Roger Lippé

EEA1 links PI(3)K function to Rab5 regulation of endosome fusion.

GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidyl-inositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5 (ref. 8). Excess Rab5–GTP complex restores endosome fusion when PI(3)K is inhibited,. Here we identify the early-endosomal autoantigen EEA1 (refs 10–12) which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion. The association of EEA1 with the endosomal membrane requires Rab5–GTP and PI(3)K activity, and excess Rab5–GTP stabilizes the membrane association of EEA1 even when PI(3)K is inhibited. The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.

A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function

The small GTPase Rab5 plays an essential role in endocytic traffic. Rab GDP dissociation inhibitor delivers Rab5 to the membrane, where a nucleotide exchange activity allows recruitment of an effector protein, Rabaptin-5. Here we uncovered a novel 60 kDa Rab5-binding protein, Rabex-5. Rabex-5 forms a tight physical complex with Rabaptin-5, and this complex is essential for endocytic membrane fusion. Sequencing of mammalian Rabex-5 by nanoelectrospray mass spectrometry and cloning revealed striking homology to Vps9p, a yeast protein implicated in endocytic traffic. Rabex-5 displays GDP/GTP exchange activity on Rab5 upon delivery of the GTPase to the membrane. This demonstrates that a soluble exchange factor coupled to a Rab effector translocates from cytosol to the membrane, where the complex stabilizes the GTPase in the active state.

Comprehensive characterization of extracellular herpes simplex virus type 1 virions.

ABSTRACT The herpes simplex virus type 1 (HSV-1) genome is contained in a capsid wrapped by a complex tegument layer and an external envelope. The poorly defined tegument plays a critical role throughout the viral life cycle, including delivery of capsids to the nucleus, viral gene expression, capsid egress, and acquisition of the viral envelope. Current data suggest tegumentation is a dynamic and sequential process that starts in the nucleus and continues in the cytoplasm. Over two dozen proteins are assumed to be or are known to ultimately be added to virions as tegument, but its precise composition is currently unknown. Moreover, a comprehensive analysis of all proteins found in HSV-1 virions is still lacking. To better understand the implication of the tegument and host proteins incorporated into the virions, highly purified mature extracellular viruses were analyzed by mass spectrometry. The method proved accurate (95%) and sensitive and hinted at 8 different viral capsid proteins, 13 viral glycoproteins, and 23 potential viral teguments. Interestingly, four novel virion components were identified (UL7, UL23, UL50, and UL55), and two teguments were confirmed (ICP0 and ICP4). In contrast, UL4, UL24, the UL31/UL34 complex, and the viral UL15/UL28/UL33 terminase were undetected, as was most of the viral replication machinery, with the notable exception of UL23. Surprisingly, the viral glycoproteins gJ, gK, gN, and UL43 were absent. Analyses of virions produced by two unrelated cell lines suggest their protein compositions are largely cell type independent. Finally, but not least, up to 49 distinct host proteins were identified in the virions.

Selective Membrane Recruitment of EEA1 Suggests a Role in Directional Transport of Clathrin-coated Vesicles to Early Endosomes

The molecular mechanisms ensuring directionality of endocytic membrane trafficking between transport vesicles and target organelles still remain poorly characterized. We have been investigating the function of the small GTPase Rab5 in early endocytic transport. In vitro studies have demonstrated a role of Rab5 in two membrane fusion events: the heterotypic fusion between plasma membrane-derived clathrin-coated vesicles (CCVs) and early endosomes and in the homotypic fusion between early endosomes. Several Rab5 effectors are required in homotypic endosome fusion, including EEA1, which mediates endosome membrane docking, as well as Rabaptin-5·Rabex-5 complex and phosphatidylinositol 3-kinase hVPS34. In this study we have examined the localization and function of Rab5 and its effectors in heterotypic fusion in vitro. We report that the presence of active Rab5 is necessary on both CCVs and early endosomes for a heterotypic fusion event to occur. This process requires EEA1 in addition to the Rabaptin-5 complex. However, whereas Rab5 and Rabaptin-5 are symmetrically distributed between CCVs and early endosomes, EEA1 is recruited selectively onto the membrane of early endosomes. Our results suggest that EEA1 is a tethering molecule that provides directionality to vesicular transport from the plasma membrane to the early endosomes.

Herpes Simplex Virus Type 1 Capsids Transit by the trans-Golgi Network, Where Viral Glycoproteins Accumulate Independently of Capsid Egress

ABSTRACT Egress of herpes capsids from the nucleus to the plasma membrane is a complex multistep transport event that is poorly understood. The current model proposes an initial envelopment at the inner nuclear membrane of capsids newly assembled in the nucleus. The capsids are then released in cytosol by fusion with the outer nuclear membrane. They are finally reenveloped at a downstream organelle before traveling to the plasma membrane for their extracellular release. Although the trans-Golgi network (TGN) is often cited as a potential site of reenvelopment, other organelles have also been proposed, including the Golgi, endoplasmic reticulum-Golgi intermediate compartment, aggresomes, tegusomes, and early or late endosomes. To clarify this important issue, we followed herpes simplex virus type 1 egress by immunofluorescence under conditions that slowed intracellular transport and promoted the accumulation of the otherwise transient reenvelopment intermediate. The data show that the capsids transit by the TGN and point to this compartment as the main reenvelopment site, although a contribution by endosomes cannot formally be excluded. Given that viral glycoproteins are expected to accumulate where capsids acquire their envelope, we examined this prediction and found that all tested could indeed be detected at the TGN. Moreover, this accumulation occurred independently of capsid egress. Surprisingly, capsids were often found immediately adjacent to the viral glycoproteins at the TGN.

Two distinct effectors of the small GTPase Rab5 cooperate in endocytic membrane fusion.

Using the yeast two‐hybrid system, we have identified a novel 62 kDa coiled‐coil protein that specifically interacts with the GTP‐bound form of Rab5, a small GTPase that regulates membrane traffic in the early endocytic pathway. This protein shares 42% sequence identity with Rabaptin‐5, a previously identified effector of Rab5, and we therefore named it Rabaptin‐5β. Like Rabaptin‐5, Rabaptin‐5β displays heptad repeats characteristic of coiled‐coil proteins and is recruited on the endosomal membrane by Rab5 in a GTP‐dependent manner. However, Rabaptin‐5β has features that distinguish it from Rabaptin‐5. The relative expression levels of the two proteins varies in different cell types. Rabaptin‐5β does not heterodimerize with Rabaptin‐5, and forms a distinct complex with Rabex‐5, the GDP/GTP exchange factor for Rab5. Immunodepletion of the Rabaptin‐5β complex from cytosol only partially inhibits early endosome fusion in vitro, whereas the additional depletion of the Rabaptin‐5 complex has a stronger inhibitory effect. Fusion activity can mostly be recovered by addition of the Rabaptin‐5 complex alone, but maximal fusion efficiency requires the presence of both Rabaptin‐5 and Rabaptin‐5β complexes. Our results suggest that Rab5 binds to at least two distinct effectors which cooperate for optimal endocytic membrane docking and fusion.

Protein Kinase D-Dependent Trafficking of the Large Herpes simplex Virus Type 1 Capsids from the TGN to Plasma Membrane

The biosynthetic pathway carries cargos from the endoplasmic reticulum (ER) to the trans Golgi network (TGN) via a typical passage through the Golgi. Interestingly, large particles such as procollagen, chylomicrons and some viruses all reach the TGN by atypical routes. Given this dichotomy, we anticipated that such cargos might rely on non‐classical machineries downstream of the TGN. Using Herpes simplex virus type 1 (HSV‐1) as a model and a synchronized infection protocol that focuses on TGN to plasma membrane transport, the present study revealed the surprising implication of the cellular serine‐threonine protein kinase D in HSV‐1 egress. These findings, confirmed by a variety of complementary means [pharmacological inhibitors, dominant negative mutant, RNA interference and electron microscopy (EM)], identify one of possibly several cellular factors that modulate the egress of viruses transiting at the TGN. Moreover, the involvement of this kinase, previously known to regulate the transport of small basolateral cargos, highlights the trafficking of both small and exceptionally large entities by a common machinery downstream of the TGN, in sharp contrast to earlier steps of transport. Conceptually, this indicates the TGN is not only a sorting station from which cargos can depart towards different destinations but also a meeting point where conventional and unconventional routes can meet along the biosynthetic pathway. Lastly, given the apical release of HSV‐1 in neurons, it opens up the possibility that this kinase might regulate some apical sorting.

Analysis of virion-incorporated host proteins required for herpes simplex virus type 1 infection through a RNA interference screen.

Viruses are strictly dependent on cells to propagate and many incorporate host proteins in their viral particles, but the significance of this incorporation is poorly understood. Recently, we performed the first comprehensive characterization of the mature herpes simplex virus type 1 (HSV-1) in which up to 49 distinct cellular proteins were identified by mass spectrometry. In the present study, we sought to identify if these cellular factors are relevant for the HSV-1 life cycle. To this end, we performed a small interfering RNA functional screen and found that 15 of these host proteins altered HSV-1 proliferation in cell culture, without any significant effect on cell viability. Moreover, the siRNA used had no negative consequences for Adenovirus type 5 propagation (with one exception) indicating that the modulation was specific for HSV-1 and not merely due to unhealthy cells. The positive host proteins include several Rab GTPases and other intracellular transport components as well as proteins involved in signal transduction, gene regulation and immunity. Remarkably, in most cases when virions were depleted for one of the above proteins, they replicated more poorly in subsequent infections in wild type cells. This highlights for the first time that both the cellular and virion-associated pools of many of these proteins actively contribute to viral propagation. Altogether, these findings underscore the power and biological relevance of combining proteomics and RNA interference to identify novel host-pathogen interactions.

Herpesviruses Exploit Several Host Compartments for Envelopment

Enveloped viruses acquire their host‐derived membrane at a variety of intracellular locations. Herpesviruses are complex entities that undergo several budding and fusion events during an infection. All members of this large family are believed to share a similar life cycle. However, they seemingly differ in terms of acquisition of their mature envelope. Herpes simplex virus is often believed to bud into an existing intracellular compartment, while the related cytomegalovirus may acquire its final envelope from a novel virus‐induced assembly compartment. This review focuses on recent advances in the characterization of cellular compartment(s) potentially contributing to herpes virion final envelopment. It also examines the common points between seemingly distinct envelopment pathways and highlights the dynamic nature of intracellular compartments in the context of herpesvirus infections.

Deciphering novel host-herpesvirus interactions by virion proteomics.

Over the years, a vast array of information concerning the interactions of viruses with their hosts has been collected. However, recent advances in proteomics and other system biology techniques suggest these interactions are far more complex than anticipated. One particularly interesting and novel aspect is the analysis of cellular proteins incorporated into mature virions. Though sometimes considered purification contaminants in the past, their repeated detection by different laboratories suggests that a number of these proteins are bona fide viral components, some of which likely contribute to the viral life cycles. The present mini review focuses on cellular proteins detected in herpesviruses. It highlights the common cellular functions of these proteins, their potential implications for host–pathogen interactions, discusses technical limitations, the need for complementing methods and probes potential future research avenues.