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 (U _L 7, U _L 23, U _L 50, and U _L 55), and two teguments were confirmed (ICP0 and ICP4). In contrast, U _L 4, U _L 24, the U _L 31/U _L 34 complex, and the viral U _L 15/U _L 28/U _L 33 terminase were undetected, as was most of the viral replication machinery, with the notable exception of U _L 23. Surprisingly, the viral glycoproteins gJ, gK, gN, and U _L 43 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.

Rab GTPases are central elements of the vesicular transport machinery. An emerging view is that downstream effectors of these GTPases are multiprotein complexes that include nucleotide exchange factors to ensure coupling between GTPase activation and effector function. We have previously shown that Rab5, which regulates various steps of transport along the early endocytic pathway, is activated by a complex consisting of Rabex-5, a Rab5 nucleotide exchange factor, and the effector Rabaptin-5. We postulated that the physical association of these two proteins is necessary for their activity in Rab5-dependent endocytic membrane transport. To evaluate the functional implications of such complex formation, we have reconstituted it with the use of recombinant proteins and characterized its properties. First, we show that Rabaptin-5 increases the exchange activity of Rabex-5 on Rab5. Second, Rab5-dependent recruitment of Rabaptin-5 to early endosomes is completely dependent on its physical association with Rabex-5. Third, complex formation between Rabaptin-5 and Rabex-5 is essential for early endosome homotypic fusion. These results reveal a functional synergy between Rabaptin-5 and Rabex-5 in the complex and have implications for the function of analogous complexes for Rab and Rho GTPases.

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.