Maria Joao Amorim

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.

Lipid Raft‐Dependent Targeting of the Influenza A Virus Nucleoprotein to the Apical Plasma Membrane

Influenza virus acquires a lipid raft‐containing envelope by budding from the apical surface of epithelial cells. Polarised budding involves specific sorting of the viral membrane proteins, but little is known about trafficking of the internal virion components. We show that during the later stages of virus infection, influenza nucleoprotein (NP) and polymerase (the protein components of genomic ribonucleoproteins) localised to apical but not lateral or basolateral membranes, even in cell types where haemagglutinin was found on all external membranes. Other cytosolic components of the virion either distributed throughout the cytoplasm (NEP/NS2) or did not localise solely to the apical plasma membrane in all cell types (M1). NP localised specifically to the apical surface even when expressed alone, indicating intrinsic targeting. A similar proportion of NP associated with membrane fractions in flotation assays from virus‐infected and plasmid‐transfected cells. Detergent‐resistant flotation at 4 °C suggested that these membranes were lipid raft microdomains. Confirming this, cholesterol depletion rendered NP detergent‐soluble and furthermore, resulted in its partial redistribution throughout the cell. We conclude that NP is independently targeted to the apical plasma membrane through a mechanism involving lipid rafts and propose that this helps determine the polarity of influenza virus budding.

Global coordination of transcriptional control and mRNA decay during cellular differentiation

The function of transcription in dynamic gene expression programs has been extensively studied, but little is known about how it is integrated with RNA turnover at the genome‐wide level. We investigated these questions using the meiotic gene expression program of Schizosaccharomyces pombe. We identified over 80 transcripts that co‐purify with the meiotic‐specific Meu5p RNA‐binding protein. Their levels and half‐lives were reduced in meu5 mutants, demonstrating that Meu5p stabilizes its targets. Most Meu5p‐bound RNAs were also targets of the Mei4p transcription factor, which induces the transient expression of ∼500 meiotic genes. Although many Mei4p targets showed sharp expression peaks, Meu5p targets had broad expression profiles. In the absence of meu5, all Mei4p targets were expressed with similar kinetics, indicating that Meu5p alters the global features of the gene expression program. As Mei4p activates meu5 transcription, Mei4p, Meu5p and their common targets form a feed‐forward loop, a motif common in transcriptional networks but not studied in the context of mRNA decay. Our data provide insight into the topology of regulatory networks integrating transcriptional and posttranscriptional controls.

Exosome Biogenesis, Regulation, and Function in Viral Infection

Exosomes are extracellular vesicles released upon fusion of multivesicular bodies (MVBs) with the cellular plasma membrane. They originate as intraluminal vesicles (ILVs) during the process of MVB formation. Exosomes were shown to contain selectively sorted functional proteins, lipids, and RNAs, mediating cell-to-cell communications and hence playing a role in the physiology of the healthy and diseased organism. Challenges in the field include the identification of mechanisms sustaining packaging of membrane-bound and soluble material to these vesicles and the understanding of the underlying processes directing MVBs for degradation or fusion with the plasma membrane. The investigation into the formation and roles of exosomes in viral infection is in its early years. Although still controversial, exosomes can, in principle, incorporate any functional factor, provided they have an appropriate sorting signal, and thus are prone to viral exploitation. This review initially focuses on the composition and biogenesis of exosomes. It then explores the regulatory mechanisms underlying their biogenesis. Exosomes are part of the endocytic system, which is tightly regulated and able to respond to several stimuli that lead to alterations in the composition of its sub-compartments. We discuss the current knowledge of how these changes affect exosomal release. We then summarize how different viruses exploit specific proteins of endocytic sub-compartments and speculate that it could interfere with exosome function, although no direct link between viral usage of the endocytic system and exosome release has yet been reported. Many recent reports have ascribed functions to exosomes released from cells infected with a variety of animal viruses, including viral spread, host immunity, and manipulation of the microenvironment, which are discussed. Given the ever-growing roles and importance of exosomes in viral infections, understanding what regulates their composition and levels, and defining their functions will ultimately provide additional insights into the virulence and persistence of infections.

Microparticles for delivering therapeutic peptides and proteins to the lumen of the small intestine

Several different peptides and proteins, such as the pancreatic trypsin inhibitor, growth factors and trefoil peptides, are known to play important roles in maintaining the structure and function of the gastrointestinal wall. With the advent of recombinant biotechnology, it has become feasible to test some of these proteins as therapeutics in different inflammatory conditions of the intestines. However, the harsh pH and enzymatic conditions of the stomach can lead to their inactivation. This research was aimed at the development of particulate, gastric-resistant pharmaceutical forms, incorporating those bioactive molecules. Mixtures of proteins in powder form were coated with cellulose acetate phthalate, Eudragit S100 or Eudragit RS PO, using simple preparation techniques based on single emulsion/solvent evaporation. Using aprotinin as a model drug, it was found that these procedures were effective in microencapsulating protein in the solid form without affecting its biological activity. Furthermore, and in particular with the first two polymers above, particles showed adequate in vitro release patterns for the applications envisioned.

Nucleozin Targets Cytoplasmic Trafficking of Viral Ribonucleoprotein-Rab11 Complexes in Influenza A Virus Infection

ABSTRACT Novel antivirals are needed to supplement existing control strategies for influenza A virus (IAV). A promising new class of drug, exemplified by the compound nucleozin, has recently been identified that targets the viral nucleoprotein (NP). These inhibitors are thought to act as “molecular staples” that stabilize interactions between NP monomers, promoting the formation of nonfunctional aggregates. Here we detail the inhibitory mechanism of nucleozin, finding that the drug has both early- and late-acting effects on the IAV life cycle. When present at the start of infection, it inhibited viral RNA and protein synthesis. However, when added at later time points, it still potently blocked the production of infectious progeny but without affecting viral macromolecular synthesis. Instead, nucleozin blocked the cytoplasmic trafficking of ribonucleoproteins (RNPs) that had undergone nuclear export, promoting the formation of large perinuclear aggregates of RNPs along with cellular Rab11. This effect led to the production of much reduced amounts of often markedly smaller virus particles. We conclude that the primary target of nucleozin is the viral RNP, not NP, and this work also provides proof of the principle that IAV replication can be effectively inhibited by blocking cytoplasmic trafficking of the viral genome.

'Genome gating'; polarized intranuclear trafficking of influenza virus RNPs

Many viruses exploit cellular polarity to constrain the assembly and release of progeny virions to a desired surface. Influenza virus particles are released only from the apical surface of epithelial cells and this polarization is partly owing to specific targeting of the viral membrane proteins to the apical plasma membrane. The RNA genome of the virus is transcribed and replicated in the nucleus, necessitating nuclear export of the individual ribonucleoprotein (RNP) segments before they can be incorporated into budding virus particles. We show that the process of polarized virus assembly begins in the nucleus with the RNPs adopting a novel asymmetric distribution at the inner nuclear membrane prior to their export to the cytoplasm. The viral nucleoprotein, the major protein component of RNPs, displays the same polarized intranuclear distribution in the absence of other influenza virus components, suggesting the existence of a hitherto unrecognized polarity within the mammalian cell nucleus.

Rng3, a member of the UCS family of myosin co‐chaperones, associates with myosin heavy chains cotranslationally

The production of functional myosin heavy chains in many eukaryotic organisms requires the function of proteins containing UCS domains (UNC‐45/CRO1/She4), which bind to the myosin head domain and stimulate its folding. UCS proteins are essential for myosin‐related functions such as muscle formation, RNA localization and cytokinesis. Here, we show that the Schizosaccharomyces pombe UCS protein Rng3 associates with polysomes, suggesting that UCS proteins might assist myosin folding cotranslationally. To identify Rng3 cotranslational targets systematically, we purified Rng3‐associated RNAs and used DNA microarrays to identify the transcripts. Rng3 copurified with only seven transcripts (around 0.1% of S. pombe genes), including all five messenger RNAs encoding myosin heavy chains. These results suggest that every myosin heavy chain in S. pombe is a cotranslational target of Rng3. Furthermore, our data suggest that microarray‐based approaches allow the genome‐wide identification of cotranslational chaperone targets, and thus pave the way for the dissection of translation‐linked chaperone networks.

Recycling Endosomes and Viral Infection.

Many viruses exploit specific arms of the endomembrane system. The unique composition of each arm prompts the development of remarkably specific interactions between viruses and sub-organelles. This review focuses on the viral–host interactions occurring on the endocytic recycling compartment (ERC), and mediated by its regulatory Ras-related in brain (Rab) GTPase Rab11. This protein regulates trafficking from the ERC and the trans-Golgi network to the plasma membrane. Such transport comprises intricate networks of proteins/lipids operating sequentially from the membrane of origin up to the cell surface. Rab11 is also emerging as a critical factor in an increasing number of infections by major animal viruses, including pathogens that provoke human disease. Understanding the interplay between the ERC and viruses is a milestone in human health. Rab11 has been associated with several steps of the viral lifecycles by unclear processes that use sophisticated diversified host machinery. For this reason, we first explore the state-of-the-art on processes regulating membrane composition and trafficking. Subsequently, this review outlines viral interactions with the ERC, highlighting current knowledge on viral-host binding partners. Finally, using examples from the few mechanistic studies available we emphasize how ERC functions are adjusted during infection to remodel cytoskeleton dynamics, innate immunity and membrane composition.

Influenza A virus ribonucleoproteins modulate host recycling by competing with Rab11 effectors

ABSTRACT Influenza A virus assembly is an unclear process, whereby individual virion components form an infectious particle. The segmented nature of the influenza A genome imposes a problem to assembly because it requires packaging of eight distinct RNA particles (vRNPs). It also allows genome mixing from distinct parental strains, events associated with influenza pandemic outbreaks. It is important to public health to understand how segmented genomes assemble, a process that is dependent on the transport of components to assembly sites. Previously, it has been shown that vRNPs are carried by recycling endosome vesicles, resulting in a change of Rab11 distribution. Here, we describe that vRNP binding to recycling endosomes impairs recycling endosome function, by competing for Rab11 binding with family-interacting proteins, and that there is a causal relationship between Rab11 ability to recruit family-interacting proteins and Rab11 redistribution. This competition reduces recycling sorting at an unclear step, resulting in clustering of single- and double-membraned vesicles. These morphological changes in Rab11 membranes are indicative of alterations in protein and lipid homeostasis during infection. Vesicular clustering creates hotspots of the vRNPs that need to interact to form an infectious particle. Summary: Binding of influenza A virus ribonucleoproteins to the recycling endosome modulates its function, decreasing its sorting efficiency and inducing vesicular clustering.