Lawrence A. Hunt

Antibodies to the Major Linear Neutralizing Domains of Cytomegalovirus Glycoprotein B Among Natural Seropositives and CMV Subunit Vaccine Recipients

The gB protein (gpUL55) of human cytomegalovirus (CMV) contains C-terminal (AD-1) and N-terminal (AD-2) linear immunodominant neutralizing domains. To measure antibodies to these epitopes, a modified protein (delta-gB) lacking heavily glycosylated intervening regions, the transmembrane domain, and the cytoplasmic domain, was expressed in recombinant baculovirus-infected cells. Eighty-six percent of 600 naturally CMV-seropositive individuals and 93% of 121 gB vaccine recipients had antibodies to delta-gB as detected by enzyme-linked immunosorbent assay (ELISA). The antibody level in vaccinees (median optical density [OD] = 1.73) exceeded that in natural seropositives (median OD = 0.94; p < .0001). Eleven percent of 95 natural seropositives and 7% of 120 gB vaccinees lacked A-gB antibodies but had neutralizing activity. Among subjects with delta-gB antibody, there were weak correlations between antibody level and neutralizing titer. These data suggest that antibodies to linear neutralizing gB domains are highly prevalent in naturally-infected individuals and regularly develop in gB vaccinees. However, for some individuals, discontinuous and/or linear epitopes not represented on delta-gB may be more important in the generation of neutralizing responses.

PROCESSING OF HUMAN CYTOMEGALOVIRUS GLYCOPROTEIN B IN RECOMBINANT ADENOVIRUS-INFECTED CELLS

Intracellular processing of human cytomegalovirus (HCMV) glycoprotein B (gB; gpUL55) expressed by a recombinant adenovirus (Ad-gB) was studied in human A549 cells as processing events could affect immunogenicity when such viruses are used as live-recombinant vaccines. Cleavage of [35S]methionine-labelled gp13O into gp93 and gp55 reached a maximum after a 3 h chase. Cleavage was completely inhibited by brefeldin A, suggesting that processing normally occurs as a late Golgi or post-Golgi event. Uncleaved gp 130 remained completely sensitive to endo-beta-N-acetylglucosaminidase H (Endo-H) in untreated cells following long chase periods, indicating high-mannose oligosaccharides at all of the 18 N-linked glycosylation sites (Asn-X-Ser/Thr) and retention in the endoplasmic reticulum. Endo-H analysis of gp55 from swainsonine-treated and untreated cells was consistent with glycosylation at all three potential sites, with two oligosaccharides remaining sensitive to Endo-H and one being processed to Endo-H resistance. The heavily glycosylated N-terminal gp93 subunit was not detected by [35S]methionine-labelling but was easily detected along with gp55 after labelling with [3H]mannose. No cleavage of gp 130 was observed in analogous pulse-chase radiolabelling of Ad-gB-infected human fibroblasts, even though these cells are permissive for HCMV replication and can process the native gB molecule. Processing of gB in recombinant adenovirus-infected A549 cells was generally similar to that previously reported for native gB in HCMV-infected fibroblasts.

Unusual heterogeneity in the glycosylation of the G protein of the Hazelhurst strain of vesicular stomatitis virus

The asparagine-linked oligosaccharides of the G protein of the Hazelhurst subtype of the New Jersey serotype of vesicular stomatitis virus (VSV) have been compared with the oligosaccharides from the G protein of the well-characterized Indiana serotype of VSV, with baby hamster kidney cells in monolayer culture as the host for both viruses. [3H]Glucosamine- and [3H]mannose-labeled glycopeptides from the G protein of purified virus were analyzed by the combined techniques of endo-beta-N-acetylglucosaminidase H (ENDO-H) digestion, concanavalin A and lentil lectin affinity chromatography, and Bio-Gel P-4 chromatography. Although almost all of the Indiana G protein oligosaccharides were acidic-type structures, as expected from previous studies; the Hazelhurst G protein contained a mixture of acidic-type, hybrid-type containing sialic acid, and neutral-type (predominantly Man5-6GlcNAc2-Asn) structures. The vast majority of acidic-type oligosaccharides from both the Hazelhurst and Indiana G proteins were diantennary structures, with less than half containing fucose linked to the innermost N-acetylglucosamine. Additional analysis of the Hazelhurst G protein by ENDO-H digestion and gel electrophoresis suggested that some of the mature G polypeptides contained acidic-type structures at both glycosylation sites, whereas the remainder contained an ENDO-H-resistant, acidic-type structure at one site and an ENDO-H-sensitive, hybrid- or neutral-type structure at the other site.

Sindbis virus glycoproteins acquire unusual neutral oligosaccharides in both normal and lectin-resistant chinese hamster ovary cell lines

Abstract The glycosylation of the Sindbis virus envelope glycoproteins was investigated in both normal (CHO-Parent) and lectin-resistant CHO cells (CHO-Pha R and CHO-Pha R ConA R ) with defects in both early and late stages of glycosylation. [ 3 H]Mannose-labeled glycopeptides from mature virions and cell-associated viral glycoprotein were analyzed by gel filtration combined with specific exoglycosidase and endoglycosidase (ENDO-H and ENDO-D) digestions. The mature viral glycoprotein synthesized in the normal cells contained a heterogeneous array of ENDO-H sensitive, neutral-type structures (Man 5–9 -GlcNAc 2 -ASN) and ENDO-H resistant, acidic-type structures [(NeuNAc±Gal-GlcNAc-) n Man 3 GlcNAc 2 -ASN], whereas the major virion glycopeptides synthesized in the two mutant cell lines were identical to those observed previously with the vesicular stomatitis virus glycoprotein (Man 5 GlcNAc 2 -ASN for CHO-Pha R and Man 4 GlcNAc 2 -ASN for CHOPha R Con R ) In addition, unusual small neutral-type structures (Man 3 GlcNAc 2 -ASN; ENDO-H resistant and ENDO-D sensitive) were present in the virion and cell-associated viral glycoproteins from all three CHO host cell lines, but not detected in significant quantities in the cell membrane glycoproteins of the uninfected cells or the glycoprotein of VSV grown in the same host cell lines.

Unusual Neutral Oligosaccharides in Mature Sindbis Virus Glycoproteins are Synthesized from Truncated Precursor Oligosaccharides in Chinese Hamster Ovary Cells

We have previously demonstrated the presence of unusual small asparaginyl-oligosaccharides [(Man)3GlcNAc2-ASN] in the mature glycoproteins of Sindbis virus released from both wild-type and lectin-resistant Chinese hamster ovary cells, but the mechanism of synthesis of these structures was not determined. Gel filtration and endo-beta-N-acetylglucosaminidase analyses of Pronase-digested glycopeptides from [3H]mannose-labelled Sindbis virus released at different times after infection of a phytohaemagglutinin-resistant line of Chinese hamster ovary cells demonstrated that these small asparaginyl-oligosaccharides were present in similar relative amounts in virus released throughout the virus infection, rather than arising primarily at late times when cytopathic effects were maximal. Similar analyses of pulse-labelled, cell-associated viral glycopeptides suggested that these small oligosaccharides on mature virus glycoprotein resulted from the normal alpha 1,2-mannosidase processing of truncated precursor oligosaccharides (containing five rather than nine mannoses), rather than from aberrant processing or degradation of the full-size precursor oligosaccharides or normal intermediates.

Oligosaccharides of the Hazelhurst vesicular stomatitis virus glycoprotein are more extensively processed in Rous sarcoma virus-transformed baby hamster kidney cells

Because of the extensive oligosaccharide heterogeneity of the membrane glycoprotein (G) from the Hazelhurst strain of vesicular stomatitis virus, this virus has been used as a specific intracellular probe of altered protein glycosylation in Rous sarcoma virus-transformed versus normal baby hamster kidney cells. Over 70% of G protein from virus released from the transformed cells had acidic-type oligosaccharides at both glycosylation sites, compared to less than 50% from the corresponding normal host cells. The remaining G protein contained an acidic-type oligosaccharide at one site and an endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide at the other. The major endoglycosidase-sensitive species were sialylated hybrid-type (NeuNAc-Gal-GlcNAc-Man5GlcNAc2-Asn) from the transformed and neutral-type (Man5-6GlcNAc2-Asn) from the normal host cells. The degree of branching of the acidic-type oligosaccharides was not increased in the transformed cells (approx. 80% biantennary for viral G protein from both cell types). At a reduced growth temperature (24 versus 37 degrees C), the G protein oligosaccharides were more extensively processed in both cell types (approximately 85-95% of G protein contained acidic-type structures at both sites), even though the level of viral protein synthesis and virus release was decreased. Essentially all of the minor, endoglycosidase-sensitive oligosaccharides on mature viral G protein were sialic acid-containing hybrid-type structures. At 24 degrees C the branching of the acidic-type oligosaccharides was increased in the virus released from the transformed cells versus normal cells.