Imaging poliovirus entry in live cells

Viruses initiate infection by transferring their genetic material across a cellular membrane and into the appropriate compartment of the cell. The mechanisms by which animal viruses, especially nonenveloped viruses, deliver their genomes are only poorly understood. This is due in part to technical difficulties involved in direct visualization of viral gene delivery and to uncertainties in distinguishing productive and nonproductive pathways caused by the high particle-to–plaque forming unit ratio of most animal viruses. Here, we combine an imaging assay that simultaneously tracks the viral capsid and genome in live cells with an infectivity-based assay for RNA release to characterize the early events in the poliovirus (PV) infection. Effects on RNA genome delivery from inhibitors of cell trafficking pathways were probed systematically by both methods. Surprisingly, we observe that genome release by PV is highly efficient and rapid, and thus does not limit the overall infectivity or the infection rate. The results define a pathway in which PV binds to receptors on the cell surface and enters the cell by a clathrin-, caveolin-, flotillin-, and microtubule-independent, but tyrosine kinase- and actin-dependent, endocytic mechanism. Immediately after the internalization of the virus particle, genome release takes place from vesicles or tightly sealed membrane invaginations located within 100–200 nm of the plasma membrane. These results settle a long-lasting debate of whether PV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell. We expect this imaging assay to be broadly applicable to the investigation of entry mechanisms for nonenveloped viruses.     

Cholesterol depletion by methyl-beta-cyclodextrin blocks cholera toxin transport from endosomes to the Golgi apparatus in hippocampal neurons

We recently demonstrated that although cholera toxin (CT) is found in detergent-insoluble domains/rafts at the cell surface of cultured hippocampal neurons, it is internalized via a raft-independent mechanism. Thus, cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) did not affect the rate of CT internalization from the plasma membrane, but did affect the rate of CT degradation, which occurs in lysosomes. In the current study, we analyze which step of CT intracellular transport is inhibited by MbetaCD. Whereas pre-incubation with MbetaCD completely blocked CT degradation, it had no effect on the degradation of wheat germ agglutinin (WGA) or bovine serum albumin (BSA), which are internalized by receptor-mediated and fluid phase endocytosis, respectively. Brefeldin A also completely blocked CT degradation but had no effect on WGA or BSA degradation. In contrast, MbetaCD did not affect CT degradation, or CT-mediated cAMP generation, when added to neurons after CT had been transported to the Golgi apparatus. We conclude that CT transport from endosomes to the Golgi apparatus is cholesterol-dependent, whereas CT transport from the Golgi apparatus to lysosomes is cholesterol-independent.     

Cholesterol effectively blocks entry of flavivirus

Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2) are enveloped flaviviruses that enter cells through receptor-mediated endocytosis and low pH-triggered membrane fusion and then replicate in intracellular membrane structures. Lipid rafts, cholesterol-enriched lipid-ordered membrane domains, are platforms for a variety of cellular functions. In this study, we found that disruption of lipid raft formation by cholesterol depletion with methyl-beta-cyclodextrin or cholesterol chelation with filipin III reduces JEV and DEN-2 infection, mainly at the intracellular replication steps and, to a lesser extent, at viral entry. Using a membrane flotation assay, we found that several flaviviral nonstructural proteins are associated with detergent-resistant membrane structures, indicating that the replication complex of JEV and DEN-2 localizes to the membranes that possess the lipid raft property. Interestingly, we also found that addition of cholesterol readily blocks flaviviral infection, a result that contrasts with previous reports of other viruses, such as Sindbis virus, whose infectivity is enhanced by cholesterol. Cholesterol mainly affected the early step of the flavivirus life cycle, because the presence of cholesterol during viral adsorption greatly blocked JEV and DEN-2 infectivity. Flavirial entry, probably at fusion and RNA uncoating steps, was hindered by cholesterol. Our results thus suggest a stringent requirement for membrane components, especially with respect to the amount of cholesterol, in various steps of the flavivirus life cycle.     

New (fluorescent) light on poliovirus entry

To initiate infection, poliovirus must release its RNA genome into the cytoplasm of a target cell, a process called 'uncoating'. How this occurs has remained uncertain, despite studies over several decades. Two new studies re-address the question of poliovirus entry. The results suggest that poliovirus enters different cells by different mechanisms, and point to a role for virus-induced intracellular signals in the process.     

Cholesterol dependence of Newcastle Disease Virus entry

Lipid rafts are membrane microdomains enriched in cholesterol, sphingolipids, and glycolipids that have been implicated in many biological processes. Since cholesterol is known to play a key role in the entry of some other viruses, we investigated the role of cholesterol and lipid rafts in the host cell plasma membrane in Newcastle Disease Virus (NDV) entry. We used methyl-β-cyclodextrin (MβCD) to deplete cellular cholesterol and disrupt lipid rafts. Our results show that the removal of cellular cholesterol partially reduces viral binding, fusion and infectivity. MβCD had no effect on the expression of sialic acid containing molecule expression, the NDV receptors in the target cell. All the above-described effects were reversed by restoring cholesterol levels in the target cell membrane. The HN viral attachment protein partially localized to detergent-resistant membrane microdomains (DRMs) at 4°C and then shifted to detergent-soluble fractions at 37°C. These results indicate that cellular cholesterol may be required for optimal cell entry in NDV infection cycle.     

Bidirectional entry of poliovirus into polarized epithelial cells.

The interactions of viruses with polarized epithelial cells are of some significance to the pathogenesis of disease because these cell types comprise the primary barrier to many virus infections and also serve as the sites for virus release from the host. Poliovirus-epithelial cell interactions are of particular interest since this virus is an important enteric pathogen and the host cell receptor has been identified. In this study, poliovirus was observed to adsorb to both the apical and basolateral surfaces of polarized monkey kidney (Vero C1008) and human intestinal (Caco-2) epithelial cells but exhibited preferential binding to the basolateral surfaces of both cell types. Localization of the poliovirus receptor by a receptor-specific monoclonal antibody (D171) revealed a similar distribution predominantly on basolateral membranes, and treatment of cells with antibody D171 inhibited virus adsorption to both membrane surfaces. Poliovirus was able to initiate infection with similar efficiency following adsorption to either surface, and infection was blocked at both surfaces by D171, indicating that functional receptor molecules are expressed on both surfaces at sufficient density to mediate efficient infection at the apical and basolateral plasma membranes. Poliovirus infection resulted in a decrease in transepithelial resistance which was inhibited by prior treatment with monoclonal antibody D171 and occurred prior to other visible cytopathic effects. These results have interesting implications for viral pathogenesis in the human gut.     

Cholesterol sensing by CD81 is important for hepatitis C virus entry

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81''s function as a molecular scaffold; these insights are relevant to CD81''s varied roles in both health and disease.     

Stimulation of Xenopus laevis oocyte maturation by methyl-beta-cyclodextrin

The cholesterol-depleting drug methyl-beta-cyclodextrin (Me-beta-CD) was tested for its effects on amphibian oocyte maturation, cholesterol depletion, and low-density membrane recovery. Progesterone-induced oocyte maturation was accelerated by pretreatment of cells with 5-50 mM Me-beta-CD in a dose-dependent manner. Treatment of oocytes with 50 mM Me-beta-CD alone was sufficient to induce germinal vesicle breakdown, stimulate formation of meiotic spindles, and stimulate phosphorylation of mitogen-activated protein kinase over time courses longer than those observed after progesterone treatment. After short-term (30 min) labeling of oocytes with [(3)H]cholesterol, 30-90 min of treatment with 5-50 mM Me-beta-CD removed 50%-70% of cell- associated label, and cholesterol depletion was not observed with alpha-cyclodextrin. After long-term (20-23 h) labeling of oocytes with [(3)H]cholesterol, Me-beta-CD treatment resulted in dose- dependent cholesterol depletion in the 5-50 mM range, and 50 mM Me-beta-CD removed approximately 50% of cell-associated label after 9 h. Treatment of oocytes with 5-50 mM Me-beta-CD also decreased recovery of low-density membrane by detergent-free sucrose gradient centrifugation. These results implicate cholesterol and low-density membrane domains in the signaling mechanisms leading to germinal vesicle breakdown in amphibian oocytes.     

Minimum internal ribosome entry site required for poliovirus infectivity.

Translation initiation by internal ribosome binding is a recently discovered mechanism of eukaryotic viral and cellular protein synthesis in which ribosome subunits interact with the mRNAs at internal sites in the 5' untranslated RNA sequences and not with the 5' methylguanosine cap structure present at the extreme 5' ends of mRNA molecules. Uncapped poliovirus mRNAs harbor internal ribosome entry sites (IRES) in their long and highly structured 5' noncoding regions. Such IRES sequences are required for viral protein synthesis. In this study, a novel poliovirus was isolated whose genomic RNA contains two gross deletions removing approximately 100 nucleotides from the predicted IRES sequences within the 5' noncoding region. The deletions originated from previously in vivo-selected viral revertants displaying non-temperature-sensitive phenotypes. Each revertant had a different predicted stem-loop structure within the 5' noncoding region of their genomic RNAs deleted. The mutant poliovirus (Se1-5NC-delta DG) described in this study contains both stem-loop deletions in a single RNA genome, thereby creating a minimum IRES. Se1-5NC-delta DG exhibited slow growth and a pinpoint plaque phenotype following infection of HeLa cells, delayed onset of protein synthesis in vivo, and defective initiation during in vitro translation of the mutated poliovirus mRNAs. Interestingly, the peak levels of viral RNA synthesis in cells infected with Se1-5NC-delta DG occurred at slightly later times in infection than those achieved by wild-type poliovirus, but these mutant virus RNAs accumulated in the host cells during the late phases of virus infection. UV cross-linking assays with the 5' noncoding regions of wild-type and mutated RNAs were carried out in cytoplasmic extracts from HeLa cells and neuronal cells and in reticulocyte lysates to identify the cellular factors that interact with the putative IRES elements. The cellular proteins that were cross-linked to the minimum IRES may represent factors playing an essential role in internal translation initiation of poliovirus mRNAs.     

Characterization of the nucleoside triphosphatase activity of poliovirus protein 2C reveals a mechanism by which guanidine inhibits poliovirus replication

The highly conserved non-structural protein 2C of picornaviruses is involved in viral genome replication and encapsidation and in the rearrangement of intracellular structures. 2C binds RNA, has nucleoside triphosphatase activity, and shares three motifs with superfamily III helicases. Motifs "A" and "B" are involved in nucleotide triphosphate (NTP) binding and hydrolysis, whereas a function for motif "C" has not yet been demonstrated. Poliovirus RNA replication is inhibited by millimolar concentrations of guanidine hydrochloride (GdnHCl). Resistance and dependence to GdnHCl map to 2C. To characterize the nucleoside triphosphatase activity of 2C, we purified poliovirus recombinant 2C fused to glutathione S-transferase (GST-2C) from Escherichia coli. GST-2C hydrolyzed ATP with a Km of 0.7 mM. Other NTPs, including GTP, competed with ATP for binding to 2C but were poor substrates for hydrolysis. Mutation of conserved residues in motif A and B abolished ATPase activity, as did mutation of the conserved asparagine residue in motif C, an observation indicating the involvement of this motif in ATP hydrolysis. GdnHCl at millimolar concentrations inhibited ATP hydrolysis. Mutations in 2C that confer poliovirus resistant to or dependent on GdnHCl increased the tolerance to GdnHCl up to 100-fold.     

Cholesterol dependence of varicella-zoster virion entry into target cells

The entry of inhaled virions into airway cells is presumably the initiating step of varicella-zoster infection. In order to characterize viral entry, we studied the relative roles played by lipid rafts and clathrin-mediated transport. Virus and target cells were pretreated with agents designed to perturb selected aspects of endocytosis and membrane composition, and the effects of these perturbations on infectious focus formation were monitored. Infectivity was exquisitely sensitive to methyl-beta-cyclodextrin (M beta CD) and nystatin, which disrupt lipid rafts by removing cholesterol. These agents inhibited infection by enveloped, but not cell-associated, varicella-zoster virus (VZV) in a dose-dependent manner and exerted these effects on both target cell and viral membranes. Inhibition by M beta CD, which could be reversed by cholesterol replenishment, rapidly declined as a function of time after exposure of target cells to VZV, suggesting that an early step in viral infection requires cholesterol. No effect of cholesterol depletion, however, was seen on viral binding; moreover, there was no reduction in the surface expression or internalization of mannose 6-phosphate receptors, which are required for VZV entry. Viral entry was energy dependent and showed concentration-dependent inhibition by chlorpromazine, which, among other actions, blocks clathrin-mediated endocytosis. These data suggest that both membrane lipid composition and clathrin-mediated transport are critical for VZV entry. Lipid rafts are likely to contribute directly to viral envelope integrity and, in the host membrane, may influence endocytosis, evoke downstream signaling, and/or facilitate membrane fusion.     

Replication of poliovirus RNA with complete internal ribosome entry site deletions

cis-acting RNA sequences and structures in the 5' and 3' nontranslated regions of poliovirus RNA interact with host translation machinery and viral replication proteins to coordinately regulate the sequential translation and replication of poliovirus RNA. The poliovirus internal ribosome entry site (IRES) in the 5' nontranslated region (NTR) has been implicated as a cis-active RNA required for both viral mRNA translation and viral RNA replication. To evaluate the role of the IRES in poliovirus RNA replication, we exploited the advantages of cell-free translation-replication reactions and preinitiation RNA replication complexes. Genetic complementation with helper mRNAs allowed us to create preinitiation RNA replication complexes containing RNA templates with defined deletions in the viral open reading frame and the IRES. A series of deletions revealed that no RNA elements of either the viral open reading frame or the IRES were required in cis for negative-strand RNA synthesis. The IRES was dispensable for both negative- and positive-strand RNA syntheses. Intriguingly, although small viral RNAs lacking the IRES replicated efficiently, the replication of genome length viral RNAs was stimulated by the presence of the IRES. These results suggest that RNA replication is not directly dependent on a template RNA first functioning as an mRNA. These results further suggest that poliovirus RNA replication is not absolutely dependent on any protein-RNA interactions involving the IRES.     

Cholesterol inhibits TCR signaling by directly restricting TCR-CD3 core tunnel motility

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IFITM3 inhibits influenza A virus infection by preventing cytosolic entry

To replicate, viruses must gain access to the host cell's resources. Interferon (IFN) regulates the actions of a large complement of interferon effector genes (IEGs) that prevent viral replication. The interferon inducible transmembrane protein family members, IFITM1, 2 and 3, are IEGs required for inhibition of influenza A virus, dengue virus, and West Nile virus replication in vitro. Here we report that IFN prevents emergence of viral genomes from the endosomal pathway, and that IFITM3 is both necessary and sufficient for this function. Notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by IFN, and IFITM3 was required and sufficient for this action. We further demonstrate that IFN expands Rab7 and LAMP1-containing structures, and that IFITM3 overexpression is sufficient for this phenotype. Moreover, IFITM3 partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. Collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to IFITM3's actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. Multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by IFN. Therefore these findings are likely to have significance for the intrinsic immune system's neutralization of a diverse array of threats.     

Cholesterol removal by peritoneal lavage with phospholipid-HDL apoprotein mixtures in hypercholesterolemic hamsters

Syrian hamsters were rendered hypercholesterolemic by supplementation of their diet with 1% cholesterol and 15% butter. The hamsters were injected intraperitoneally (i.p.) with about 20 mg of phospholipid liposomes containing trace amounts of [3H]cholesteryl linoleyl ether ([ 3H]CLE) alone or combined with 10 mg delipidated high-density lipoprotein (apoHDL). After 2 h the peritoneal cavity was washed repeatedly with up to 15 ml phosphate-buffered saline. 60%-70% of [3H]CLE were retained after i.p. injection without apoHDL, 30-50% in the presence of apoHDL. The amount of free cholesterol recovered in the peritoneal lavage was significantly higher when apoHDL was combined with 18:2 sphingomyelin or dilinoleyl phosphatidylcholine liposomes, when compared to either liposomes or apoHDL alone. It is suggested that supplementation of dialysate with HDL apolipoproteins and phospholipids in patients undergoing continuous peritoneal dialysis could be of use in a cholesterol depletion regimen.     

Membrane-anchored peptide inhibits human immunodeficiency virus entry

Peptides derived from the heptad repeats of human immunodeficiency virus (HIV) gp41 envelope glycoprotein, such as T20, can efficiently inhibit HIV type 1 (HIV-1) entry. In this study, replication of HIV-1 was inhibited more than 100-fold in a T-helper cell line transduced with a retrovirus vector expressing membrane-anchored T20 on the cell surface. Inhibition was independent of coreceptor usage.     

Host range phenotype induced by mutations in the internal ribosomal entry site of poliovirus RNA.

Most poliovirus strains infect only primates. The host range (HR) of poliovirus is thought to be primarily determined by a cell surface molecule that functions as poliovirus receptor (PVR), since it has been shown that transgenic mice are made poliovirus sensitive by introducing the human PVR gene into the genome. The relative levels of neurovirulence of polioviruses tested in these transgenic mice were shown to correlate well with the levels tested in monkeys (H. Horie et al., J. Virol. 68:681-688, 1994). Mutants of the virulent Mahoney strain of poliovirus have been generated by disruption of nucleotides 128 to 134, at stem-loop II within the 5' noncoding region, and four of these mutants multiplicated well in human HeLa cells but poorly in mouse TgSVA cells that had been established from the kidney of the poliovirus-sensitive transgenic mouse. Neurovirulence tests using the two animal models revealed that these mutants were strongly attenuated only in tests with the mouse model and were therefore HR mutants. The virus infection cycle in TgSVA cells was restricted by an internal ribosomal entry site (IRES)-dependent initiation process of translation. Viral protein synthesis and the associated block of cellular protein synthesis were not observed in TgSVA cells infected with three of four HR mutants and was evident at only a low level in the remaining mutant. The mutant RNAs were functional in a cell-free protein synthesis system from HeLa cells but not in those from TgSVA and mouse neuroblastoma NS20Y cells. These results suggest that host factor(s) affecting IRES-dependent translation of poliovirus differ between human and mouse cells and that the mutant IRES constructs detect species differences in such host factor(s). The IRES could potentially be a host range determinant for poliovirus infection.     

Cholesterol removal by some lactic acid bacteria that can be used as probiotic

In the present study, the relationship between exopolysaccharide production and cholesterol removal rates of five strains of Lactobacillus delbrueckii subsp. bulgaricus isolated from home‐made yoghurt was studied. Test strains were selected according to their exopolysaccharide production capacity. Influence of different bile concentrations on cholesterol removal was investigated. It was confirmed that B3, ATCC 11842 and G11 strains which produce high amounts of exopolysaccharide (211, 200 and 159 mg/l, respectively) were able to remove more cholesterol from the medium compared to those that produce low amounts of exopolysaccharide (B2, A13). The highest cholesterol removal (31%) was observed by strain L. delbrueckii subsp. bulgaricus B3, producing a high amount of exopolysaccharide, in 3 mg/ml bile concentration. Cholesterol removal by resting and dead cells was investigated and it was found to be 4%–14% and 3%–10%, respectively. Cholesterol removal by immobilized and free cells of the B3 strain was studied and it was determined that immobilized cells are more effective. Influence of cholesterol on exopolysaccharide production has also been tested and it was found that cholesterol increased the production of EPS. The results indicated that: (i) there is a correlation between cholesterol removal and EPS production; and (ii) L. delbrueckii subsp. bulgaricus B3 is regarded as a suitable candidate probiotic and adjunct culture.