Jason Mercer

Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host Cells

Viruses employ many different strategies to enter host cells. Vaccinia virus, a prototype poxvirus, enters cells in a pH-dependent fashion. Live cell imaging showed that fluorescent virus particles associated with and moved along filopodia to the cell body, where they were internalized after inducing the extrusion of large transient membrane blebs. p21-activated kinase 1 (PAK1) was activated by the virus, and the endocytic process had the general characteristics of macropinocytosis. The induction of blebs, the endocytic event, and infection were all critically dependent on the presence of exposed phosphatidylserine in the viral membrane, which suggests that vaccinia virus uses apoptotic mimicry to enter cells.

Virus entry by macropinocytosis.

As obligatory intracellular parasites, viruses rely on host-cell functions for most aspects of their replication cycle. This is born out during entry, when most viruses that infect vertebrate and insect cells exploit the endocytic activities of the host cell to move into the cytoplasm. Viruses belonging to vaccinia, adeno, picorna and other virus families have been reported to take advantage of macropinocytosis, an endocytic mechanism normally involved in fluid uptake. The virus particles first activate signalling pathways that trigger actin-mediated membrane ruffling and blebbing. Usually, this is followed by the formation of large vacuoles (macropinosomes) at the plasma membrane, internalization of virus particles and penetration by the viruses or their capsids into the cytosol through the limiting membrane of the macropinosomes. We review the molecular machinery involved in macropinocytosis and describe what is known about its role in virus entry.

Subversion of CtBP1‐controlled macropinocytosis by human adenovirus serotype 3

Endocytosis supports cell communication, growth, and pathogen infection. The species B human adenovirus serotype 3 (Ad3) is associated with epidemic conjunctivitis, and fatal respiratory and systemic disease. Here we show that Ad3 uses dynamin‐independent endocytosis for rapid infectious entry into epithelial and haematopoietic cells. Unlike Ad5, which uses dynamin‐dependent endocytosis, Ad3 endocytosis spatially and temporally coincided with enhanced fluid‐phase uptake. It was sensitive to macropinocytosis inhibitors targeting F‐actin, protein kinase C, the sodium–proton exchanger, and Rac1 but not Cdc42. Infectious Ad3 macropinocytosis required viral activation of p21‐activated kinase 1 (PAK1) and the C‐terminal binding protein 1 of E1A (CtBP1), recruited to macropinosomes. These macropinosomes also contained the Ad3 receptors CD46 and αv integrins. CtBP1 is a phosphorylation target of PAK1, and is bifunctionally involved in membrane traffic and transcriptional repression of cell cycle, cancer, and innate immunity pathways. Phosphorylation‐defective S147A‐CtBP1 blocked Ad3 but not Ad5 infection, providing a direct link between PAK1 and CtBP1. The data show that viruses induce macropinocytosis for infectious entry, a pathway used in antigen presentation and cell migration.

Gulping rather than sipping: macropinocytosis as a way of virus entry

Macropinocytosis has emerged as a major endocytic mechanism in the cell entry of animal viruses. The process differs fundamentally from other endocytic mechanisms involved in virus internalization. By activating growth factor receptors or other signaling molecules, plasma membrane-bound viruses trigger the activation of a signaling pathway. When amplified, this causes a transient, global change in cell behavior. The consequences of this change include the actin-dependent formation of membrane protrusions, the elevation of non-specific uptake of fluid, and the internalization of membrane together with surface-bound ligands and particles including viruses. Recent studies show that this strategy is used by a variety of enveloped and non-enveloped viruses.

Vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry

To enter host cells, vaccinia virus, a prototype poxvirus, can induce transient macropinocytosis followed by endocytic internalization and penetration through the limiting membrane of pinosomes by membrane fusion. Although mature virions (MVs) of the Western reserve (WR) strain do this in HeLa cells by activating transient plasma membrane blebbing, MVs from the International Health Department-J strain were found to induce rapid formation (and lengthening) of filopodia. When the signaling pathways underlying these responses were compared, differences were observed at the level of Rho GTPases. Key to the filopodial formation was the virus-induced activation of Cdc42, and for the blebbing response the activation of Rac1. In addition, unlike WR, International Health Department-J MVs did not rely on genistein-sensitive tyrosine kinase and PI(3)K activities. Only WR MVs had membrane fusion activity at low pH. Inhibitor profiling showed that MVs from both strains entered cells by macropinocytosis and that this was induced by virion-exposed phosphatidylserine. Both MVs relied on the activation of epidermal growth factor receptor, on serine/threonine kinases, protein kinase C, and p21-activated kinase 1. The results showed that different strains of the same virus can elicit dramatically different responses in host cells during entry, and that different macropinocytic mechanisms are possible in the same cell line through subtle differences in the activating ligand.

Single-cell analysis of population context advances RNAi screening at multiple levels

Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cells microenvironment, in image‐based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single‐cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome‐wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell‐based screens at this depth reveals widespread RNAi‐induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell‐to‐cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large‐scale RNAi screens are increasingly performed to reach a systems‐level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single‐cell microenvironment.

RNAi screening reveals proteasome- and Cullin3-dependent stages in vaccinia virus infection.

A two-step, automated, high-throughput RNAi silencing screen was used to identify host cell factorsxa0required during vaccinia virus infection. Validation and analysis of clustered hits revealed previously unknown processes during virus entry, including a mechanism for genome uncoating. Viral core proteins were found to be already ubiquitinatedxa0during virus assembly. After entering the cytosol of an uninfected cell, the viral DNA was released from the core through the activity of the cells proteasomes. Next, a Cullin3-based ubiquitin ligase mediated a further round of ubiquitination and proteasome action. This was needed in order to initiate viral DNA replication. The results accentuate the value of large-scale RNAi screens in providing directions for detailed cell biological investigation of complex pathways. The list of cell functions required during poxvirus infection will, moreover, provide a resource for future virus-host cell interaction studies and for the discovery of antivirals.

Tracking Viral Genomes in Host Cells at Single-Molecule Resolution

Viral DNA trafficking in cells has large impacts onxa0physiology and disease development. Current methods lack the resolution and accuracy to visualize and quantify viral DNA trafficking at single-molecule resolution. We developed a noninvasive protocol for accurate quantification of viral DNA-genome (vDNA) trafficking in single cells. Ethynyl-modified nucleosides were used to metabolically label newly synthesized adenovirus, herpes virus, and vaccinia virus vDNA, without affecting infectivity. Superresolution microscopy and copper(I)-catalyzed azide-alkyne cycloaddition (click) reactions allowed visualization of infection at single vDNA resolution within mammalian cells. Analysis of adenovirus infection revealed that a large pool of capsid-free vDNA accumulated in the cytosol upon virus uncoating, indicating that nuclear import of incoming vDNA is a bottleneck. The method described here isxa0applicable for the entire replication cycle of DNA viruses and offers opportunities to localize cellular and viral effector machineries on newly replicated viral DNA, or innate immune sensors on cytoplasmic viral DNA.

Vaccinia extracellular virions enter cells by macropinocytosis and acid‐activated membrane rupture

Vaccinia virus (VACV), the model poxvirus, produces two types of infectious particles: mature virions (MVs) and extracellular virions (EVs). EV particles possess two membranes and therefore require an unusual cellular entry mechanism. By a combination of fluorescence and electron microscopy as well as flow cytometry, we investigated the cellular processes that EVs required to infect HeLa cells. We found that EV particles were endocytosed, and that internalization and infection depended on actin rearrangements, activity of Na+/H+ exchangers, and signalling events typical for the macropinocytic mechanism of endocytosis. To promote their internalization, EVs were capable of actively triggering macropinocytosis. EV infection also required vacuolar acidification, and acid exposure in endocytic vacuoles was needed to disrupt the outer EV membrane. Once exposed, the underlying MV‐like particle presumably fused its single membrane with the limiting vacuolar membrane. Release of the viral core into the host cell cytosol allowed for productive infection.

Virus interactions with endocytic pathways in macrophages and dendritic cells

Macrophages and dendritic cells (DCs) are at the front line of defence against fungi, bacteria, and viruses. Together with physical barriers, such as mucus and a range of antimicrobial compounds, they constitute a major part of the intrinsic and innate immune systems. They have elaborate features, including pattern recognition receptors (PRRs) and specialized endocytic mechanisms, cytokines and chemokines, and the ability to call on reserves. As masters of manipulation and counter-attack, viruses shunt intrinsic and innate recognition, enter immune cells, and spread from these cells throughout an organism. Here, we review mechanisms by which viruses subvert endocytic and pathogen-sensing functions of macrophages and DCs, while highlighting possible strategic advantages of infecting cells normally tuned into pathogen destruction.