Amanda D. Stuart

A Novel Cell Entry Pathway for a DAF-Using Human Enterovirus Is Dependent on Lipid Rafts

ABSTRACT The glycosylphosphatidylinositol (GPI)-anchored complement regulatory protein decay-accelerating factor (DAF) is used by a number of enteroviruses as a receptor during infection. DAF and other GPI-anchored proteins can be found in cholesterol-rich ordered domains within the plasma membrane that are known as “lipid rafts.” We have shown, by using drugs to specifically inhibit various endocytosis routes, that infection by a DAF-using strain of echovirus 11 (EV11) is dependent upon cholesterol and an intact cytoskeleton, whereas a non-DAF-using mutant derived from it was unaffected by these drugs. Using RNA transfection and virus-binding assays, we have shown that this requirement for cholesterol, the actin cytoskeleton, and the microtubule network occurs postbinding of the virus but prior to uncoating of the RNA, indicating a role during virus entry. Confocal microscopy of virus infection supported the role of cholesterol and the cytoskeleton during entry. In addition, [35S]methionine-labeled DAF-using EV11, but not the non-DAF-using EV11, could be copurified with lipid raft components during infection after Triton X-100 extraction. These data indicate that DAF usage by EV11 enables the virus to associate with lipid rafts and enter cells through this novel route.

Identification of a Novel Splice Variant Form of the Influenza A Virus M2 Ion Channel with an Antigenically Distinct Ectodomain

Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42) with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.

The Rab11 Pathway Is Required for Influenza A Virus Budding and Filament Formation

ABSTRACT Influenza A virus buds through the apical plasma membrane, forming enveloped virus particles that can take the shape of pleomorphic spheres or vastly elongated filaments. For either type of virion, the factors responsible for separation of viral and cell membranes are not known. We find that cellular Rab11 (a small GTP-binding protein involved in endocytic recycling) and Rab11-family interacting protein 3 ([FIP3] which plays a role in membrane trafficking and regulation of actin dynamics) are both required to support the formation of filamentous virions, while Rab11 is additionally involved in the final budding step of spherical particles. Cells transfected with Rab11 GTP-cycling mutants or depleted of Rab11 or FIP3 content by small interfering RNA treatment lost the ability to form virus filaments. Depletion of Rab11 resulted in up to a 100-fold decrease in titer of spherical virus released from cells. Scanning electron microscopy of Rab11-depleted cells showed high densities of virus particles apparently stalled in the process of budding. Transmission electron microscopy of thin sections confirmed that Rab11 depletion resulted in significant numbers of abnormally formed virus particles that had failed to pinch off from the plasma membrane. Based on these findings, we see a clear role for a Rab11-mediated pathway in influenza virus morphogenesis and budding.

A Rab11- and Microtubule-Dependent Mechanism for Cytoplasmic Transport of Influenza A Virus Viral RNA

ABSTRACT The viral RNA (vRNA) genome of influenza A virus is replicated in the nucleus, exported to the cytoplasm as ribonucleoproteins (RNPs), and trafficked to the plasma membrane through uncertain means. Using fluorescent in situ hybridization to detect vRNA as well as the live cell imaging of fluorescently labeled RNPs, we show that an early event in vRNA cytoplasmic trafficking involves accumulation near the microtubule organizing center in multiple cell types and viral strains. Here, RNPs colocalized with Rab11, a pericentriolar recycling endosome marker. Cytoplasmic RNP localization was perturbed by inhibitors of vesicular trafficking, microtubules, or the short interfering RNA-mediated depletion of Rab11. Green fluorescent protein (GFP)-tagged RNPs in living cells demonstrated rapid, bidirectional, and saltatory movement, which is characteristic of microtubule-based transport, and also cotrafficked with fluorescent Rab11. Coprecipitation experiments showed an interaction between RNPs and the GTP-bound form of Rab11, potentially mediated via the PB2 subunit of the polymerase. We propose that influenza virus RNPs are routed from the nucleus to the pericentriolar recycling endosome (RE), where they access a Rab11-dependent vesicular transport pathway to the cell periphery.

A LC3-Interacting Motif in the Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability

Summary Autophagy recycles cellular components and defends cells against intracellular pathogens. While viruses must evade autophagocytic destruction, some viruses can also subvert autophagy for their own benefit. The ability of influenza A virus (IAV) to evade autophagy depends on the Matrix 2 (M2) ion-channel protein. We show that the cytoplasmic tail of IAV M2 interacts directly with the essential autophagy protein LC3 and promotes LC3 relocalization to the unexpected destination of the plasma membrane. LC3 binding is mediated by a highly conserved LC3-interacting region (LIR) in M2. The M2 LIR is required for LC3 redistribution to the plasma membrane in virus-infected cells. Mutations in M2 that abolish LC3 binding interfere with filamentous budding and reduce virion stability. IAV therefore subverts autophagy by mimicking a host short linear protein-protein interaction motif. This strategy may facilitate transmission of infection between organisms by enhancing the stability of viral progeny.

Budding of filamentous and non-filamentous influenza A virus occurs via a VPS4 and VPS28-independent pathway

The mechanism of membrane scission during influenza A virus budding has been the subject of controversy. We confirm that influenza M1 binds VPS28, a subunit of the ESCRT-1 complex. However, confocal microscopy of infected cells showed no marked colocalisation between M1 and VPS28 or VPS4 ESCRT proteins, or relocalisation of the cellular proteins. Trafficking of HA and M1 appeared normal when endosomal sorting was impaired by expression of inactive VPS4. Overexpression of either isoform of VPS28 or wildtype or dominant negative VPS4 proteins did not alter production of filamentous virions. SiRNA depletion of endogenous VPS28 had no significant effect on influenza virus replication. Furthermore, cells expressing wildtype or dominant-negative VPS4 replicated filamentous and non-filamentous strains of influenza to similar titres, indicating that influenza release is VPS4-independent. Overall, we see no role for the ESCRT pathway in influenza virus budding and the significance of the M1-VPS28 interaction remains to be determined.

Role of the Rab11 pathway in negative-strand virus assembly

As intracellular pathogens, enveloped viruses must usurp the host cell machinery for many stages of the viral life cycle in order to produce a new generation of infectious virions. In one of the less understood steps of viral assembly, viral components including the transmembrane glycoproteins, structural proteins and the viral genome must be targeted to the site of viral budding, where they assemble and are incorporated into a newly formed virion that gains a lipid envelope from a cellular membrane. Recent work has revealed that the cellular recycling endosome pathway, in particular Rab11, plays an important role in the assembly of negative-strand RNA viruses such as respiratory syncytial virus, influenza A virus, Andes virus and Sendai virus. The present mini-review discusses this emerging field and explores the potential roles of the Rab11 pathway in the trafficking, assembly and budding steps of these viruses.

Phospholipase D Facilitates Efficient Entry of Influenza Virus, Allowing Escape from Innate Immune Inhibition

Background: Identifying host factors used by influenza can aid in the defense against pandemics that threaten public health. Results: Phospholipase D (PLD) contributes to viral infection and innate immune evasion strategies. Conclusion: Inhibition of PLD activity reduces influenza reproduction. Significance: PLD inhibition presents a novel approach to restrict influenza infection and viral escape. Lipid metabolism plays a fundamental role during influenza virus replication, although key regulators of lipid-dependent trafficking and virus production remain inadequately defined. This report demonstrates that infection by influenza virus stimulates phospholipase D (PLD) activity and that PLD co-localizes with influenza during infection. Both chemical inhibition and RNA interference of PLD delayed viral entry and reduced viral titers in vitro. Although there may be contributions by both major isoenzymes, the effects on viral infectivity appear to be more dependent on the PLD2 isoenzyme. In vivo, PLD2 inhibition reduced virus titer and correlated with significant increases in transcription of innate antiviral effectors. The reduction in viral titer downstream of PLD2 inhibition was dependent on Rig-I (retinoic acid-inducible gene-1), IRF3, and MxA (myxovirus resistance gene A) but not IRF7. Inhibition of PLD2 accelerated the accumulation of MxA in foci as early as 30 min postinfection. Together these data suggest that PLD facilitates the rapid endocytosis of influenza virus, permitting viral escape from innate immune detection and effectors that are capable of limiting lethal infection.

Decay-Accelerating Factor Binding Determines the Entry Route of Echovirus 11 in Polarized Epithelial Cells

ABSTRACT The interaction between echovirus 11 strain 207 (EV11-207) and decay-accelerating factor (DAF or CD55) at the apical surface of polarized Caco-2 cells results in rapid transport of the virus to tight junctions and in its subsequent uptake. A virus mutant (EV11-207R) which differs at 6 amino acids and whose affinity for DAF is apparently significantly lower remains at the apical surface, from where its uptake occurs. Binding of EV11-207 to DAF and its transport to tight junctions result in a loss of function of the junctions. In contrast, the mutant virus EV11-207R is not transferred to tight junctions, nor does it impair the integrity of these junctions. Cholesterol depletion from the apical membrane leads to DAF aggregation and, presumably, internalization and inhibits infection by EV11-207. However, infection by EV11-207R is significantly less sensitive to cholesterol depletion than infection by EV11-207, confirming the DAF requirement for EV11-207, but not EV11-207R, to infect cells. These data strongly indicate that in the case of infection of polarized epithelial cells by echovirus 11, DAF binding appears be a key determinant in the choice of entry pathway, at least in cell culture.

Echovirus 11 infection induces dramatic changes in the actin cytoskeleton of polarized Caco-2 cells

Binding of echovirus 11 strain 207 (EV11-207) to Caco-2 monolayers results in rapid transfer of the virus to tight junctions prior to uptake. Using a confocal microscopy based-method, this study quantified the spatiotemporal distribution of actin during the time course of infection by EV11-207 in Caco-2 polarized cells. It was found that binding of EV11-207 to the apical surface resulted in rapid rearrangement of the actin cytoskeleton, concomitant with transport of the virus particles to tight junctions. By interfering with the actin network dynamics, the virus remained trapped at the cell surface, leading to abortion of infection. In addition, it was observed that at 4 h post-infection, concomitant with the detection of virus replication, actin filament was depolymerized and degraded. Finally, it was shown that the mechanisms leading to loss of actin were independent of viral genome synthesis, indicating a potential role for the viral protein synthesis seen in late infection. These data confirmed a previous study on the requirement for an intact actin cytoskeleton for EV11-207 to infect cells and reinforce the notion of actin cytoskeleton subversion by picornaviruses during infection in polarized epithelial cells.