Lipidome analysis of rotavirus-infected cells confirms the close interaction of lipid droplets with viroplasms.

Eleanor R Gaunt,Winsome Cheung,Ulrich Desselberger,Qifeng Zhang,Andrew M L Lever,Michael J O Wakelam

doi:10.1099/vir.0.049635-0

Eleanor R Gaunt, Winsome Cheung + Show 4 more

Open Access

https://doi.org/10.1099/vir.0.049635-0

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Abstract

Rotaviruses (RVs) cause acute gastroenteritis in infants and young children, and are globally distributed. Within the infected host cell, RVs establish replication complexes in viroplasms (‘viral factories’) to which lipid droplet organelles are recruited. To further understand this recently discovered phenomenon, the lipidomes of RV-infected and uninfected MA104 cells were investigated. Cell lysates were subjected to equilibrium ultracentrifugation through iodixanol gradients. Fourteen different classes of lipids were differentiated by mass spectrometry. The concentrations of virtually all lipids were elevated in RV-infected cells. Fractions of low density (1.11–1.15 g ml−1), in which peaks of the RV dsRNA genome and lipid droplet- and viroplasm-associated proteins were observed, contained increased amounts of lipids typically found concentrated in the cellular organelle lipid droplets, confirming the close interaction of lipid droplets with viroplasms. A decrease in the ratio of the amounts of surface to internal components of lipid droplets upon RV infection suggested that the lipid droplet–viroplasm complexes became enlarged.

Highlights

Rotaviruses (RVs) form a genus of the family Reoviridae and cause significant morbidity and mortality in infants and young children, in the developing world (Estes & Kapikian, 2007; Tate et al, 2012).RVs possess a genome of 11 segments of ds RNA
Lipids associated with the rotavirus viroplasm additional sets of paired samples were subsequently fractionated, including samples treated with anti-mitochondrial antibody
The results of these analyses are presented. Gradient fractions from the latter two experiments were tested for the presence of viral dsRNAs (Fig. 1), which were previously shown to co-fractionate with NSP2/NSP5 and with perilipin A (Cheung et al, 2010)

Summary

Introduction

Rotaviruses (RVs) form a genus of the family Reoviridae and cause significant morbidity and mortality in infants and young children, in the developing world (Estes & Kapikian, 2007; Tate et al, 2012).RVs possess a genome of 11 segments of ds RNA. RVs lose their outer protein layer, and the resulting double-layered particles (DLPs) immediately start to transcribe ssRNAs of positive polarity, which are released into the cytoplasm where they are translated. Two virusencoded non-structural proteins (NSPs), NSP2 and NSP5, are necessary for the formation of the cytoplasmic inclusion bodies known as ‘viroplasms’ (Fabbretti et al, 1999). Viral core particles containing proteins VP1, VP2, VP3 and viral RNAs are formed and viral RNA replication takes place. VP6 is acquired in the viroplasm leading to the formation of DLPs which are released into the cytoplasm. DLPs acquire the outer proteins VP7 and VP4 to become infectious triple-layered particles (TLPs)

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