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).

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).

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.

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).

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).

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).

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.

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.

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.