The cytoplasmic compartments occupied by exocytosing herpes simplex virus (HSV) are poorly defined. It is unclear which organelles contain the majority of trafficking virions and which are occupied by virions on a productive rather than defective assembly pathway. These problems are compounded by the fact that HSVinfected cells produce virus continuously over many hours. All stages in viral assembly and export therefore coexist, making it impossible to determine the sequence of events and their kinetics. To address these problems, we have established assays to monitor the presence of capsids and enveloped virions in cell extracts and prepared HSV-containing organelles from normally infected cells and from cells undergoing a single synchronized wave of viral egress. We find that, in both cases, HSV particles exit the nucleus and accumulate in organelles which cofractionate with the trans-Golgi network (TGN) and endosomes. In addition to carrying enveloped infectious virions in their lumen, HSV-bearing organelles also displayed nonenveloped capsids attached to their cytoplasmic surface. Neutralization of organellar pH by chloroquine or bafilomycin A resulted in the accumulation of noninfectious enveloped particles. We conclude that the organelles of the TGN/endocytic network play a key role in the assembly and trafficking of infectious HSV.The mechanisms of envelopment and intracellular trafficking of herpes simplex virus (HSV) are poorly understood. A number of ultrastructural studies have shown that viral nucleocapsids containing packaged DNA are assembled in the infected cell nucleus and then bud through the inner nuclear membrane into the perinuclear space, acquiring an envelope (62,63,66). However, the interpretation of images representing subsequent stages in virus egress has been controversial. Electron microscopy reveals that the infected cell cytoplasm contains capsids partially wrapped by adjacent membranes, enveloped virions completely enclosed within membranous vesicles, and "naked" capsids entirely lacking envelopes (4,14,38,41,72).Two principal HSV egress models have been proposed to account for these various cytoplasmic structures (27,80). In one model, it has been suggested that perinuclear enveloped virions enter transport vesicles budding from the outer nuclear membrane or endoplasmic reticulum (ER) and then traffic as normal secretory pathway cargo through the Golgi apparatus and are eventually secreted from the cell (25,38,66,72). In this model, naked cytoplasmic capsids arise due to nonproductive, erroneous deenvelopment, wherein the envelope of trafficking virions becomes fused with the surrounding lipid bilayer of the transport vesicle. Increased accumulation of naked capsids in the cytoplasm of cells infected by an HSV strain mutant in glycoprotein gD supports the view that premature fusion can generate nonenveloped capsids (14); however, it is not clear if such a phenomenon is the source of naked capsids during a wild-type viral infection.In an alternative model (65), perinuclear virions u...

During Herpes simplex virus envelopment, capsids, tegument polypeptides, and membrane proteins assemble at the site of budding and a cellular lipid bilayer becomes refashioned into a spherical envelope. Though the molecular interactions driving these events are poorly understood, several lines of evidence suggest that associations between envelope protein cytoplasmic tails and tegument polypeptides may play important roles. Consistent with this hypothesis, we show here that a fusion of the cytoplasmic tail of gH with Glutathione-S-Transferase binds to VP16 in a temperature-dependent manner. VP16 prepared by in vitro translation behaves in a similar fashion, demonstrating that the interaction is not dependent on other viral polypeptides. Mutational analysis of the gH tail has also enabled us to identify amino acid residues critical for VP16 binding in vitro. A fusion protein in which the gH tail is fused to the carboxy-terminus of GFP coimmunoprecipitates with VP16 in infected cells, indicating that VP16 can interact with the gH tail in vivo.

The determinants of transmembrane protein insertion orientation at the endoplasmic reticulum have been investigated in Saccharomyces cerevisiae using variants of a Type III (naturally exofacial N terminus (N exo )) transmembrane fusion protein derived from the N terminus of Ste2p, the ␣-factor receptor. Small positive and negative charges adjacent to the transmembrane segment had equal and opposite effects on orientation, and this effect was independent of N-or C-terminal location, consistent with a purely electrostatic interaction with response mechanisms. A 3:1 bias toward N exo insertion, observed in the absence of a charge difference, was shown to reflect the N exo bias conferred by longer transmembrane segments. Orientation correlated best with total hydrophobicity rather than length, but it was also strongly affected by the distribution of hydrophobicity within the transmembrane segment. The most hydrophobic terminus was preferentially translocated. Insertion orientation thus depends on integration of responses to at least three parameters: charge difference across a transmembrane segment, its total hydrophobicity, and its hydrophobicity gradient. Relative signal strengths were estimated, and consequences for topology prediction are discussed. Responses to transmembrane sequence may depend on protein-translocon interactions, but responses to charge difference may be mediated by the electrostatic field provided by anionic phospholipids.

Herpes simplex virus (HSV) capsids assemble, mature and package their viral genome in the nucleoplasm. They then exit the nucleus into the cytoplasm, where they acquire their final tegument and envelope. The molecular mechanism of cytoplasmic envelopment is unclear, but evidence suggests that the viral glycoprotein tails play an important role in the recruitment of tegument and capsids at the final envelopment site. However, due to redundancy in protein-protein interactions among the viral glycoproteins, genetic analysis of the role of individual glycoproteins in assembly has been difficult. To overcome this problem, a glutathione S-transferase fusion protein-binding assay was used in this study to test the interaction between the cytoplasmic tail of one specific viral glycoprotein, gD, and tegument proteins. The study demonstrated that the 38 kDa tegument protein VP22 bound specifically to the gD tail. This association was dependent on arginine and lysine residues at positions 5 and 6 in the gD tail. In addition, HSV-1 capsids bound the gD tail and exhibited a similar sequence dependence. It is concluded that VP22 may serve as a linker protein, mediating the interaction of the HSV capsid with gD.

Yeast protein insertion orientation (PIO) mutants were isolated by selecting for growth on sucrose in cells in which the only source of invertase is a C-terminal fusion to a transmembrane protein.Only the fraction with an exocellular C terminus can be processed to secreted invertase and this fraction is constrained to 2-3% by a strong charge difference signal. Identified pio mutants increased this to 9 -12%. PIO1 is SPF1, encoding a P-type ATPase located in the endoplasmic reticulum (ER) or Golgi. spf1-null mutants are modestly sensitive to EGTA. Sensitivity is considerably greater in an spf1 pmr1 double mutant, although PIO is not further disturbed. Pmr1p is the Golgi Ca 2ϩ ATPase and Spf1p may be the equivalent ER pump. PIO2 is STE24, a metalloprotease anchored in the ER membrane. Like Spf1p, Ste24p is expressed in all yeast cell types and belongs to a highly conserved protein family. The effects of ste24-and spf1-null mutations on invertase secretion are additive, cell generation time is increased 60%, and cells become sensitive to cold and to heat shock. Ste24p and Rce1p cleave the C-AAX bond of farnesylated CAAX box proteins. The closest paralog of SPF1 is YOR291w. Neither rce1-null nor yor291w-null mutations affected PIO or the phenotype of spf1-or ste24-null mutants. Mutations in PIO3 (unidentified) cause a weaker Pio phenotype, enhanced by a null mutation in BMH1, one of two yeast 14-3-3 proteins.