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

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.

Inhibition of rotavirus replication by downregulation of fatty acid synthesis.

Recently the recruitment of lipid droplets (LDs) to sites of rotavirus (RV) replication was reported. LDs are polymorphic organelles that store triacylglycerols, cholesterol and cholesterol esters. The neutral fats are derived from palmitoyl-CoA, synthesized via the fatty acid biosynthetic pathway. RV-infected cells were treated with chemical inhibitors of the fatty acid biosynthetic pathway, and the effects on viral replication kinetics were assessed. Treatment with compound C75, an inhibitor of the fatty acid synthase enzyme complex (FASN), reduced RV infectivity 3.2-fold (P = 0.07) and modestly reduced viral RNA synthesis (1.2-fold). Acting earlier in the fatty acid synthesis pathway, TOFA [5-(Tetradecyloxy)-2-furoic acid] inhibits the enzyme acetyl-CoA carboxylase 1 (ACC1). TOFA reduced the infectivity of progeny RV 31-fold and viral RNA production 6-fold. The effect of TOFA on RV infectivity and RNA replication was dose-dependent, and infectivity was reduced by administering TOFA up to 4 h post-infection. Co-treatment of RV-infected cells with C75 and TOFA synergistically reduced viral infectivity. Knockdown by siRNA of FASN and ACC1 produced findings similar to those observed by inhibiting these proteins with the chemical compounds. Inhibition of fatty acid synthesis using a range of approaches uniformly had a more marked impact on viral infectivity than on viral RNA yield, inferring a role for LDs in virus assembly and/or egress. Specific inhibitors of fatty acid metabolism may help pinpoint the critical structural and biochemical features of LDs that are essential for RV replication, and facilitate the development of antiviral therapies.

Rotaviruses associate with cellular lipid droplet components to replicate in viroplasms, and compounds disrupting or blocking lipid droplets inhibit viroplasm formation and viral replication.

Rotaviruses are a major cause of acute gastroenteritis in children worldwide. Early stages of rotavirus assembly in infected cells occur in viroplasms. Confocal microscopy demonstrated that viroplasms associate with lipids and proteins (perilipin A, ADRP) characteristic of lipid droplets (LDs). LD-associated proteins were also found to colocalize with viroplasms containing a rotaviral NSP5-enhanced green fluorescent protein (EGFP) fusion protein and with viroplasm-like structures in uninfected cells coexpressing viral NSP2 and NSP5. Close spatial proximity of NSP5-EGFP and cellular perilipin A was confirmed by fluorescence resonance energy transfer. Viroplasms appear to recruit LD components during the time course of rotavirus infection. NSP5-specific siRNA blocked association of perilipin A with NSP5 in viroplasms. Viral double-stranded RNA (dsRNA), NSP5, and perilipin A cosedimented in low-density gradient fractions of rotavirus-infected cell extracts. Chemical compounds interfering with LD formation (isoproterenol plus isobutylmethylxanthine; triacsin C) decreased the number of viroplasms and inhibited dsRNA replication and the production of infectious progeny virus; this effect correlated with significant protection of cells from virus-associated cytopathicity. Rotaviruses represent a genus of another virus family utilizing LD components for replication, pointing at novel therapeutic targets for these pathogens.

Rotaviruses and rotavirus vaccines.

BACKGROUND: Rotaviruses (RVs) are an important cause of acute gastroenteritis in infants and young children worldwide, resulting in more than 600 000 deaths per annum, mainly in developing countries. Since the 1980s, there has been intensive research on the development of RV vaccine candidates, and since 2006 two vaccines have been licensed in many countries. SOURCES OF DATA: The scientific literature since the 1970s has been consulted, and the results of original research carried out in authors' laboratories were used. AREAS OF AGREEMENT: There are firmly established data on virus particle structure, genome composition, gene-protein assignment, protein-function assignment (incomplete), virus classification, the mechanisms of several steps of the replication cycle (adsorption, primary transcription, virus maturation-all partial), several mechanisms of pathogenesis, aspects of the immune response, diagnosis, illness and treatment, epidemiology and vaccine development. AREAS OF CONTROVERSY: Research on the following areas is still in full flux and in part not generally accepted: several steps of the replication cycle (mechanism of viral entry into host cells, mechanisms of packaging and reassortment of viral RNAs, morphogenesis of subviral particles in viroplasms and maturation of virus particles in the rough endoplasmic reticulum (RER) with temporary acquisition and subsequent loss of an envelope), the true correlates of protection and the long-term effectiveness of RV vaccines. GROWING RESEARCH: Recently, a system that allows carrying out reverse genetics with some of the RV genes has been established which, however, has limitations. There is intensive research ongoing, which is trying to develop better and universally applicable reverse genetics systems. There is broad research on the molecular mechanisms of the immune response and on which immunological parameter correlates best with lasting protection from severe RV disease. Research into other than live attenuated vaccines is growing. AREAS TIMELY FOR DEVELOPING RESEARCH: The establishment of better reverse genetics systems for RVs is the most important research goal for both the understanding of the molecular biology of RVs and the development of new and safe RV vaccines. The black boxes of our knowledge on aspects of RV replication (RNA packaging, RNA replication, control of reassortment and functions of the non-structural RV proteins) are under intensive research.

The secondary structure of the 5' end of the FIV genome reveals a long-range interaction between R/U5 and gag sequences, and a large, stable stem-loop.

Feline immunodeficiency virus (FIV) is a lentivirus that infects cats and is related to human immunodeficiency virus (HIV). Although it is a common worldwide infection, and has potential uses as a human gene therapy vector and as a nonprimate model for HIV infection, little detail is known of the viral life cycle. Previous experiments have shown that its packaging signal includes two or more regions within the first 511 nucleotides of the genomic RNA. We have undertaken a secondary structural analysis of this RNA by minimal free-energy structural prediction, biochemical mapping, and phylogenetic analysis, and show that it contains five conserved stem-loops and a conserved long-range interaction between heptanucleotide sequences 5'-CCCUGUC-3' in R/U5 and 5'-GACAGGG-3' in gag. This long-range interaction is similar to that seen in primate lentiviruses where it is thought to be functionally important. Along with strains that infect domestic cats, this heptanucleotide interaction can also occur in species-specific FIV strains that infect pumas, lions, and Pallas' cats where the heptanucleotide sequences involved vary. We have analyzed spliced and genomic FIV RNAs and see little structural change or sequence conservation within single-stranded regions of the 5' UTR that are important for viral packaging, suggesting that FIV may employ a cotranslational packaging mechanism.