A functional YNKI motif in the short cytoplasmic tail of varicella-zoster virus glycoprotein gH mediates clathrin-dependent and antibody-independent endocytosis

The trafficking of varicella-zoster virus (VZV) gH was investigated under both infection and transfection conditions. In initial endocytosis assays performed in infected cells, the three glycoproteins gE, gI, and gB served as positive controls for internalization from the plasma membrane. Subsequently, we discovered that gH in VZV-infected cells was also internalized and followed a similar trafficking pattern. This observation was unexpected because all herpesvirus gH homologues have short endodomains not known to contain trafficking motifs. Further investigation demonstrated that VZV gH, when expressed alone with its chaperone gL, was capable of endocytosis in a clathrin-dependent manner, independent of gE, gI, or gB. Upon inspection of the short gH cytoplasmic tail, we discovered a putative tyrosine-based endocytosis motif (YNKI). When the tyrosine was replaced with an alanine, endocytosis of gH was blocked. Utilizing an endocytosis assay dependent on biotin labeling, we further documented that endocytosis of VZV gH was antibody independent. In control experiments, we showed that gE, gI, and gB also internalized in an antibody-independent manner. Alignment analysis of the VZV gH cytoplasmic tail to other herpesvirus gH homologues revealed two important findings: (i) herpes simplex virus type 1 and 2 homologues lacked an endocytosis motif, while all other alphaherpesvirus gH homologues contained a potential motif, and (ii) the VZV gH and simian varicella virus gH cytoplasmic tails were likely longer in length (18 amino acids) than predicted in the original sequence analyses (12 and 16 amino acids, respectively). The longer tails provided the proper context for a functional endocytosis motif.     

Endocytosis and recycling of varicella-zoster virus Fc receptor glycoprotein gE: internalization mediated by a YXXL motif in the cytoplasmic tail.

Varicella-zoster virus (VZV) encodes a cell surface Fc receptor, glycoprotein gE. VZV gE has previously been shown to display several features common to nonviral cell surface receptors. Most recently, VZV gE was reported to be tyrosine phosphorylated on a dimeric form (J. K. Olson, G. A. Bishop, and C. Grose, J. Virol. 71:110-119, 1997). Thereafter, attention focused on the ability of VZV gE to undergo receptor-mediated endocytosis. The current transient transfection studies demonstrated by confocal microscopy and internalization assays that VZV gE was endocytosed when expressed in HeLa cells. Endocytosis of gE was shown to be dependent on clathrin-coated vesicle formation within the cells. Subsequent colocalization studies showed that endocytosis of VZV gE closely mimicked endocytosis of the transferrin receptor. The gE cytoplasmic tail and more specifically tyrosine residue 582 were determined by mutagenesis studies to be important for efficient internalization of the protein; this tyrosine residue is part of a conserved YXXL motif. The amount of gE internalized at any given time reached a steady state of 32%. In addition, like the transferrin receptor, internalized gE recycled to the cell surface. The finding of gE endocytosis provided insight into earlier documentation of gE serine/threonine and tyrosine phosphorylation, since these phosphorylation events may serve as sorting signals for internalized receptors. Taken together with the previous discovery that both human and simian immunodeficiency virus envelope proteins can undergo endocytosis, the gE findings suggest that endocytosis of envelope components may be a posttranslational regulatory mechanism among divergent families of enveloped viruses.     

Varicella-Zoster Virus Fc Receptor Component gI Is Phosphorylated on Its Endodomain by a Cyclin-Dependent Kinase

Varicella-zoster virus (VZV) glycoprotein gI is a type 1 transmembrane glycoprotein which is one component of the heterodimeric gE:gI Fc receptor complex. Like VZV gE, VZV gI was phosphorylated in both VZV-infected cells and gI-transfected cells. Preliminary studies demonstrated that a serine 343-proline 344 sequence located within the gI cytoplasmic tail was the most likely phosphorylation site. To determine which protein kinase catalyzed the gI phosphorylation event, we constructed a fusion protein, consisting of glutathione-S-transferase (GST) and the gI cytoplasmic tail, called GST-gI-wt. When this fusion protein was used as a substrate for gI phosphorylation in vitro, the results demonstrated that GST-gI-wt fusion protein was phosphorylated by a representative cyclin-dependent kinase (CDK) called P-TEFb, a homologue of CDK1 (cdc2). When serine 343 within the serine-proline phosphorylation site was replaced with an alanine residue, the level of phosphorylation of the gI fusion protein was greatly reduced. Subsequent experiments with individually immunoprecipitated mammalian CDKs revealed that the VZV gI fusion protein was phosphorylated best by CDK1, to a lesser degree by CDK2, and not at all by CDK6. Transient-transfection assays carried out in the presence of the specific CDK inhibitor roscovitine strongly supported the prior results by demonstrating a marked decrease in gI phosphorylation while gI protein expression was unaffected. Finally, the possibility that VZV gI contained a CDK phosphorylation site in its endodomain was of further interest because its partner, gE, contains a casein kinase II phosphorylation site in its endodomain; prior studies have established that CDK1 can phosphorylate casein kinase II.     

Regulation of varicella-zoster virus-induced cell-to-cell fusion by the endocytosis-competent glycoproteins gH and gE

The gH glycoprotein of varicella-zoster virus (VZV) is a major fusogen. The realigned short cytoplasmic tail of gH (18 amino acids) harbors a functional endocytosis motif (YNKI) that mediates internalization in both VZV-infected and transfected cells (T. J. Pasieka, L. Maresova, and C. Grose, J. Virol. 77: 4194-4202, 2003). During subsequent confocal microscopy studies of endocytosis-deficient gH mutants, we observed that cells transfected with the gH tail mutants exhibited marked fusion. Therefore, we postulated that VZV gH endocytosis served to regulate cell-to-cell fusion. Subsequent analyses of gH+gL transfection fusion assays by the Kolmogorov-Smirnov statistical test demonstrated that expression of the endocytosis-deficient gH mutants resulted in a statistically significant enhancement of cell-to-cell fusion (P < 0.0001) compared to wild-type gH. On the other hand, coexpression of VZV gE, another endocytosis-competent VZV glycoprotein, was able to temper the fusogenicity of the gH endocytosis mutants by facilitating internalization of the mutant gH protein from the cell surface. When the latter results were similarly analyzed, there was no longer any enhanced fusion by the endocytosis-deficient gH mutant protein. In summary, these studies support a role for gH endocytosis in regulating the cell surface expression of gH and thereby regulating gH-mediated fusion. The data also confirm and extend prior observations of a gE-gH interaction during viral glycoprotein trafficking in a VZV transfection system.     

Mutation of the YXXL endocytosis motif in the cytoplasmic tail of pseudorabies virus gE

The role of alphaherpesvirus membrane protein internalization during the course of viral infection remains a matter of speculation. To determine the role of internalization of the pseudorabies virus (PRV) gE and gI proteins, we constructed viral mutants encoding specific mutations in the cytoplasmic tail of the gE gene that inhibited internalization of the gE-gI complex. We used these mutants to assess the role of gE-gI endocytosis in incorporation of the proteins into the viral envelope and in gE-mediated spread or gE-promoted virulence. In addition, we report that another viral mutant, PRV 25, which encodes a gE protein defective in endocytosis, contains an additional, previously uncharacterized mutation in the gE gene. We compared PRV 25 to another viral mutant, PRV 107, that does not express the cytoplasmic tail of the gE protein. The gE protein encoded by PRV 107 is also defective in endocytosis. We conclude that efficient endocytosis of gE is not required for gE incorporation into virions, gE-mediated virulence, or spread of virus in the rat central nervous system. However, we do correlate the defect in endocytosis to a small-plaque phenotype in cultured cells.     

Unusual phosphorylation sequence in the gpIV (gI) component of the varicella-zoster virus gpI-gpIV glycoprotein complex (VZV gE-gI complex).

Varicella-zoster virus (VZV) glycoprotein gpIV, to be renamed VZV gI, forms a heterodimer with glycoprotein gpI (gE) which functions as an Fc receptor in virus-infected cells. Like VZV gpI (gE), this viral glycoprotein is phosphorylated in cell culture during biosynthesis. In this report, we investigated the nature and specificity of the phosphorylation event involving VZV gpIV (gI). Phosphoamino acid analysis indicated that gpIV (gI) was modified mainly on serine residues. To identify the precise location of the phosphorylation site on the 64-kDa protein, a step-by-step mutagenesis procedures was followed. Initially a tailless mutant was generated, and this truncated product was no longer phosphorylated. Thereafter, point mutations were made within the cytoplasmic tail of gpIV (gI) at potential phosphorylation sites. The phosphorylation site was localized to the following sequence: Ser-Pro-Pro (amino acids 343 to 345). Examination of the point mutants established that serine 343 in the cytoplasmic tail was the major phosphoacceptor. In addition, we found that the prolines located immediately to the C terminus of serine 343 were an integral part of the kinase recognition sequence. This site was located immediately N terminal to a predicted beta-turn secondary structure. By comparison with known substrate consensus sequences for various protein kinases, these data suggested that the phosphorylation of VZV gpIV (gI) was catalyzed by a proline-directed protein kinase. Computer homology analysis of other alphaherpesviruses demonstrated that a similar potential phosphorylation site was highly conserved in the cytoplasmic tails of herpes simplex virus type 1 gI, equine herpesvirus type 1 gI, and pseudorabies virus gp63.     

Intracellular Traffic of Herpes Simplex Virus Glycoprotein gE: Characterization of the Sorting Signals Required for Its trans-Golgi Network Localization

Herpes simplex virus (HSV) and varicella-zoster virus (VZV) are two pathogenic human alphaherpesviruses whose intracellular assembly is thought to follow different pathways. VZV presumably acquires its envelope in the trans-Golgi network (TGN), and it has recently been shown that its major envelope glycoprotein, VZV-gE, accumulates in this compartment when expressed alone. In contrast, the envelopment of HSV has been proposed to occur at the inner nuclear membrane, although to which compartment the gE homolog (HSV-gE) is transported is unknown. For this reason, we have studied the intracellular traffic of HSV-gE and have found that this glycoprotein accumulates at steady state in the TGN, both when expressed from cloned cDNA and in HSV-infected cells. In addition, HSV-gE cycles between the TGN and the cell surface and requires a conserved tyrosine-containing motif within its cytoplasmic tail for proper trafficking. These results show that VZV-gE and HSV-gE have similar intracellular trafficking pathways, probably reflecting the presence of similar sorting signals in the cytoplasmic domains of both molecules, and suggest that the respective viruses, VZV and HSV, could use the same subcellular organelle, the TGN, for their envelopment.     

Incorporation of three endocytosed varicella-zoster virus glycoproteins, gE, gH, and gB, into the virion envelope

The cytoplasmic tails of all three major varicella-zoster virus (VZV) glycoproteins, gE, gH, and gB, harbor functional tyrosine-based endocytosis motifs that mediate internalization. The aim of the present study was to examine whether endocytosis from the plasma membrane is a cellular route by which VZV glycoproteins are delivered to the final envelopment compartment. In this study, we demonstrated that internalization of the glycoproteins occurred in the first 24 h postinfection but was reduced later in infection. Using surface biotinylation of VZV-infected cells followed by a glutathione cleavage assay, we showed that endocytosis was independent of antibody binding to gE, gH, and gB. Subsequently, with this assay, we demonstrated that biotinylated gE, gH, and gB retrieved from the cell surface were incorporated into nascent virus particles isolated after density gradient sedimentation. To confirm and extend this finding, we repeated the above sedimentation step and specifically detected envelopes decorated with Streptavidin-conjugated gold beads on a majority of complete virions through examination by transmission electron microscopy. In addition, a gE-gI complex and a gE-gH complex were found on the virions. Therefore, the above studies established that VZV subsumed a postendocytosis trafficking pathway as one mechanism by which to deliver viral glycoproteins to the site of virion assembly in the cytoplasm. Furthermore, since a recombinant VZV genome lacking only endocytosis-competent gE cannot replicate, these results supported the conclusion that the endocytosis-envelopment pathway is an essential component of the VZV life cycle.     

Varicella-Zoster Virus Glycoprotein I Is Essential for Spread in Dorsal Root Ganglia and Facilitates Axonal Localization of Structural Virion Components in Neuronal Cultures

Neurons of the sensory ganglia are the major site of varicella-zoster virus (VZV) latency and may undergo productive infection during reactivation. Although the VZV glycoprotein E/glycoprotein I (gE/gI) complex is known to be critical for neurovirulence, few studies have assessed the roles of these proteins during infection of dorsal root ganglia (DRG) due to the high human specificity of the virus. Here, we show that the VZV glycoprotein I gene is an important neurotropic gene responsible for mediating the spread of virus in neuronal cultures and explanted DRG. Inoculation of differentiated SH-SY5Y neuronal cell cultures with a VZV gI gene deletion strain (VZV rOkaΔgI) showed a large reduction in the percentage of cells infected and significantly smaller plaque sizes in a comparison with cultures infected with the parental strain (VZV rOka). In contrast, VZV rOkaΔgI was not significantly attenuated in fibroblast cultures, demonstrating a cell type-specific role for VZV gI. Analysis of rOkaΔgI protein localization by immunofluorescent staining revealed aberrant localization of viral glycoprotein and capsid proteins, with little or no staining present in the axons of differentiated SH-SY5Y cells infected with rOkaΔgI, yet axonal vesicle trafficking was not impaired. Further studies utilizing explanted human DRG indicated that VZV gI is required for the spread of virus within DRG. These data demonstrate a role for VZV gI in the cell-to-cell spread of virus during productive replication in neuronal cells and a role in facilitating the access of virion components to axons.     

Glycoprotein E of varicella-zoster virus enhances cell-cell contact in polarized epithelial cells

Varicella-zoster virus (VZV) infection involves the cell-cell spread of virions, but how viral proteins interact with the host cell membranes that comprise intercellular junctions is not known. Madin-Darby canine kidney (MDCK) cells were constructed to express the glycoproteins gE, gI, or gE/gI constitutively and were used to examine the effects of these VZV glycoproteins in polarized epithelial cells. At low cell density, VZV gE induced partial tight junction (TJ) formation under low-calcium conditions, whether expressed alone or with gI. Although most VZV gE was intracellular, gE was also shown to colocalize with the TJ protein ZO-1 with or without concomitant expression of gI. Freeze fracture electron microscopy revealed normal TJ strand morphology in gE-expressing MDCK cells. Functionally, the expression of gE was associated with a marked acceleration in the establishment of maximum transepithelial electrical resistance (TER) in MDCK-gE cells; MDCK-gI and MDCK-gE/gI cells exhibited a similar pattern of early TER compared to MDCK cells, although peak resistances were lower than those of gE alone. VZV gE expression altered F-actin organization and lipid distribution, but coexpression of gI modulated these effects. Two regions of the gE ectodomain, amino acids (aa) 278 to 355 and aa 467 to 498, although lacking Ca(2+) binding motifs, exhibit similarities with corresponding regions of the cell adhesion molecules, E-cadherin and desmocollin. These observations suggest that VZV gE and gE/gI may contribute to viral pathogenesis by facilitating epithelial cell-cell contacts.     

Deletion of the first cysteine-rich region of the varicella-zoster virus glycoprotein E ectodomain abolishes the gE and gI interaction and differentially affects cell-cell spread and viral entry

Varicella-zoster virus (VZV) glycoprotein E (gE) is the most abundant glycoprotein in infected cells and, in contrast to those of other alphaherpesviruses, is essential for viral replication. The gE ectodomain contains a unique N-terminal region required for viral replication, cell-cell spread, and secondary envelopment; this region also binds to the insulin-degrading enzyme (IDE), a proposed VZV receptor. To identify new functional domains of the gE ectodomain, the effect of mutagenesis of the first cysteine-rich region of the gE ectodomain (amino acids 208 to 236) was assessed using VZV cosmids. Deletion of this region was compatible with VZV replication in vitro, but cell-cell spread of the rOka-ΔCys mutant was reduced significantly. Deletion of the cysteine-rich region abolished the binding of the mutant gE to gI but not to IDE. Preventing gE binding to gI altered the pattern of gE expression at the plasma membrane of infected cells and the posttranslational maturation of gI and its incorporation into viral particles. In contrast, deletion of the first cysteine-rich region did not affect viral entry into human tonsil T cells in vitro or into melanoma cells infected with cell-free VZV. These experiments demonstrate that gE/gI heterodimer formation is essential for efficient cell-cell spread and incorporation of gI into viral particles but that it is dispensable for infectious varicella-zoster virion formation and entry into target cells. Blocking gE binding to gI resulted in severe impairment of VZV infection of human skin xenografts in SCIDhu mice in vivo, documenting the importance of cell fusion mediated by this complex for VZV virulence in skin.     

Induction of immunoglobulin G Fc receptors by recombinant vaccinia viruses expressing glycoproteins E and I of herpes simplex virus type 1.

Glycoprotein E (gE) of herpes simplex virus type 1 (HSV-1) will bind immunoglobulin G (IgG) (Fc) affinity columns (R. B. Bauke and P. G. Spear, J. Virol. 32:779-789, 1979), but recent evidence suggests that the HSV-1 Fc receptor is composed of a complex of gE and glycoprotein I (gI) and that both gI and gE are required for Fc receptor activity (D. C. Johnson and V. Feenstra, J. Virol. 61:2208-2216, 1987; D. C. Johnson, M. C. Frame, M. W. Ligas, A. M. Cross, and N. D. Stow, J. Virol. 62:1347-1354, 1988). We have expressed gE and gI, either alone or in combination, on the surface of HeLa cells by using recombinant vaccinia viruses and have measured Fc receptor activity by Fc-rosetting or IgG-binding assays. Expression of gE alone resulted in the induction of Fc receptor activity, while expression of gI alone gave no detectable Fc binding. Coexpression of gE and gI resulted in higher levels of IgG binding than did expression of gE alone, despite the fact that under conditions of coexpression, the levels of surface gE were reduced. We propose that gE and gI together form a receptor of higher affinity than gE alone and that HSV-1 therefore has the potential to induce two Fc receptors of different affinities.     

Role of Envelope Protein gE Endocytosis in the Pseudorabies Virus Life Cycle

Several groups have reported that certain herpesvirus envelope proteins do not remain on the surface of cells that express them but rather are internalized by endocytosis in a recycling process. The biological function of membrane protein endocytosis in the virus life cycle remains a matter of speculation and debate. In this report, we demonstrate that some, but not all, membrane proteins encoded by the alphaherpesvirus pseudorabies virus (PRV) are internalized after reaching the plasma membrane. Glycoproteins gE and gB are internalized from the plasma membrane of cells, while gI and gC are not internalized efficiently. We show for gE that the cytoplasmic domain of the protein is required for endocytosis. While the gI protein is incapable of endocytosis on its own, it can be internalized when complexed with gE. We demonstrate that endocytosis of the gE-gI complex and gB occurs early after infection of tissue culture cells but that this process stops completely after 6 h of infection, a time that correlates with significant shutoff of host protein synthesis. We also show that gE protein internalized at 4 h postinfection is not present in virions formed at a later time. We discuss the differences in PRV gE and gI endocytosis compared to that of the varicella-zoster virus homologs and the possible roles of glycoprotein endocytosis in the virus life cycle.     

Mutational analysis of the role of glycoprotein I in varicella-zoster virus replication and its effects on glycoprotein E conformation and trafficking.

The contributions of the glycoproteins gI (ORF67) and gE (ORF68) to varicella-zoster virus (VZV) replication were investigated in deletion mutants made by using cosmids with VZV DNA derived from the Oka strain. Deletion of both gI and gE prevented virus replication. Complete deletion of gI or deletions of 60% of the N terminus or 40% of the C terminus of gI resulted in a small plaque phenotype as well as reduced yields of infectious virus. Melanoma cells infected with gI deletion mutants formed abnormal polykaryocytes with a disrupted organization of nuclei. In the absence of intact gI, gE became localized in patches on the cell membrane, as demonstrated by confocal microscopy. A truncated N-terminal form of gI was transported to the cell surface, but its expression did not restore plaque morphology or infectivity. The fusogenic function of gH did not compensate for gI deletion or the associated disruption of the gE-gI complex. These experiments demonstrated that gI was dispensable for VZV replication in vitro, whereas gE appeared to be required. Although VZV gI was dispensable, its deletion or mutation resulted in a significant decrease in infectious virus yields, disrupted syncytium formation, and altered the conformation and distribution of gE in infected cells. Normal cell-to-cell spread and replication kinetics were restored when gI was expressed from a nonnative locus in the VZV genome. The expression of intact gI, the ORF67 gene product, is required for efficient membrane fusion during VZV replication.     

The extracellular domain of herpes simplex virus gE is sufficient for accumulation at cell junctions but not for cell-to-cell spread

Herpes simplex virus (HSV) expresses a number of membrane glycoproteins, including gB, gD, and gH/gL, that function in both entry of virus particles and movement of virus from an infected cell to an uninfected cell (cell-to-cell spread). However, a complex of HSV glycoproteins gE and gI (gE/gI) is required for efficient cell-to-cell spread, especially between cells that form extensive cell junctions, yet it is not necessary for entry of extracellular virions. We previously showed that gE/gI has the capacity to localize specifically to cell junctions; the glycoprotein complex was found at lateral surfaces of cells in contact with other cells but not at those lateral surfaces not forming junctions or at apical surfaces. By virtue of these properties, gE/gI is an important molecular handle on the poorly understood process of cell-to-cell spread. Here, we show that the cytoplasmic domain of gE is important for the proper delivery of gE/gI to lateral surfaces of cells. Without this domain, gE/gI is found on the apical surface of epithelial cells, and more uniformly in the cytoplasm, although incorporation into the virion envelope is unaffected. However, even without proper trafficking signals, a substantial fraction of gE/gI retained the capacity to accumulate at cell junctions. Therefore, the extracellular domain of gE can mediate accumulation of gE/gI at cell junctions, if the glycoprotein can be delivered there, probably through interactions with ligands on the opposing cell. The role of phosphorylation of the cytoplasmic domain of gE was also studied. A second mutant HSV type 1 was constructed in which three serine residues that form a casein kinase II phosphorylation site were changed to alanine residues, reducing phosphorylation by 70 to 80%. This mutation did not affect accumulation at cell junctions or cell-to-cell spread.     

Structure-function analysis of the gE-gI complex of feline herpesvirus: mapping of gI domains required for gE-gI interaction, intracellular transport, and cell-to-cell spread.

Alphaherpesvirus glycoproteins gE and gI form a noncovalently associated hetero-oligomeric complex, which is involved in cell-to-cell spread. In the absence of gI, feline herpesvirus (FHV) gE is transport incompetent and fully retained in the endoplasmic reticulum. Here, we assess the effect of progressive C-terminal truncations of FHV gI on the biosynthesis, intracellular transport, and function of the gE-gI complex. The truncated gI proteins were coexpressed with gE in the vaccinia virus-based vTF7-3 expression system. The results were corroborated and extended by studying FHV recombinants expressing truncated gI derivatives. The following conclusions can be drawn. (i) Deletion of the cytoplasmic tail, the transmembrane region plus the C-terminal half of the ectodomain of gI, does not affect intracellular transport of gE. Apparently, the N-terminal 166 residues of gI constitute a domain involved in gE-gI interaction. (ii) A region mediating stable association with gE is located within the N-terminal 93 residues of gI. (iii) The cytoplasmic domain of gI is not essential for gE-gI-mediated cell-to-cell transmission of FHV, as judged from plaque morphology. Deletion of the cytoplasmic tail of gI reduced plaque size by only 35%. (iv) Recombinants expressing the N-terminal 166 residues of gI display a small-plaque phenotype but produce larger plaques than recombinants with a disrupted gI gene. Thus, a complex consisting of gE and the N-terminal half of the gI ectodomain may retain residual biological activity. The implications of these findings for gE-gI interaction and function are discussed.     

Role of the pseudorabies virus gI cytoplasmic domain in neuroinvasion, virulence, and posttranslational N-linked glycosylation

The glycoproteins I and E of pseudorabies virus are important mediators of cell-to-cell spread and virulence in all animal models tested. Although these two proteins form a complex with one another, ascribing any function to the individual proteins has been difficult. We have shown previously, using nonsense mutations, that the N-terminal ectodomain of the gE protein is sufficient for gE-mediated transsynaptic spread whereas the cytoplasmic domain of the protein is required for full expression of virulence. These same studies demonstrated that the cytoplasmic domain of gE is also required for endocytosis of the protein. In this report, we describe the construction of viruses with nonsense mutations in gI that allowed us to determine the contributions of the gI cytoplasmic domain to protein expression as well as virus neuroinvasion and virulence after infection of the rat eye. We also constructed double mutants with nonsense mutations in both gE and gI so that the contributions of both the gE and gI cytoplasmic domains could be determined. We observed that the gI cytoplasmic domain is required for efficient posttranslational modification of the gI protein. The gE cytoplasmic domain has no effect on gE posttranslational glycosylation. In addition, we found that infection of all gE-gI-dependent anterograde circuits projecting from the rat retina requires both ectodomains and at least one of the cytoplasmic domains of the proteins. The gI cytoplasmic domain promotes transsynaptic spread of virus better than the gE cytoplasmic domain. Interestingly, both gE and gI cytoplasmic tails are required for virulence; lack of either one or both results in an attenuated infection. These data suggest that gE and gI play differential roles in mediating directional neuroinvasion of the rat; however, the gE and gI cytoplasmic domains most likely function together to promote virulence.     

Role of an acidic cluster/dileucine motif in cation-independent mannose 6-phosphate receptor traffic

The endocytic trafficking of the cation-independent mannose 6-phosphate receptor (CI-MPR) involves multiple sorting steps. A cluster of acidic amino acids followed by a dileucine motif in the cytoplasmic tail has been proposed to mediate receptor sorting from the trans Golgi network (TGN) to late endosomes. Mutations in this motif impair lysosomal enzyme sorting by preventing association of CI-MPR with coat proteins. The role of the acidic cluster/dileucine motif in the post-endocytic transport of the receptor was examined using the CI-MPR mutants, AC01 and D160E (Chen HJ, Yuan J, Lobel P. J Biol Chem 1997;272:7003-7012). Following internalization, wild type (WT) CI-MPR is transported through sorting endosomes into the endocytic recycling compartment (ERC), after which it traffics to the TGN and other organelles. However, the mutants localize mostly to the ERC and only a small portion reaches the TGN, suggesting that the sorting of the CI-MPR mutants from the ERC into the TGN is severely impaired. We observed no defect in receptor internalization or in the rate of tail mutant recycling to the cell surface compared to the WT. These results demonstrate that the acidic cluster/dileucine motif of CI-MPR is critical for receptor sorting at early stages of intracellular transport following endocytosis.     

Herpes simplex virus gE/gI sorts nascent virions to epithelial cell junctions, promoting virus spread

Alphaherpesviruses spread rapidly through dermal tissues and within synaptically connected neuronal circuitry. Spread of virus particles in epithelial tissues involves movement across cell junctions. Herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseudorabies virus (PRV) all utilize a complex of two glycoproteins, gE and gI, to move from cell to cell. HSV gE/gI appears to function primarily, if not exclusively, in polarized cells such as epithelial cells and neurons and not in nonpolarized cells or cells that form less extensive cell junctions. Here, we show that HSV particles are specifically sorted to cell junctions and few virions reach the apical surfaces of polarized epithelial cells. gE/gI participates in this sorting. Mutant HSV virions lacking gE or just the cytoplasmic domain of gE were rarely found at cell junctions; instead, they were found on apical surfaces and in cell culture fluids and accumulated in the cytoplasm. A component of the AP-1 clathrin adapter complexes, mu1B, that is involved in sorting of proteins to basolateral surfaces was involved in targeting of PRV particles to lateral surfaces. These results are related to recent observations that (i) HSV gE/gI localizes specifically to the trans-Golgi network (TGN) during early phases of infection but moves out to cell junctions at intermediate to late times (T. McMillan and D. C. Johnson, J. Virol., in press) and (ii) PRV gE/gI participates in envelopment of nucleocapsids into cytoplasmic membrane vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004-4016, 2000). Therefore, interactions between the cytoplasmic domains of gE/gI and the AP-1 cellular sorting machinery cause glycoprotein accumulation and envelopment into specific TGN compartments that are sorted to lateral cell surfaces. Delivery of virus particles to cell junctions would be expected to enhance virus spread and enable viruses to avoid host immune defenses.     

Herpes simplex virus type 1 encodes two Fc receptors which have different binding characteristics for monomeric immunoglobulin G (IgG) and IgG complexes.

Two herpes simplex virus type 1 glycoproteins, gE and gI, have been shown to form a complex that binds the Fc domain of immunoglobulin G (IgG). We demonstrate that this complex is required for the binding of monomeric nonimmune IgG but that gE alone is sufficient for binding polymeric IgG in the form of IgG complexes. Evidence that gE but not gI is required for binding IgG complexes is as follows. IgG complexes bound equally well to cells infected with gI-negative mutants or with wild-type virus, whereas cells infected with gE-negative mutants did not bind IgG complexes. Furthermore, L cells transiently transfected to express gE bound IgG complexes. Additional evidence that gI fails to augment binding of IgG complexes comes from experiments in which the gI gene was inducibly expressed in cells after infection. Inducible gI expression failed to increase binding of IgG complexes to infected cells in comparison with cells not capable of inducible gI expression. In contrast, expression of both gE and gI was necessary for binding of monomeric IgG, as demonstrated by flow cytometry using cells infected with gE-negative and gI-negative mutants. These observations demonstrate that herpes simplex virus type 1 Fc receptors (FcRs) have different binding characteristics for monomeric IgG and IgG complexes. Furthermore, it appears that gE is the FcR for IgG complexes and that gE and gI form the FcR for monomeric IgG.