Herpes simplex virus type 1 (HSV-1) acquires its mature virus envelope by budding into the lumen of cytoplasmic membranous compartments carrying the viral glycoproteins. In a cellular context, a budding process with identical topology occurs during the formation of intraluminal vesicles in multivesicular bodies. The cellular machinery that mediates this budding process is composed of four protein complexes termed endosomal sorting complexes required for transport (ESCRTs) and several associated proteins, including the ATPase VPS4. We have recently shown that functional VPS4 is specifically required for the cytoplasmic envelopment of HSV-1. We now demonstrate that, consistent with a role of VPS4 in virus envelopment, dominant-negative ESCRT-III proteins potently block HSV-1 production. Retroviruses are known to recruit the ESCRT machinery by small peptide motifs termed late domains. These late domains interact with various ESCRT components and thereby promote ESCRT recruitment. The best-characterized late-domain interacting ESCRT proteins are ALIX and TSG101. The presence of potential ALIX and TSG101 binding sequence motifs in various structural HSV-1 proteins suggested a functional role of these proteins in HSV-1 envelopment. We therefore used a set of dominant-negative proteins, as well as RNA interference, to characterize the contribution of ALIX and TSG101 to HSV-1 production. Interestingly, despite the strict requirement for a functional ESCRT-III complex, our data suggest that HSV-1 production is independent of ALIX and TSG101 expression. In line with these data, we also find that ESCRT-III proteins and VPS4A/B are specifically incorporated into mature HSV-1 virions.Herpes simplex virus type 1 (HSV-1), a human pathogen, is a member of the large family of Herpesviridae. Herpesviruses are pleiomorphic enveloped particles that contain a doublestranded DNA genome. The viral genome is surrounded by an icosahedral capsid shell that is wrapped in a proteinaceous layer named the tegument. The tegumented capsid is enveloped by a cell-derived lipid bilayer in which numerous virally encoded envelope proteins are embedded.Herpesviruses acquire their final envelope in the cytoplasm of the host cell in a process that is termed secondary envelopment. During secondary envelopment tegument-coated nucleocapsids bud into the lumen of vesicles that are thought to be derived from the trans-Golgi network. Mature virus particles are then released from the cell in an exocytic manner by fusion of this virus-containing compartment with the plasma membrane (reviewed in reference 26).Cellular processes that are topologically related to secondary envelopment of herpesviruses are (i) the formation of intraluminal vesicles within multivesicular bodies and (ii) the final abscission step of cytokinesis. Furthermore, the secondary envelopment of herpesviruses is topologically related to the budding process of many enveloped RNA viruses. All of the above-mentioned processes involve membrane deformation and fission events that are mediated by...

Focal adhesions are macromolecular complexes that connect the actin cytoskeleton to the extracellular matrix. Dynamic turnover of focal adhesions is crucial for cell migration. Paxillin is a multiadaptor protein that plays an important role in regulating focal adhesion dynamics. Here, we identify TRIM15, a member of the tripartite motif protein family, as a paxillin-interacting factor and a component of focal adhesions. TRIM15 localizes to focal contacts in a myosin-II-independent manner by an interaction between its coiled-coil domain and the LD2 motif of paxillin. Unlike other focal adhesion proteins, TRIM15 is a stable focal adhesion component with restricted mobility due to its ability to form oligomers. TRIM15-depleted cells display impaired cell migration and reduced focal adhesion disassembly rates, in addition to enlarged focal adhesions. Thus, our studies demonstrate a cellular function for TRIM15 as a regulatory component of focal adhesion turnover and cell migration.

Focal adhesions are macromolecular complexes that connect the actin cytoskeleton to the extracellular matrix. Dynamic turnover of focal adhesions is crucial for cell migration. Paxillin is a multiadaptor protein that plays an important role in regulating focal adhesion dynamics. Here, we identify TRIM15, a member of the tripartite motif protein family, as a paxillin-interacting factor and a component of focal adhesions. TRIM15 localizes to focal contacts in a myosin-II-independent manner by an interaction between its coiled-coil domain and the LD2 motif of paxillin. Unlike other focal adhesion proteins, TRIM15 is a stable focal adhesion component with restricted mobility due to its ability to form oligomers. TRIM15depleted cells display impaired cell migration and reduced focal adhesion disassembly rates, in addition to enlarged focal adhesions. Thus, our studies demonstrate a cellular function for TRIM15 as a regulatory component of focal adhesion turnover and cell migration.