Synaptojanin is a nerve terminal protein of relative molecular mass 145,000 which appears to participate with dynamin in synaptic vesicle recycling. The central region of synaptojanin defines it as a member of the inositol-5-phosphatase family, which includes the product of the gene that is defective in the oculocerebrorenal syndrome of Lowe. Synaptojanin has 5-phosphatase activity and its amino-terminal domain is homologous with the yeast protein Sac1 (Rsd1), which is genetically implicated in phospholipid metabolism and in the function of the actin cytoskeleton. The carboxy terminus, which is of different lengths in adult and developing neurons owing to the alternative use of two termination sites, is proline-rich, consistent with the reported interaction of synaptojanin with the SH3 domains of Grb2 (refs 1, 2). Synaptojanin is the only other major brain protein besides dynamin that binds the SH3 domain of amphiphysin, a presynaptic protein with a putative function in endocytosis. Our results suggest a link between phosphoinositide metabolism and synaptic vesicle recycling.

Amphiphysin I is an abundant presynaptic protein that interacts via its COOH-terminal src homology 3 (SH3) domain with the GTPase dynamin I and the inositol-5-phosphatase synaptojanin. Both dynamin I and synaptojanin I have a putative role in synaptic vesicle recycling and undergo rapid dephosphorylation in rat brain synaptosomes stimulated to secrete by a depolarizing stimulus. We show here that amphiphysin I also undergoes constitutive phosphorylation and stimulationdependent dephosphorylation. Dephosphorylation of amphiphysin I requires extracellular Ca 2؉ and is unaffected by pretreatment of synaptosomes with tetanus toxin. Thus, Ca 2؉ influx, but not synaptic vesicle exocytosis, is required for dephosphorylation. Dephosphorylation of amphiphysin I, like dephosphorylation of dynamin I and synaptojanin I, is inhibited by cyclosporin A and FK-506 (0.5 M), two drugs that specifically block the Ca 2؉ /calmodulin-dependent phosphatase 2B calcineurin, but not by okadaic acid (1 M), which blocks protein phosphatases 1 and 2B. We also show by immunogold electron microscopy immunocytochemistry that amphiphysin I is localized in the nerve terminal cytomatrix and is partially associated with endocytic intermediates. These include the clathrin-coated buds and dynamin-coated tubules, which accumulate in nerve terminal membranes incubated in the presence of guanosine 5-3-O-(thio)triphosphate. These data support the hypothesis that amphiphysin I, dynamin I, and synaptojanin I are physiological partners in some step(s) of synaptic vesicle endocytosis. We hypothesize that the parallel Ca 2؉

SummaryAs the inner tegument proteins pUL36 and pUL37 of alphaherpesviruses may contribute to efficient intracellular transport of viral particles, we investigated their role in cytosolic capsid motility during assembly of herpes simplex virus type 1 (HSV1). As reported previously for pUL36, untagged pUL37 and UL37GFP bound to cytosolic capsids before these acquired outer tegument and envelope proteins. Capsids tagged with CheVP26 analysed by live cell imaging were capable of directed long-distance cytoplasmic transport during the assembly of wild-type virions, while capsids of the HSV1-DUL37 or HSV1-DUL36 deletion mutants showed only random, undirected motion. The HSV1-DUL37 phenotype was restored when UL37GFP had been overexpressed prior to infection. Quantitative immunoelectron microscopy revealed that capsids of HSV1-DUL37 still recruited pUL36, whereas pUL37 did not colocalize with capsids of HSV1-DUL36. Nevertheless, the cytosolic capsids of neither mutant could undergo secondary envelopment. Our data suggest that pUL36 and pUL37 are important prior to their functions in linking the inner to the outer tegument. Efficient capsid transport to the organelle of secondary envelopment requires recruitment of pUL37 onto capsids, most likely via its interaction with pUL36, while capsid-associated pUL36 alone is insufficient.

The largest tegument protein of herpes simplex virus type 1 (HSV1), pUL36, is a multivalent cross-linker between the viral capsids and the tegument and associated membrane proteins during assembly that upon subsequent cell entry releases the incoming capsids from the outer tegument and viral envelope. Here we show that pUL36 was recruited to cytosolic progeny capsids that later colocalized with membrane proteins of herpes simplex virus type 1 (HSV1) and the trans-Golgi network. During cell entry, pUL36 dissociated from viral membrane proteins but remained associated with cytosolic capsids until arrival at the nucleus. HSV1 UL36 mutants lacking C-terminal portions of increasing size expressed truncated pUL36 but could not form plaques. Cytosolic capsids of mutants lacking the C-terminal 735 of the 3,164 amino acid residues accumulated in the cytosol but did not recruit pUL36 or associate with membranes. In contrast, pUL36 lacking only the 167 C-terminal residues bound to cytosolic capsids and subsequently colocalized with viral and host membrane proteins. Progeny virions fused with neighboring cells, but incoming capsids did not retain pUL36, nor could they target the nucleus or initiate HSV1 gene expression. Our data suggest that residues 2430 to 2893 of HSV1 pUL36, containing one binding site for the capsid protein pUL25, are sufficient to recruit pUL36 onto cytosolic capsids during assembly for secondary envelopment, whereas the 167 residues of the very C terminus with the second pUL25 binding site are crucial to maintain pUL36 on incoming capsids during cell entry. Capsids lacking pUL36 are targeted neither to membranes for virus assembly nor to nuclear pores for genome uncoating. Infections with herpes simplex virus type 1 (HSV1; human alphaherpesvirus 1) cause the common herpes labialis, herpes keratitis, and keratoconjunctivitis, as well as life-threatening neonatal infections, herpes encephalitis in patients with primary immune deficiencies, and eczema herpeticum in patients with atopic dermatitis (46,54,101,102). The virions contain the DNA genomes of 152 kb encased in icosahedral capsids that interact with the surrounding tegument; this protein layer consists of a partially icosahedrally ordered inner portion and a less organized outer portion that connects to the viral lipid envelope (42,88,101,118). HSV1 packages up to 26 different tegument proteins that have been grouped into inner and outer tegument on the basis of their preferred association with capsids or membranes during assembly and entry as well as their fractionation behavior during virion lysis (40,60,62,68,75,96,116).Herpesvirus morphogenesis commences in the nucleus, where preassembled capsids package newly synthesized viral genomes (12,33,47,75). According to the most widely accepted secondary reenvelopment model, nuclear capsids traverse the nuclear membranes by primary envelopment at the inner nuclear membrane and primary fusion with the membranes of the endoplasmic reticulum to enter the cytosol. Inner tegument proteins may bind to nuc...