GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidylinositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5. Excess Rab5-GTP complex restores endosome fusion when PI(3)K is inhibited. Here we identify the early-endosomal autoantigen EEA1 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.

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.

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. Herpes simplex virus type 1 (HSV-1) is a multilayered particle composed of a DNA core surrounded by a capsid, a tegument, and finally an envelope. The tegument consists of several proteins that are critical for the virus. For instance, upon the virus' entry into the cell, the tegument likely directs the virus to the nucleus (20, 33, 52, 90). There, the U L 36 tegument protein anchors the capsid to the nuclear pore to enable viral DNA transfer into the nucleus (90). Three other teguments, namely, ICP0, ICP4, and U L 48 (VP16), then play an essential role in initiating viral transcription (24). Meanwhile, the U L 41 (VHS) tegument specifically degrades some mRNAs to the benefit of the virus (93, 94). During egress, passage of the newly assembled capsids across the two nuclear membranes relies on the U L 31 (tegument)/U L 34 (transmembrane protein) complex, as well as the U S 3 tegument (46,81). Interestingly, despite the involvement of all three proteins in nuclear viral egress, only U S 3 is found in mature virions (81).

Viral proteins are usually processed by the ‘classical’ major histocompatibility complex (MHC) class I presentation pathway. Here we showed that although macrophages infected with herpes simplex virus type 1 (HSV-1) initially stimulated CD8+ T cells by this pathway, a second pathway involving a vacuolar compartment was triggered later during infection. Morphological and functional analyses indicated that distinct forms of autophagy facilitated the presentation of HSV-1 antigens on MHC class I molecules. One form of autophagy involved a previously unknown type of autophagosome that originated from the nuclear envelope. Whereas interferon-γ stimulated classical MHC class I presentation, fever-like hyperthermia and the pyrogenic cytokine interleukin 1β activated autophagy and the vacuolar processing of viral peptides. Viral peptides in autophagosomes were further processed by the proteasome, which suggests a complex interaction between the vacuolar and MHC class I presentation pathways.

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 reticulumGolgi 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.The release of newly assembled herpesviruses requires passage through several host membranes by mechanisms that are poorly understood. Following their assembly and maturation in the nucleus, the capsids acquire a primary envelope by budding through the inner nuclear membrane (16,58,82) to end up in the perinuclear space, which is contiguous with the endoplasmic reticulum (ER) lumen. One model suggests these perinuclear virions escape the cell via the host biosynthetic pathway, which requires an obligatory transit through the Golgi (16, 44). However, the currently favored model proposes that the enveloped perinuclear capsids fuse with the outer nuclear membrane to produce naked cytosolic capsids (81,82). These would in turn acquire a secondary envelope downstream from an intracellular compartment, before reaching the plasma membrane and being released extracellularly by a second fusion event. This reenvelopment model appears valid for several, if not all, members of the herpesvirus family and is supported by several approaches, including electron microscopy (EM), immunofluorescence, freeze fracture, lipid content, as well as analysis of the site of tegument addition and the use of various viral mutants (23,53,54).Herpes simplex virus type 1 (HSV-1) is a member of the herpes family that has extensively been studied for egress. Unfortunately, its relatively short life cycle makes it difficult to analyze the vectorial movement of the virus during its rapid egress. Furthermore, EM analysis often gives a static snapshot without detailed information ...