Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Montana State University

Princeton University

Cellular autophagy: surrender, avoidance and subversion by microorganisms.

Infection of human cells with poliovirus induces the proliferation of double-membraned cytoplasmic vesicles whose surfaces are used as the sites of viral RNA replication and whose origin is unknown. Here, we show that several hallmarks of cellular autophagosomes can be identified in poliovirus-induced vesicles, including colocalization of LAMP1 and LC3, the human homolog of Saccharomyces cerevisiae Atg8p, and staining with the fluorophore monodansylcadaverine followed by fixation. Colocalization of LC3 and LAMP1 was observed early in the poliovirus replicative cycle, in cells infected with rhinoviruses 2 and 14, and in cells that express poliovirus proteins 2BC and 3A, known to be sufficient to induce double-membraned vesicles. Stimulation of autophagy increased poliovirus yield, and inhibition of the autophagosomal pathway by 3-methyladenine or by RNA interference against mRNAs that encode two different proteins known to be required for autophagy decreased poliovirus yield. We propose that, for poliovirus and rhinovirus, components of the cellular machinery of autophagosome formation are subverted to promote viral replication. Although autophagy can serve in the innate immune response to microorganisms, our findings are inconsistent with a role for the induced autophagosome-like structures in clearance of poliovirus. Instead, we argue that these double-membraned structures provide membranous supports for viral RNA replication complexes, possibly enabling the nonlytic release of cytoplasmic contents, including progeny virions, from infected cells.

Subversion of cellular autophagosomal machinery by RNA viruses.

Cells infected with poliovirus exhibit a rapid inhibition of protein secretion and disruption of the Golgi complex. Neither the precise step at which the virus inhibits protein secretion nor the fate of the Golgi complex during infection has been determined. We find that transport-vesicle exit from the endoplasmic reticulum (ER) and trafficking to the ER-Golgi intermediate compartment (ERGIC) are unaffected in the poliovirus-infected cell. By contrast, poliovirus infection blocks transport from the ERGIC to the Golgi complex. Poliovirus infection also induces fragmentation of the Golgi complex resulting in diffuse distribution of both large and small vesicles throughout the cell. Pre-treatment with nocodazole prevents complete fragmentation, indicating that microtubules are required for poliovirus-induced Golgi dispersion. However, virally induced inhibition of the secretory pathway is not affected by nocodazole, and Golgi dispersion was found to occur during infection with mutant viruses with reduce ability to inhibit protein secretion. We conclude that the dispersion of the Golgi complex is not in itself the cause of inhibition of traffic between the ERGIC and the Golgi. Instead, these phenomena are independent effects of poliovirus infection on the host secretory complex.

Poliovirus infection blocks ERGIC-to-Golgi trafficking and induces microtubule-dependent disruption of the Golgi complex.

Poliovirus infection remodels intracellular membranes, creating a large number of membranous vesicles on which viral RNA replication occurs. Poliovirus-induced vesicles display hallmarks of cellular autophagosomes, including delimiting double membranes surrounding the cytosolic lumen, acquisition of the endosomal marker LAMP-1, and recruitment of the 18-kDa host protein LC3. Autophagy results in the covalent lipidation of LC3, conferring the property of membrane association to this previously microtubule-associated protein and providing a biochemical marker for the induction of autophagy. Here, we report that a similar modification of LC3 occurs both during poliovirus infection and following expression of a single viral protein, a stable precursor termed 2BC. Therefore, one of the early steps in cellular autophagy, LC3 modification, can be genetically separated from the induction of double-membraned vesicles that contain the modified LC3, which requires both viral proteins 2BC and 3A. The existence of viral inducers that promote a distinct aspect of the formation of autophagosome-like membranes both facilitates the dissection of this cellular process and supports the hypothesis that this branch of the innate immune response is directly subverted by poliovirus.

Modification of cellular autophagy protein LC3 by poliovirus.

The RNA replication complexes of small positive-strand RNA viruses such as poliovirus are known to form on the surfaces of membranous vesicles in the cytoplasm of infected mammalian cells. These membranes resemble cellular autophagosomes in their double-membraned morphology, cytoplasmic lumen, lipid-rich composition and the presence of cellular proteins LAMP 1 and LC3. Furthermore, LC3 protein is covalently modified during poliovirus infection in a manner indistinguishable from that observed during bona fide autophagy. This covalent modification can also be induced by the expression of viral protein 2BC in isolation. However, differences between poliovirus-induced vesicles and autophagosomes also exist: the viral-induced membranes are smaller, at 200-400 nm in diameter, and can be induced by the combination of two viral proteins, termed 2BC and 3A. Experimental suppression of expression of proteins in the autophagy pathway was found to reduce viral yield, arguing that this pathway facilitates viral infection, rather than clearing it. We have hypothesized that, in addition to providing membranous surfaces for assembly of viral RNA replication complexes, double-membraned vesicles provide a topological mechanism to deliver cytoplasmic contents, including mature virus, to the extracellular milieu without lysing the cell.

Potential subversion of autophagosomal pathway by picornaviruses.

Cellular autophagy, a process that directs cytosolic contents to the endosomal and lysosomal pathways via the formation of double-membraned vesicles, is a crucial aspect of innate immunity to many intracellular pathogens. However, evidence is accumulating that certain RNA viruses, such as poliovirus, subvert this pathway to facilitate viral growth. The autophagosome-like membranes induced during infection with wild-type poliovirus were found to be, unlike cellular autophagosomes, relatively immobile. Their mobility increased upon nocodazole treatment, arguing that vesicular tethering is microtubule dependent. In cells infected with a mutant virus that is defective in its interaction with the host cytoskeleton and secretory pathway, vesicle movement increased, indicating reduced tethering. In all cases, the release of tethering correlated with increased amounts of extracellular virus, which is consistent with the hypothesis that small amounts of cytosol and virus entrapped by double-membraned structures could be released via fusion with the plasma membrane. We propose that this extracellular delivery of cytoplasmic contents be termed autophagosome-mediated exit without lysis (AWOL). This pathway could explain the observed exit, in the apparent absence of cellular lysis, of other cytoplasmic macromolecular complexes, including infectious agents and complexes of aggregated proteins.

Role of microtubules in extracellular release of poliovirus.

Autophagy is acknowledged as an important cellular defense mechanism against intracellular pathogens. As with other innate immune responses, pathogens have adapted to evade autophagy and in some cases, subvert the pathway to promote their own replication. Poliovirus, a prototypical small positive-strand RNA virus that replicates and assembles in the cytoplasm of the host cell, utilizes membranes derived from the autophagic pathway to aid viral replication and egress from the cell. Recently we made the surprising discovery that GFP-LC3-staining vesicles are physically immobilized during poliovirus infection. Here we discuss our model for the mechanism of vesicle immobilization and the predictions it makes for pathogens that subvert the autophagic pathway to their own ends.

Viruses and arrested autophagosome development.

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Viral infection converts the normal functions of a cell to optimize viral replication and virion production. One striking observation of this conversion is the reconfiguration and reorganization of cellular actin, affecting every stage of the viral life cycle, from entry through assembly to egress. The extent and degree of cytoskeletal reorganization varies among different viral infections, suggesting the evolution of myriad viral strategies. In this Review, we describe how the interaction of viral proteins with the cell modulates the structure and function of the actin cytoskeleton to initiate, sustain and spread infections. The molecular biology of such interactions continues to engage virologists in their quest to understand viral replication and informs cell biologists about the role of the cytoskeleton in the uninfected cell.

Subversion of the actin cytoskeleton during viral infection.

L&#x26;#39;infection virale convertit les fonctions normales d&#x26;#39;une cellule &#xE0; optimiser la r&#xE9;plication virale et la production du virion. Une observation frappante de cette conversion est la reconfiguration et la r&#xE9;organisation de l&#x26;#39;actine cellulaire, affectant toutes les &#xE9;tapes du cycle viral, de l&#x26;#39;entr&#xE9;e jusqu&#x26;#39;&#xE0; l&#x26;#39;assemblage &#xE0; l&#x26;#39;&#xE9;vacuation. La mesure et le degr&#xE9; de la r&#xE9;organisation du cytosquelette varie entre les diff&#xE9;rentes infections virales, ce qui sugg&#xE8;re l&#x26;#39;&#xE9;volution d&#x26;#39;une myriade de strat&#xE9;gies virales. Dans cette revue, nous d&#xE9;crivons comment l&#x26;#39;interaction des prot&#xE9;ines virales avec la cellule module la structure et la fonction du cytosquelette d&#x26;#39;actine &#xE0; initier, soutenir et propager les infections. La biologie mol&#xE9;culaire de ces interactions continue &#xE0; s&#x26;#39;engager virologues dans leur qu&#xEA;te pour comprendre la r&#xE9;plication virale et informe les biologistes cellulaires sur le r&#xF4;le du cytosquelette dans la cellule infect&#xE9;e.

Subversion du cytosquelette d&#x26;#39;actine dans une infection virale

Virale Infektion konvertiert die normalen Funktionen einer Zelle Virusreplikation und Virion Produktion zu optimieren. Eine auffallende Beobachtung dieser Konvertierung ist den Umbau und die Reorganisation der zellul&auml;ren Actin, Auswirkungen auf jeder Stufe des viralen Lebenszyklus, von der Eingabe bis zu egress Assembly. Das Ausma&szlig; und den Grad der Zytoskelett Reorganisation unterschiedlich verschiedene virale Infektionen, die Entwicklung der unz&auml;hligen virale Strategien vorschlagen. In diesem Bericht beschreiben wir, wie die Interaktion der viralen Proteine mit der Zelle moduliert, die Struktur und Funktion des Aktin-Zytoskeletts zu initiieren, zu erhalten und Infektionen zu verbreiten. Der Molekularbiologie solche Interaktionen weiterhin Virologen in ihrem Streben nach Verst&auml;ndnis der Virusreplikation zu engagieren und die Rolle des Zytoskeletts in der nicht infizierten Zelle Zelle Biologen informiert.

Subversion des Aktin-Zytoskeletts w&auml;hrend virale Infektion.

Infezione virale converte le normali funzioni di un cellulare per ottimizzare la replicazione virale e la produzione del virione. Un&#39;osservazione sorprendente di questa conversione &egrave; la riconfigurazione e la riorganizzazione dell&#39;actina cellulare, che colpisce ogni fase del ciclo di vita virale, dall&#39;ingresso attraverso il montaggio di uscita. La misura e il grado di riorganizzazione del citoscheletro varia tra le diverse infezioni virali, suggerendo l&#39;evoluzione delle strategie virali una miriade. In questa recensione, si descrive come l&#39;interazione delle proteine virali con la cellula modula la struttura e la funzione del citoscheletro di actina per avviare, sostenere e diffondere le infezioni. La biologia molecolare di tali interazioni continua ad impegnarsi virologi nella loro ricerca per capire la replicazione virale e informa biologi cellula circa il ruolo del citoscheletro nella cella non infetto.

Sovversione del citoscheletro di actina durante l&#39;infezione virale.

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La infecci&#xF3;n viral convierte las funciones normales de una c&#xE9;lula para optimizar la replicaci&#xF3;n viral y la producci&#xF3;n de viriones. Una observaci&#xF3;n sorprendente de esta conversi&#xF3;n es la reconfiguraci&#xF3;n y la reorganizaci&#xF3;n de la actina celular, que afecta a todas las etapas del ciclo vital del virus, a partir de la entrada a trav&#xE9;s del conjunto de salida. La extensi&#xF3;n y el grado de reorganizaci&#xF3;n del citoesqueleto var&#xED;a entre los diferentes infecciones virales, lo que sugiere la evoluci&#xF3;n de la mir&#xED;ada de las estrategias virales. En esta revisi&#xF3;n, se describe c&#xF3;mo la interacci&#xF3;n de las prote&#xED;nas virales con la c&#xE9;lula modula la estructura y funci&#xF3;n del citoesqueleto de actina para iniciar, mantener y propagar infecciones. La biolog&#xED;a molecular de estas interacciones sigue manteniendo con vir&#xF3;logos en su b&#xFA;squeda para entender la replicaci&#xF3;n viral e informa a los bi&#xF3;logos celulares sobre el papel del citoesqueleto en la c&#xE9;lula infectada.

La subversi&#xF3;n del citoesqueleto de actina durante la infecci&#xF3;n viral

The spread of viral infection within a host can be restricted by bottlenecks that limit the size and diversity of the viral population. An essential process for alphaherpesvirus infection is spread from axons of peripheral nervous system neurons to cells in peripheral epithelia (anterograde-directed spread, ADS). ADS is necessary for the formation of vesicular lesions characteristic of reactivated herpesvirus infections; however, the number of virions transmitted is unknown. We have developed two methods to quantitate ADS events using a compartmentalized neuronal culture system. The first method uses HSV-1 and pseudorabies virus recombinants that express one of three different fluorescent proteins. The fluorescence profiles of cells infected with the virus mixtures are used to quantify the number of expressed viral genomes. Strikingly, although epithelial or neuronal cells express 3-10 viral genomes after infection by free virions, epithelial cells infected by HSV-1 or pseudorabies virus following ADS express fewer than two viral genomes. The second method uses live-cell fluorescence microscopy to track individual capsids involved in ADS. We observed that most ADS events involve a single capsid infecting a target epithelial cell. Together, these complementary analyses reveal that ADS events are restricted to small numbers of viral particles, most often a single virion, resulting in a single viral genome initiating infection.

Alphaherpesvirus axon-to-cell spread involves limited virion transmission.

During infection of the nervous system, alphaherpesviruses-including pseudorabies virus (PRV)-use retrograde axonal transport to travel toward the neuronal cell body and anterograde transport to traffic back to the cell periphery upon reactivation from latency. The PRV protein Us9 plays an essential but unknown role in anterograde viral spread. To determine Us9 function, we identified viral and host proteins that interact with Us9 and explored the role of KIF1A, a microtubule-dependent kinesin-3 motor involved in axonal sorting and transport. Viral particles are cotransported with KIF1A in axons ofÂ primary rat superior cervical ganglion neurons, and overexpression or disruption of KIF1A function, respectively, increases and reduces anterograde capsid transport. Us9 and KIF1A interact early during infection with the aid of additional viral protein(s) but exhibit diminished binding at later stages, when capsids typically stall in axons. Thus, alphaherpesviruses repurpose the axonal transport and sorting pathway to spread within their hosts.

Kinesin-3 mediates axonal sorting and directional transport of alphaherpesvirus particles in neurons.

Alphaherpes viruses, such as pseudorabies virus (PRV), undergo anterograde transport in neuronal axons to facilitate anterograde spread within hosts. Axonal sorting and anterograde transport of virions is dependent on the viral membrane protein Us9, which interacts with the host motor protein Kif1A to direct transport. Us9-Kif1A interactions are necessary but not sufficient for these processes, indicating that additional cofactors or post-translational modifications are needed. In this study, we characterized two conserved serine phosphorylation sites (S51 and S53) in the PRV Us9 protein that are necessary for anterograde spread in vivo. We assessed the subcellular localization of phospho-Us9 subspecies during infection of neurons and found that the phospho-form is detectable on the majority, but not all, of axonal vesicles containing Us9 protein. In biochemical assays, phospho-Us9 was enriched in lipid raft membrane microdomains, though Us9 phosphorylation did not require prior lipid raft association. During infections of chambered neuronal cultures, we observed only a modest reduction in anterograde spread capacity for diserine mutant Us9, and no defect for monoserine mutants. Conversely, mutation of the kinase recognition sequence residues adjacent to the phosphorylation sites completely abrogated anterograde spread. In live-cell imaging analyses, anterograde transport of virions was reduced during infection with a recombinant PRV strain expressing GFP-tagged diserine mutant Us9. Phosphorylation was not required for Us9-Kif1A interaction, suggesting that Us9-Kif1A binding is a distinct step from the activation and/or stabilization of the transport complex. Taken together, our findings indicate that, while not essential, Us9 phosphorylation enhances Us9-Kif1A-based transport of virions in axons to modulate the overall efficiency of long-distance anterograde spread of infection.

Role of Us9 phosphorylation in axonal sorting and anterograde transport of pseudorabies virus.

Alphaherpesviruses, including pseudorabies virus (PRV), spread directionally within the nervous systems of their mammalian hosts. Three viral membrane proteins are required for efficient anterograde-directed spread of infection in neurons, including Us9 and a heterodimer composed of the glycoproteins gE and gI. We previously demonstrated that the kinesin-3 motor KIF1A mediates anterograde-directed transport of viral particles in axons of cultured peripheral nervous system (PNS) neurons. The PRV Us9 protein copurifies with KIF1A, recruiting the motor to transport vesicles, but at least one unidentified additional viral protein is necessary for this interaction. Here we show that gE/gI are required for efficient anterograde transport of viral particles in axons by mediating the interaction between Us9 and KIF1A. In the absence of gE/gI, viral particles containing green fluorescent protein (GFP)-tagged Us9 are assembled in the cell body but are not sorted efficiently into axons. Importantly, we found that gE/gI are necessary for efficient copurification of KIF1A with Us9, especially at early times after infection. We also constructed a PRV recombinant that expresses a functional gE-GFP fusion protein and used affinity purification coupled with mass spectrometry to identify gE-interacting proteins. Several viral and host proteins were found to associate with gE-GFP. Importantly, both gI and Us9, but not KIF1A, copurified with gE-GFP. We propose that gE/gI are required for efficient KIF1A-mediated anterograde transport of viral particles because they indirectly facilitate or stabilize the interaction between Us9 and KIF1A.

Glycoproteins gE and gI Are Required for Efficient KIF1A-Dependent Anterograde Axonal Transport of Alphaherpesvirus Particles in Neurons.

The use of genetically encodable calcium indicator proteins to monitor neuronal activity is hampered by slow response times and a narrow Ca(2+)-sensitive range. Here we identify three performance-limiting features of GCaMP3, a popular genetically encodable calcium indicator protein. First, we find that affinity is regulated by the calmodulin domain's Ca(2+)-chelating residues. Second, we find that off-responses to Ca(2+) are rate-limited by dissociation of the RS20 domain from calmodulin's hydrophobic pocket. Third, we find that on-responses are limited by fast binding to the N-lobe at high Ca(2+) and by slow binding to the C-lobe at lower Ca(2+). We develop Fast-GCaMPs, which have up to 20-fold accelerated off-responses and show that they have a 200-fold range of KD, allowing coexpression of multiple variants to span an expanded range of Ca(2+) concentrations. Finally, we show that Fast-GCaMPs track natural song in Drosophila auditory neurons and generate rapid responses in mammalian neurons, supporting the utility of our approach.

Matthew P. Taylor

Department of Immunology and Infectious Diseases

Montana State University

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Matthew P. Taylor

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Montana State University

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Department of Immunology and Infectious Diseases