Herpes simplex virus glycoprotein D can bind to poliovirus receptor-related protein 1 or herpesvirus entry mediator, two structurally unrelated mediators of virus entry

Several cell membrane proteins have been identified as herpes simplex virus (HSV) entry mediators (Hve). HveA (formerly HVEM) is a member of the tumor necrosis factor receptor family, whereas the poliovirus receptor-related proteins 1 and 2 (PRR1 and PRR2, renamed HveC and HveB) belong to the immunoglobulin superfamily. Here we show that a truncated form of HveC directly binds to HSV glycoprotein D (gD) in solution and at the surface of virions. This interaction is dependent on the native conformation of gD but independent of its N-linked glycosylation. Complex formation between soluble gD and HveC appears to involve one or two gD molecules for one HveC protein. Since HveA also mediates HSV entry by interacting with gD, we compared both structurally unrelated receptors for their binding to gD. Analyses of several gD variants indicated that structure and accessibility of the N-terminal domain of gD, essential for HveA binding, was not necessary for HveC interaction. Mutations in functional regions II, III, and IV of gD had similar effects on binding to either HveC or HveA. Competition assays with neutralizing anti-gD monoclonal antibodies (MAbs) showed that MAbs from group Ib prevented HveC and HveA binding to virions. However, group Ia MAbs blocked HveC but not HveA binding, and conversely, group VII MAbs blocked HveA but not HveC binding. Thus, we propose that HSV entry can be mediated by two structurally unrelated gD receptors through related but not identical binding with gD.     

Antigenic structure of soluble herpes simplex virus (HSV) glycoprotein D correlates with inhibition of HSV infection

Soluble forms of herpes simplex virus (HSV) glycoprotein D (gD) block viral penetration. Likewise, most HSV strains are sensitive to gD-mediated interference by cells expressing gD. The mechanism of both forms of gD-mediated inhibition is thought to be at the receptor level. We analyzed the ability of different forms of soluble, truncated gD (gDt) to inhibit infection by different strains of HSV-1 and HSV-2. Strains that were resistant to gD-mediated interference were also resistant to inhibition by gDt, thereby suggesting a link between these two phenomena. Virion gD was the major viral determinant for resistance to inhibition by gDt. An insertion-deletion mutant, gD-1(delta 290-299t), had an enhanced inhibitory activity against most strains tested. The structure and function of gDt proteins derived from the inhibition-resistant viruses rid1 and ANG were analyzed. gD-1(ridlt) and gD-1(ANGt) had a potent inhibitory effect on plaque formation by wild-type strains of HSV but, surprisingly, little or no effect on their parental strains. As measured by quantitative enzyme-linked immunosorbent assay with a diverse panel of monoclonal antibodies, the antigenic structures of gD-1(rid1t) and gD-1(ANGt) were divergent from that of the wild type yet were similar to each other and to that of gD-1 (delta 290-299t). Thus, three different forms of gD have common antigenic changes that correlate with enhanced inhibitory activity against HSV. We conclude that inhibition of HSV infectivity by soluble gD is influenced by the antigenic conformation of the blocking gDt as well as the form of gD in the target virus.     

Examination of the kinetics of herpes simplex virus glycoprotein D binding to the herpesvirus entry mediator, using surface plasmon resonance

Previously, we showed that truncated soluble forms of herpes simplex virus (HSV) glycoprotein D (gDt) bound directly to a truncated soluble form of the herpesvirus entry mediator (HveAt, formerly HVEMt), a cellular receptor for HSV. The purpose of the present study was to determine the affinity of gDt for HveAt by surface plasmon resonance and to compare and contrast the kinetics of an expanded panel of gDt variants in binding to HveAt in an effort to better understand the mechanism of receptor binding and virus entry. Both HveAt and gDt are dimers in solution and interact with a 2:1 stoichiometry. With HveAt, gD1(306t) (from the KOS strain of HSV-1) had a dissociation constant (KD) of 3.2 x 10(-6) M and gD2(306t) had a KD of 1.5 x 10(-6) M. The interaction between gDt and HveAt fits a 1:1 Langmuir binding model, i.e., two dimers of HveAt may act as one binding unit to interact with one dimer of gDt as the second binding unit. A gD variant lacking all signals for N-linked oligosaccharides had an affinity for HveAt similar to that of gD1(306t). A variant lacking the bond from cysteine 1 to cysteine 5 had an affinity for HveAt that did not differ from that of the wild type. However, variants with double cysteine mutations that eliminated either of the other two disulfide bonds showed decreased affinity for HveAt. This result suggests that two of the three disulfide bonds of gD are important for receptor binding. Four nonfunctional gDt variants, each representing one functional domain of gD, were also studied. Mutations in functional regions I and II drastically decreased the affinity of gDt for HveAt. Surprisingly, a variant with an insertion in functional region III had a wild-type level of affinity for HveAt, suggesting that this domain may function in virus entry at a step other than receptor binding. A variant with a deletion in functional region IV [gD1(Delta290-299t)] exhibited a 100-fold enhancement in affinity for HveAt (KD = 3.3 x 10(-8) M) due mainly to a 40-fold increase in its kinetic on rate. This agrees with the results of other studies showing the enhanced ability of gD1(Delta290-299t) to block infection. Interestingly, all the variants with decreased affinities for HveAt exhibited decreased kinetic on rates but only minor changes in their kinetic off rates. The results suggest that once the complex between gDt and HveAt forms, its stability is unaffected by a variety of changes in gD.     

Children''s emerging regulation of conduct: restraint, compliance, and internalization from infancy to the second year

Receptor-mediated endocytosis of circulating collagen is a major physiological scavenger function of the liver endothelial cell and an important catabolic event in the complete turnover of this abundant connective tissue protein. In the present study, transport of collagen through the endocytic pathway was investigated in cultured liver endothelial cells. Collagen conjugated to fluorescein isothiocyanate, to allow detection of the ligand by fluorescence and immunoelectron microscopy, was found sequentially in three different organelles that compose the basic degradative endocytic pathway of eukaryotic cells: early endosomes, late endosomes, and terminal lysosomes. Early endosomes were identified as vesicles positive for early endosome antigen 1 (EEA1). Late endosomes were distinguished as structures positive for the late endosomal/lysosomal marker rat lysosomal membrane glycoprotein 120, but negative for EEA1 and lysosomally targeted BSA-gold. Lysosomes were defined by their content of BSA-gold, injected 24 hours before isolation of cells. Coated pits and coated vesicles mediated an extremely rapid internalization. Shortly after internalization and during the first 20 minutes, ligand was found in early endosomes. From 20 minutes on, ligand started to appear in late endosomes (23%), and by 2 hours the transfer was largely complete (82.5%). Only 2.5% of ligand was transferred to the lysosomes after 2 hours, and this number slowly increased to 21% and 53% after 6 and 16 hours, respectively. We conclude that 1) EEA1 is a useful marker for tracing early events of endocytosis in liver endothelial cells; 2) in contrast to the rapid internalization, transit of internalized ligand through early sorting endosomes generally takes from 20 minutes to 2 hours; and 3) exit from the late endosomes is very slow, requiring several hours.     

Biliary tract infections: a guide to drug treatment

The genes encoding the respiratory syncytial virus (RSV) attachment (G) and fusion (F) envelope glycoproteins were expressed separately as additional genes in recombinant vesicular stomatitis viruses (VSV). Cells infected with the VSV-RSV F recombinant formed large syncytia illustrating the fusion activity of F in absence of other RSV proteins. Both F and G glycoproteins were expressed at the cell surface and incorporated into virions. Incorporation of these proteins did not require cytoplasmic tail sequences of VSV G. Using a compound, ammonium chloride, that raises the endosomal pH, we showed that presence of the RSV F glycoprotein in the envelope of recombinant VSV allowed for infectivity through a low-pH-independent pathway. Recombinant VSV expressing RSV glycoproteins could be useful as an RSV vaccine.     

The 46-kDa mannose 6-phosphate receptor contains multiple binding sites for clathrin adaptors

The two known mannose 6-phosphate receptors (MPR46 and MPR300) both mediate the transport of Man-6-P-containing lysosomal proteins to lysosomes. However, the MPRs cannot be detected in lysosomes, instead they recycle between the plasma membrane and endosomes and between endosomes and the trans-Golgi network. Both, endocytosis from the plasma membrane and budding of transport vesicles from the trans-Golgi network involves the interaction of the receptor with the clathrin-coated vesicles-associated protein complexes AP1 and AP2. We have analyzed this interaction between the Golgi-restricted AP1 complex and the plasma membrane-restricted AP2 complex with the MPR46 tail in vitro by using a biosensor. AP1 and AP2 both bind to and dissociate from the MPR46 tail with similar kinetics. Using synthetic peptides corresponding to different MPR receptor tail regions in inhibition and binding studies, a common high affinity binding site for AP1 and AP2 and two separate high affinity binding sites for AP1 and AP2, respectively, were identified.     

Functional region IV of glycoprotein D from herpes simplex virus modulates glycoprotein binding to the herpesvirus entry mediator

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry and has four functional regions (I to IV) important for this process. We previously showed that a truncated form of a functional region IV variant, gD1(Delta290-299t), had an enhanced ability to block virus entry and to bind to the herpesvirus entry mediator (HveAt; formerly HVEMt), a cellular receptor for HSV. To explore this phenotype further, we examined other forms of gD, especially ones with mutations in region IV. Variant proteins with deletions of amino acids between 277 and 300 (region IV), as well as truncated forms lacking C-terminal residues up to amino acid 275 of gD, were able to block HSV entry into Vero cells 1 to 2 logs better than wild-type gD1(306t). In contrast, gD truncated at residue 234 did not block virus entry into Vero cells. Using optical biosensor technology, we recently showed that gD1(Delta290-299t) had a 100-fold-higher affinity for HveAt than gD1(306t) (3.3 x 10(-8) M versus 3.2 x 10(-6) M). Here we found that the affinities of other region IV variants for HveAt were similar to that of gD1(Delta290-299t). Thus, the affinity data follow the same hierarchy as the blocking data. In each case, the higher affinity was due primarily to a faster kon rather than to a slower koff. Therefore, once the gDt-HveAt complex formed, its stability was unaffected by mutations in or near region IV. gD truncated at residue 234 bound to HveAt with a lower affinity (2.0 x 10(-5) M) than did gD1(306t) due to a more rapid koff. These data suggest that residues between 234 and 275 are important for maintaining stability of the gDt-HveAt complex and that functional region IV is important for modulating the binding of gD to HveA. The binding properties of any gD1(234t)-receptor complex could account for the inability of this form of gDt to block HSV infection.     

Ligand binding specificities of the two mannose 6-phosphate receptors

Two mannose 6-phosphate (Man-6-P) receptors (MPRs) direct the vesicular transport of newly synthesized lysosomal enzymes that contain Man-6-P from the Golgi to a prelysosomal compartment. In order to understand the respective roles of the Mr = 46,000 cation-dependent (CD-) MPR and the Mr = 300,000 cation-independent (CI-) MPR in lysosomal targeting, an assay has been developed that simultaneously measures the relative affinity of each MPR for multiple ligands. Glycoproteins containing Man-6-P were affinity-purified from the metabolically labeled secretions of mutant mouse fibroblasts lacking both MPRs. They were incubated with purified MPRs, and the resulting receptor-ligand complexes were immunoprecipitated by anti-MPR monoclonal antibodies coupled to agarose beads. Ligands were eluted with Man-6-P and then quantified following SDS-polyacrylamide gel electrophoresis. Saturating concentrations of CI-MPR resulted in the complete recovery of each Man-6-P glycoprotein in receptor-ligand complexes. Apparent affinity constants ranged between 1 and 5 nM for the individual species. Ligands precipitated by the CD-MPR appeared identical to those bound by the CI-MPR, with apparent affinity constants ranging between 7 and 28 nM. The binding affinities of the two receptors for different ligands were not correlated, indicating that the two MPRs preferentially recognize different subsets of lysosomal enzymes. In addition, saturating levels of CD-MPR resulted in the precipitation of only 50% of the total input ligands, suggesting that the CD-MPR binds a subpopulation of the Man-6-P glycoproteins bound by the CI-MPR. These results provide a biochemical mechanism, which, in part, may explain the interaction of the two MPRs with overlapping yet distinct subsets of ligands in vivo.     

Uptake of poliovirus into the endosomal system of HeLa cells

To understand the topology and mechanism of poliovirus uncoating, the question of whether intact virions can be endocytosed by the host cell was studied by a combination of various techniques. In order to prevent alteration of the virus to subviral particles, Hela cells were infected at 26 degrees C. At this temperature the majority of cell-associated virions remained at the plasma membrane, whereas a smaller amount accumulated in vesicles having the same mobility (upon free-flow electrophoresis) and migration behaviour on Nycodenz density gradients as early and late endosomes. Co-localization of native poliovirions with endosomal markers was verified by peroxidase-induced diaminobenzidine density-shift of endosomal vesicles. Internalization of poliovirions into endosomes makes it likely, but does not prove that viral RNA can be released into the cytoplasm from the vesicular compartment.     

Partial resistance to gD-mediated interference conferred by mutations affecting herpes simplex virus type 1 gC and gK

Cells expressing herpes simplex virus (HSV) gD can be resistant to HSV entry as a result of gD-mediated interference. HSV strains differ in sensitivity to this interference, which blocks viral penetration but not binding. Previous studies have shown that mutations or variations in virion-associated gD can confer resistance to gD-mediated interference. Here we show that HSV-1 mutants selected for enhanced ability to bind and penetrate in the presence of inhibitory concentrations of heparin were partially resistant to gD-mediated interference. The resistance was largely due to the presence of two mutations: one in gC (the major heparin-binding glycoprotein) resulting in the absence of gC expression and the other in gK resulting in a syncytial phenotype. The results imply that heparin selected for mutants with altered postbinding requirements for entry. Resistance to gD-mediated interference conferred by mutations affecting gC and gK has not been previously described.     

Proper sorting of the cation-dependent mannose 6-phosphate receptor in endosomes depends on a pair of aromatic amino acids in its cytoplasmic tail

The 67-amino acid cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains a signal(s) that prevents the receptor from entering lysosomes where it would be degraded. To identify the key residues required for proper endosomal sorting, we analyzed the intracellular distribution of mutant forms of the receptor by Percoll density gradients. A receptor with a Trp19 --> Ala substitution in the cytoplasmic tail was highly missorted to lysosomes whereas receptors with either Phe18 --> Ala or Phe13 --> Ala mutations were partially defective in avoiding transport to lysosomes. Analysis of double and triple mutants confirmed the key role of Trp19 for sorting of the CD-MPR in endosomes, with Phe18, Phe13, and several neighboring residues contributing to this function. The addition of the Phe18-Trp19 motif of the CD-MPR to the cytoplasmic tail of the lysosomal membrane protein Lamp1 was sufficient to partially impair its delivery to lysosomes. Replacing Phe18 and Trp19 with other aromatic amino acids did not impair endosomal sorting of the CD-MPR, indicating that two aromatic residues located at these positions are sufficient to prevent the receptor from trafficking to lysosomes. However, alterations in the spacing of the diaromatic amino acid sequence relative to the transmembrane domain resulted in receptor accumulation in lysosomes. These findings indicate that the endosomal sorting of the CD-MPR depends on the correct presentation of a diaromatic amino acid-containing motif in its cytoplasmic tail. Because a diaromatic amino acid sequence is also present in the cytoplasmic tail of other receptors known to be internalized from the plasma membrane, this feature may prove to be a general determinant for endosomal sorting.     

Interaction of herpes simplex virus glycoprotein gC with mammalian cell surface molecules

The entry of herpes simplex virus (HSV) into mammalian cells is a multistep process beginning with an attachment step involving glycoproteins gC and gB. A second step requires the interaction of glycoprotein gD with a cell surface molecule. We explored the interaction between gC and the cell surface by using purified proteins in the absence of detergent. Truncated forms of gC and gD, gC1(457t), gC2(426t), and gD1(306t), lacking the transmembrane and carboxyl regions were expressed in the baculovirus system. We studied the ability of these proteins to bind to mammalian cells, to bind to immobilized heparin, to block HSV type 1 (HSV-1) attachment to cells, and to inhibit plaque formation by HSV-1. Each of these gC proteins bound to conformation-dependent monoclonal antibodies and to human complement component C3b, indicating that they maintained the same conformation of gC proteins expressed in mammalian cells. Biotinylated gC1(457t) and gC2(426t) each bind to several cell lines. Binding was inhibited by an excess of unlabeled gC but not by gD, indicating specificity. The attachment of gC to cells involves primarily heparan sulfate proteoglycans, since heparitinase treatment of cells reduced gC binding by 50% but had no effect on gD binding. Moreover, binding of gC to two heparan sulfate-deficient L-cell lines, gro2C and sog9, both of which are mostly resistant to HSV infection, was markedly reduced. Purified gD1 (306t), however, bound equally well to the two mutant cell lines. In contrast, saturating amounts of gC1(457t) interfered with HSV-1 attachment to cells but failed to block plaque formation, suggesting a role for gC in attachment but not penetration. A mutant form of gC lacking residues 33 to 123, gC1(delta 33-123t), expressed in the baculovirus system, bound significantly less well to cells than did gC1(457t) and competed poorly with biotinylated gC1(457t) for binding. These results suggest that residues 33 to 123 are important for gC attachment to cells. In contrast, both the mutant and wild-type forms of gC bound to immobilized heparin, indicating that binding of these proteins to the cell surface involves more than a simple interaction with heparin. To determine that the contribution of the N-terminal region of gC is important for HSV attachment, we compared several properties of a mutant HSV-1 which contains gC lacking amino acids 33 to 123 to those of its parental virus, which contains full-length gC. The mutant bound less well to cells than the parental virus but exhibited normal growth properties.(ABSTRACT TRUNCATED AT 400 WORDS)     

Toward a song code: evidence for a syllabic representation in the canary brain

The occurrence of clathrin-coated buds on immature granules (IGs) of the regulated secretory pathway suggests that specific transmembrane proteins are sorted into these buds through interaction with cytosolic adaptor proteins. By quantitative immunoelectron microscopy of rat endocrine pancreatic beta cells and exocrine parotid and pancreatic cells, we show for the first time that the mannose 6-phosphate receptors (MPRs) for lysosomal enzyme sorting colocalize with the AP-1 adaptor in clathrin-coated buds on IGs. Furthermore, the concentrations of both MPR and AP-1 decline by approximately 90% as the granules mature. Concomitantly, in exocrine secretory cells lysosomal proenzymes enter and then are sorted out of IGs, just as was previously observed in beta cells (Kuliawat, R., J. Klumperman, T. Ludwig, and P. Arvan. 1997. J. Cell Biol. 137:595-608). The exit of MPRs in AP-1/clathrin-coated buds is selective, indicated by the fact that the membrane protein phogrin is not removed from maturing granules. We have also made the first observation of a soluble N-ethylmaleimide-sensitive factor attachment protein receptor, syntaxin 6, which has been implicated in clathrin-coated vesicle trafficking from the TGN to endosomes (Bock, J.B., J. Klumperman, S. Davanger, and R.H. Scheller. 1997. Mol. Biol. Cell. 8:1261-1271) that enters and then exits the regulated secretory pathway during granule maturation. Thus, we hypothesize that during secretory granule maturation, MPR-ligand complexes and syntaxin 6 are removed from IGs by AP-1/clathrin-coated vesicles, and then delivered to endosomes.     

The prophylactic effect of immunization with DNA encoding herpes simplex virus glycoproteins on HSV-induced disease in guinea pigs

Plasmid expression vectors encoding herpes simplex virus type 2 (HSV-2) glycoproteins B (gB) or D (gD) were constructed and tested for their ability to immunize guinea pigs against genital HSV infection. Immunization with a plasmid expressing the aminoterminal 707 amino acids (aa) of gB induced humoral immune responses detected by ELISA and virus neutralization. When challenged by vaginal infection, immunized animals were partially protected from genital herpes, exhibiting significantly reduced primary and subsequent recurrent disease. When the gB plasmid was combined with a plasmid expressing full-length gD, immunized guinea pigs developed humoral responses to both proteins and were also significantly protected from viral challenge.     

Intracellular routes and selective retention of antigens in mildly acidic cathepsin D/lysosome-associated membrane protein-1/MHC class II-positive vesicles in immature dendritic cells

Immature dendritic cells (DC) use both macropinocytosis and mannose receptor-mediated endocytosis to internalize soluble Ags efficiently. These Ags are ultimately presented to T cells after DC maturation and migration into the lymph nodes. We have previously described the immortalized myeloid cell line FSDC as displaying the characteristics of early DC precursors that efficiently internalize soluble Ags. To describe the different routes of Ag uptake and to identify the Ag retention compartments in FSDC, we followed the intracellular fate of FITC-coupled OVA, dextran (DX), transferrin, and Lucifer Yellow using flow cytometry, confocal microscopy, and immunoelectron microscopy. OVA and DX gained access into macropinosomes and early endosomes. DX was preferentially sorted into endosomal compartments, while most of the OVA entered macropinosomes via fluid phase uptake. We found a long-lasting retention of DX and OVA of up to 24 h. After 6 h of chase, these two molecules were concentrated in common vesicular compartments. These retention compartments were distinct from endosomes and lysosomes; they were much larger, only mildly acidic, and lacked the small GTP binding protein rab7. However, they were positive for lysosome-associated membrane protein-1, the protease cathepsin D, and MHC class II molecules, thus representing matured macropinosomes. These data suggest that the activity of vacuolar proteases is reduced at the mildly acidic pH of these vesicles, which explains their specific retention of an Ag. The retention compartments might be used by nonlymphoid tissue DC to store peripheral Ags during their migration to the lymph node.     

Cells and viruses with mutations affecting viral entry are selected during persistent infections of L cells with mammalian reoviruses

Previous studies demonstrated that both cellular and viral mutants are selected during maintenance of persistent infections established in murine L cells with high-passage stocks of mammalian reoviruses. In particular, when one culture was cured of persistent infection, the resulting cells were found to support the growth of viruses isolated from persistently infected cultures (termed PI viruses here) better than that of wild-type (wt) viruses (R. Ahmed, W. M. Canning, R. S. Kauffman, A. H. Sharpe, J. V. Hallum, and B. N. Fields, Cell 25:325-332, 1981). To address the nature of cellular and viral mutations selected during maintenance of persistent reovirus infections, we established independent, persistently infected cultures with L cells and high-passage stocks of wt reovirus. These cultures served as sources of new PI viruses and cured cells for study. We found that although wt viruses grew poorly in cured cells when infection was initiated with intact virions, they grew well in cured cells when infection was initiated with infectious subvirion particles generated from virions by in vitro treatment with chymotrypsin. This finding indicates that the block to growth of wt viruses in cured cells involves an early step that is unique to infection by virions, such as proteolytic processing in an endocytic compartment. We also found that PI viruses grew better than wt viruses in L cells treated with ammonium chloride, a weak base that inhibits the pH decrease in endosomes and lysosomes. Because ammonium chloride blocks an early step in infection by intact virions, probably the proteolytic processing of viral outer capsid proteins by acid-dependent cellular proteases in late endosomes or lysosomes, this finding indicates that PI viruses differ from wt viruses with respect to viral entry into cells. Therefore, these results indicate that both cells and viruses evolve mutations that affect one or more early steps in the viral growth cycle during maintenance of L-cell cultures persistently infected with reoviruses.     

Re-expression of the mannose 6-phosphate receptors in receptor-deficient fibroblasts. Complementary function of the two mannose 6-phosphate receptors in lysosomal enzyme targeting

We have previously generated primary embryonic fibroblasts lacking either the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (MPR) or the cation-dependent MPR, two trans-membrane proteins that bind the mannose 6-phosphate (Man-6-P) recognition marker on soluble lysosomal enzymes (Ludwig, T., Munier-Lehmann, H., Bauer, U., Hollinshead, M., Ovitt, C., Lobel, P., and Hoflack, B.(1994) EMBO J. 13, 3430-3437). These two cell types partially missort phosphorylated lysosomal enzymes. Using two-dimensional gel electrophoresis, we show here that they secrete, in a large part, different phosphorylated ligands. In order to better understand the sorting function of the MPRs, we have re-expressed each MPR in MPR-negative fibroblasts. We show that the MPRs have similar capacities for transporting the bulk of the newly synthesized lysosomal enzymes and that they target individual ligands with various efficiencies. However, high levels of one MPR do not fully compensate for the absence of the other, demonstrating that the two MPRs have complementary targeting functions, perhaps by recognizing different features on lysosomal enzymes. The analysis of the phosphorylated oligosaccharides shows that the ligands missorted in the absence of the cation-dependent MPR are slightly but significantly depleted in oligosaccharides with two Man-6-P residues, when compared with those missorted in the absence of the cation-independent MPR. While these results could explain some differences between the structure and the sorting function of the two MPRs, they strongly suggest that the reason why cells express two different but related MPRs is to maintain an efficient Man-6-P-dependent targeting process that could be potentially regulated by MPR expression.     

Determinants of human immunodeficiency virus type 1 envelope glycoprotein activation by soluble CD4 and monoclonal antibodies

Infection by some human immunodeficiency virus type 1 (HIV-1) isolates is enhanced by the binding of subneutralizing concentrations of soluble receptor, soluble CD4 (sCD4), or monoclonal antibodies directed against the viral envelope glycoproteins. In this work, we studied the abilities of different antibodies to mediate activation of the envelope glycoproteins of a primary HIV-1 isolate, YU2, and identified the regions of gp120 envelope glycoprotein contributing to activation. Binding of antibodies to a variety of epitopes on gp120, including the CD4 binding site, the third variable (V3) loop, and CD4-induced epitopes, enhanced the entry of viruses containing YU2 envelope glycoproteins. Fab fragments of antibodies directed against either the CD4 binding site or V3 loop also activated YU2 virus infection. The activation phenotype was conferred on the envelope glycoproteins of a laboratory-adapted HIV-1 isolate (HXBc2) by replacing the gp120 V3 loop or V1/V2 and V3 loops with those of the YU2 virus. Infection by the YU2 virus in the presence of activating antibodies remained inhibitable by macrophage inhibitory protein 1beta, indicating dependence on the CCR5 coreceptor on the target cells. Thus, antibody enhancement of YU2 entry involves neither Fc receptor binding nor envelope glycoprotein cross-linking, is determined by the same variable loops that dictate enhancement by sCD4, and probably proceeds by a process fundamentally similar to the receptor-activated virus entry pathway.     

Herpes simplex virus 2 UL45 is a type II membrane protein

In addition to eleven glycoproteins, the herpes simplex virus type 2 (HSV-2) genome encodes several proteins with potential membrane-spanning segments but no asparagine-linked carbohydrates. One of these is UL45. Fractionation of infected cells showed that HSV-2 UL45 is an integral membrane protein, and analysis of UL45 mutants with potential glycosylation sites showed that it has a type II membrane orientation, the first HSV protein known to have this orientation. Furthermore, it is detectable in infected cells at a time similar to that when glycoproteins gB and gD are detected, consistent with a role in cell-cell fusion, which has previously been found for HSV-1 UL45.