (isoproterenol + IBMX) and TOFA should be explored in animal models of RV infection.

8. Other cellular proteins involved in rotavirus replication


Throughout its replication cycle RV depends on the interaction with many different cellular proteins. The RV surface proteins VP4 (VP8*) and VP7 interact with various cellular receptor molecules and undergo structural alterations (López and Arias, 2004; Hu et al., 2012; Díaz-Salinas et al., 2014; Abdelhakim et al., 2014) as described in detail in Chapters 2.1 and 2.2.

For viral translation the N terminus of the RV-encoded NSP3 interacts with the 3′ terminus of viral (+) ssRNA and the C terminus of NSP3 with the translation factor eIF4G which in turn interacts with the 5′ terminus of the mRNA, leading to RNA circularization (Groft and Burley, 2002). More importantly, NSP3 can displace the cellular poly A binding protein (PABP) from cellular mRNAs and thus prevent their translation (Piron et al., 1998; Montero et al., 2006; Harb et al., 2008; Rubio et al., 2013). Interestingly, after transfection into susceptible cells full length RV (+) ssRNAs are translated very poorly (or not at all), despite them being functional in in vitro translation systems. This suggests that viral mRNAs, after being extruded from DLPs into the cytoplasm, encounter ribosomes immediately and are translated without delay (Richards et al., 2013).

The cytoskeleton protein actin interacts with RV VP4 to be remodeled into actin bodies (Gardet et al., 2006; Gardet et al. 2007). Since actin rearrangements are Ca2+ dependent, they are also regulated by RV NSP4 (Berkova et al., 2007). NSP4 is a RV-encoded transmembrane glycoprotein which acts as a viroporin (Hyser et al., 2010, 2012) and also with the autophagy pathway (Crawford et al., 2012; Crawford and Estes, 2013). The cellular ubiquitin-proteasome system (UPS) plays a significant role in the replication of many viruses (Isaacson and Ploegh, 2009). For RVs it has been shown that UPS inhibition reduces recruitment of viral proteins to viroplasms and viral RNA replication (Contin et al., 2011; López et al., 2011; Crawford et al., 2012). These and other activities of NSP4 are described in detail in Chapter 2.4.

Rotavirus infection activates different cascades of the cellular innate immune response (IIR) (Sen et al., 2011; Sen et al., 2012; Angel et al., 2012). The viral NSP1 is an IIR antagonist (Barro and Patton, 2005; Barro and Patton, 2007; Arnold et al., 2013). The details of the virus–host relationship regarding the IIR are presented in Chapter 2.8.

9. Conclusions


Rotavirus replication in cells depends on an intense interplay of viral and cellular factors throughout the viral growth cycle. Here, details of the interaction of viroplasms with lipid droplets have been presented, including the demonstration that interference with lipid droplet homoeostasis reduces the amounts of synthesized rotavirus significantly. These observations may provide intriguing new therapeutic approaches to combat this ubiquitous pathogen.


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Apr 25, 2018 | Posted by in MICROBIOLOGY | Comments Off on Rotavirus Replication: the Role of Lipid Droplets
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