A cytomegalovirus glycoprotein re-routes MHC class I complexes to lysosomes for degradation

Mouse cytomegalovirus (MCMV) early gene expression interferes with the major histocompatibility complex class I (MHC class I) pathway of antigen presentation. Here we identify a 48 kDa type I transmembrane glycoprotein encoded by the MCMV early gene m06, which tightly binds to properly folded beta2-microglobulin (beta2m)-associated MHC class I molecules in the endoplasmic reticulum (ER). This association is mediated by the lumenal/transmembrane part of the protein. gp48-MHC class I complexes are transported out of the ER, pass the Golgi, but instead of being expressed on the cell surface, they are redirected to the endocytic route and rapidly degraded in a Lamp-1(+) compartment. As a result, m06-expressing cells are impaired in presenting antigenic peptides to CD8(+) T cells. The cytoplasmic tail of gp48 contains two di-leucine motifs. Mutation of the membrane-proximal di-leucine motif of gp48 restored surface expression of MHC class I, while mutation of the distal one had no effect. The results establish a novel viral mechanism for downregulation of MHC class I molecules by directly binding surface-destined MHC complexes and exploiting the cellular di-leucine sorting machinery for lysosomal degradation.     

Identification and analysis of a novel heparin-binding glycoprotein encoded by human herpesvirus 7

Human herpesvirus 6 (HHV-6) and HHV-7 are closely related betaherpesviruses that encode a number of genes with no known counterparts in other herpesviruses. The product of one such gene is the HHV-6 glycoprotein gp82-105, which is a major virion component and a target for neutralizing antibodies. A 1.7-kb cDNA clone from HHV-7 was identified which contains a large open reading frame capable of encoding a predicted primary translational product of 468 amino acids (54 kDa) with 13 cysteine residues and 9 potential N-linked glycosylation sites. This putative protein, which we have termed gp65, was homologous to HHV-6 gp105 (30% identity) and contained a single potential membrane-spanning domain located near its amino terminus. Comparison of the cDNA sequence with that of the viral genome revealed that the gene encoding gp65 contains eight exons, spanning almost 6 kb of the viral genome at the right (3') end of the HHV-7 genome. Northern (RNA) blot analysis with poly(A)(+) RNA from HHV-7-infected cells revealed that the cDNA insert hybridized to a single major RNA species of 1.7 kb. Antiserum raised against a purified, recombinant form of gp65 recognized a protein of roughly 65 kDa in sucrose density gradient-purified HHV-7 preparations; treatment with PNGase F reduced this glycoprotein to a putative precursor of approximately 50 kDa. Gp65-specific antiserum also neutralized the infectivity of HHV-7, while matched preimmune serum did not do so. Finally, analysis of the biochemical properties of recombinant gp65 revealed a specific interaction with heparin and heparan sulfate proteoglycans and not with closely related molecules such as N-acetylheparin and de-N-sulfated heparin. At least two domains of the protein were found to contribute to heparin binding. Taken together, these findings suggest that HHV-7 gp65 may contribute to viral attachment to cell surface proteoglycans.     

Nucleotide sequence and molecular characterization of the structural glycoprotein gp41 gene homologue of Spodoptera litura nucleopolyhedrosis virus (spltNPV-I)

The structural glycoprotein gene gp41 homologue of Spodoptera litura nucleopolyhedrosis virus (SpltNPV-I *) was identified in the 4.0 kb EcoRI-L fragment of the viral genome. The nucleotide sequence of 2063 bp of this fragment revealed an open reading frame of 1014 nucleotides to encode a polypeptide of 337 amino acids. Analysis of nucleotide and deduced amino acid sequences of the putative ORF indicated its identity with gp41 protein of other baculoviruses sharing maximum homology with that of Spodoptera frugiperda nucleopolyhedrosis virus (SfNPV). The coding sequence was preceded by an AT-rich region containing the consensus baculoviral late promoter motif RTAAG. The putative SpltNPV gp41 ORF was abundantly expressed as a 37 kDa apoprotein in E. coli and as a 50 kDa glycoprotein in Sf9 cells. The recombinant protein expressed in insect cells was glycosylated (20%) and has GlcNAc as the terminal sugar. The gene is conserved among baculoviruses and places SpltNPV-I close to Spodoptera frugiperda and Spodoptera exigua NPVs in phylogenetic tree.     

Nucleotide sequence and molecular characterization of the structural glycoprotein gp41 gene homologue of <Emphasis Type="Italic">Spodoptera litura</Emphasis> nucleopolyhedrosis virus (SpltNPV-I)

The structural glycoprotein gene gp41 homologue of Spodoptera litura nucleopolyhedrosis virus (SpltNPV-I *) was identified in the 4.0 kb EcoRI-L fragment of the viral genome. The nucleotide sequence of 2063 bp of this fragment revealed an open reading frame of 1014 nucleotides to encode a polypeptide of 337 amino acids. Analysis of nucleotide and deduced amino acid sequences of the putative ORF indicated its identity with gp41 protein of other baculoviruses sharing maximum homology with that of Spodoptera frugiperda nucleopolyhedrosis virus (SfNPV). The coding sequence was preceded by an AT-rich region containing the consensus baculoviral late promoter motif RTAAG. The putative SpltNPV gp41 ORF was abundantly expressed as a 37 kDa apoprotein in E. coli and as a 50 kDa glycoprotein in Sf9 cells. The recombinant protein expressed in insect cells was glycosylated (20%) and has GlcNAc as the terminal sugar. The gene is conserved among baculoviruses and places SpltNPV-I close to Spodoptera frugiperda and Spodoptera exigua NPVs in phylogenetic tree.Assigned GenBank accession no. for the nucleotide sequence data is AF445192.abbreviated as SlNPV in earlier publications and GenBank     

Intercellular gamma-herpesvirus dissemination involves co-ordinated intracellular membrane protein transport

The murine gamma-herpesvirus-68 (MHV-68) ORF27 encodes gp48, a type 2 transmembrane glycoprotein that contributes to intercellular viral spread. Gp48 is expressed on the surface of infected cells but is retained intracellularly after transfection. In this study, we show that the multimembrane spanning ORF58 gene product is both necessary and sufficient for gp48 to reach the cell surface. ORF58-deficient MHV-68 expressed ORF27 in normal amounts, but retained it in the endoplasmic reticulum (ER). Transfected ORF27 also remained in ER, whereas green fluorescent protein-tagged ORF58 localized to the ER and trans-Golgi network. When ORF27 and ORF58 were co-transfected, they formed a protein complex and reached the cell surface. Surprisingly, ORF58 rather than ORF27 mediated cell binding via a small extracellular loop. The heavily glycosylated ORF27 component of the complex may, therefore, act mainly to protect this loop against antibody. The interdependent transport of ORF27 and ORF58 transport ensures that such protection is always present.     

A gamma-herpesvirus glycoprotein complex manipulates actin to promote viral spread

Viruses lack self-propulsion. To move in multi-cellular hosts they must therefore manipulate infected cells. Herpesviruses provide an archetype for many aspects of host manipulation, but only for alpha-herpesviruses in is there much information about they move. Other herpesviruses are not necessarily the same. Here we show that Murine gamma-herpesvirus-68 (MHV-68) induces the outgrowth of long, branched plasma membrane fronds to create an intercellular network for virion traffic. The fronds were actin-based and RhoA-dependent. Time-lapse imaging showed that the infected cell surface became highly motile and that virions moved on the fronds. This plasma membrane remodelling was driven by the cytoplasmic tail of gp48, a MHV-68 glycoprotein previously implicated in intercellular viral spread. The MHV-68 ORF58 was also required, but its role was simply transporting gp48 to the plasma membrane, since a gp48 mutant exported without ORF58 did not require ORF58 to form membrane fronds either. Together, gp48/ORF58 were sufficient to induce fronds in transfected cells, as were the homologous BDLF2/BMRF2 of Epstein-Barr virus. Gp48/ORF58 therefore represents a conserved module by which gamma-herpesviruses rearrange cellular actin to increase intercellular contacts and thereby promote their spread.     

Identification of a gp63 surface glycoprotein in Leishmania tarentolae

The promastigote stage of most if not all Leishmania species possesses an abundant surface glycoprotein of 63 kDa (gp63) that has protease activity. We show that the lizard parasite Leishmania tarentolae appears to lack the surface protease activity. L. tarentolae does, however, possess an approximately 63-kDa molecule that is antigenically cross-reactive with the L. major gp63. Additionally, the genome of L. tarentolae contains sequences that hybridise at high stringency to a L. major gp63 gene probe.     

Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus

The major envelope glycoprotein (gp350) of Epstein-Barr virus has been expressed and secreted in the yeast Saccharomyces cerevisiae as a 400-kDa glycoprotein. This is the first example of the secretion of such a large, heavily glycosylated heterologous protein in yeast. Since gp350 proved highly toxic to S. cerevisiae, initial cellular growth required repression of the expression of gp350. Using temperature- or galactose-inducible promoters, cells could be grown and the expression of gp350 then induced. After induction, the glycoprotein accumulated both intracellularly as well as in the culture medium. Only the most heavily glycosylated form was secreted, suggesting a role for N-linked glycans in directing secretion. The extent of O-linked glycosylation of the yeast-derived protein was similar to that of the mature viral gp350. N-linked glycosylation varied slightly depending upon culture conditions and host strain used and was more extensive than that associated with the mature viral gp350. Although there is no evidence that more than a single mRNA for the glycoprotein was expressed from the recombinant plasmid, variously sized glycoproteins accumulated in yeast at early stages after induction, probably reflecting intermediates in glycosylation. The yeast-derived glycoproteins reacted with animal and human polyclonal antibodies to gp350 as well as with a neutralizing murine monoclonal antibody to gp350, suggesting that this glycoprotein retains several epitopes of the native glycoprotein.     

Several N-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission

The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 N-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and N-linked glycans in HIV-1 gp120, with a predominance of N-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, N-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, N-glycans at positions close to, but not immediately neighboring disulphide bridges seem to be disfavored at the N-terminal side of the involved cysteines. Such a pronounced co-localization of disulphide bridges and N-glycans was also found for the N-glycans on glycoprotein E1 of the hepatitis C virus (HCV) but not for other heavily glycosylated proteins such as E2 from HCV and the surface GP from Ebola virus. The potential functional role of the presence of N-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved N-glycans or by inserting new N-glycosylation sites near disulphide bridges. The generated HIV-1_NL4.3 mutants were subjected to an array of assays, determining the envelope glycoprotein levels in mutant viral particles, their infectivity and the capture and transmission efficiencies of mutant virus particles by DC-SIGN. Three N-glycans located nearby disulphide bridges were found to be crucial for the preservation of several of these functions of gp120. In addition, introduction of new N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the N-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection and transmission.     

Trans-splicing of an early embryo mRNA in Litomosoides carinii, coding for the major microfilarial sheath protein gp22.

Both genomic and cDNA clones have been isolated encoding the major sheath glycoprotein, gp22, of Litomosoides carinii microfilariae. The mature gp22 mRNA is shown to result from both trans-splicing of a 22-nucleotide 5'-leader sequence to an acceptor site at position 313 of the pre-mRNA, immediately upstream from the start codon, and from cis-splicing of a 117-nt intron located within the coding sequence. Cis-splicing precedes the trans-splicing reaction. The gp22 reading frame of 148 codons has the inferred structure of a prepro-protein and includes a leader peptide and a pro-segment ahead of the known N terminus of the mature, extracellular protein of 105 amino acids. The N-terminal part of that protein contains five repeats of an elastin-related pentapeptide sequence, which, together with a proline-threonine segment between two Cys clusters in the center and at its C terminus, may cause an elongated conformation with an apparent molecular size of 22 kDa in contrast to the calculated M(r) of 11,200.     

Carbohydrate structure of glycoprotein 52 encoded by the polycythemia-inducing strain of Friend spleen focus-forming virus

The primary envelope (env) gene product of the polycythemia-inducing variant of Friend spleen focus-forming virus (F-SFFVP), representing a glycoprotein with an apparent Mr of 52,000 (gp52), was isolated from F-SFFVP-infected normal rat kidney cells metabolically labeled with [2-3H]mannose in the presence or absence of glucose. Structures of the oligosaccharides present were determined by micromethylation analysis, acetolysis, and digestion with exoglycosidases. Gp52 radiolabeled in the presence of glucose contains solely oligomannosidic glycans comprising 6 to 9 mannose residues (Man6-9GlcNAc2), some of which carry additional glucose. The structures of the glycans found reflect the typical intermediates of oligosaccharide processing. The glycosylation of gp52 isolated from glucose-deprived cells (-Glc), however, is characterized by increased amounts of Man5-7 species comprising other structural isomers. Only gp52 (-Glc) glycans are, in part, further processed yielding incomplete complex-type oligosaccharides. Our results demonstrate that the limited post-translational processing of the primary F-SFFVP env gene product is neither due to aberrant trimming of its oligomannosidic glycans nor due to transfer of immature lipid-linked oligosaccharide-intermediates as observed in glucose-starved cells.     

Complete sequence of the Rous sarcoma virus env gene: identification of structural and functional regions of its product.

The amino-terminal amino acid sequences of gp85 and gp37, the envelope glycoproteins of Rous sarcoma virus (RSV), were determined. Alignment of these sequences with the amino acid sequence predicted from the complete nucleotide sequence of the Prague strain of RSV, subgroup C (PR-C), has allowed us to delineate the env gene-coding region of this virus. The coding sequences for gp85 and gp37 have been placed in an open reading frame that extends from nucleotide 5045 to nucleotide 6862 and predict sizes of 341 amino acids (36,962 molecular weight) for gp85 and 198 amino acids (21,566 molecular weight) for gp37. Carbohydrate makes a significant contribution to the observed molecular weights of these polypeptides--the amino acid sequence contains 14 potential glycosylation sites (Asn-X-Ser/Thr) in gp85 and two in gp37. Experiments aimed at estimating the number of carbohydrate side chains yielded results consistent with most or all of these sites being occupied. Although an initiation codon is located early (codon 4) in the open reading frame, it is likely that splicing yields an mRNA on which translation initiates at the same AUG as that of the gag gene to produce a nascent polypeptide in which gp85 is preceded by a 62-amino-acid-long leader peptide. This leader contains the hydrophobic sequence (signal sequence) necessary for translocation across the endoplasmic reticulum and is completely removed from the env gene product during translation. The polyprotein precursor, Pr95env, is cleaved to gp85 and gp37 at the carboxyl side of the basic sequence:-Arg-Arg-Lys-Arg-. gp85 is attached through a disulphide linkage to gp37, and although the positions of the cysteines involved in this linkage are not known, the presence of a 27-amino-acid-long hydrophobic region at the carboxy-terminus of gp37 is consistent with its role as a membrane anchor for the viral glycoprotein complex. The location of host range variable regions with respect to the possible tertiary structure of the complex is discussed.     

Pathogenicity of a Mutant Friend Spleen Focus-Forming Virus Encoding an Env-Like Membrane Glycoprotein (gp55) with Substitution by a Xenotropic Murine Leukemia Virus Env gp70 Sequence

Friend spleen focus-forming virus (F-SFFV) is a replication-defective acutely leukemogenic mouse retrovirus and encodes an envelope protein (Env)-like membrane glycoprotein (gp55) in its defective env gene, which is responsible for the early stage of the viral leukemogenesis. Gp55 is a modified Env protein and contains a polytropic mink cell focus-inducing (MCF) murine leukemia virus (MuLV) Env gp70-derived sequence in its amino-terminal region. To evaluate the possibility that the presumed binding of gp55 to an MCF MuLV receptor protein has some role in leukemogenesis, we examined the biological activities of a mutant gp55 (XE gp55), which has a xenotropic MuLV Env gp70 amino-terminal region. XE gp55 displayed almost the same biological activities as the wild-type gp55, excluding the above possibility.     

Mutational Analysis of the N-Linked Glycans on Autographa californica Nucleopolyhedrovirus gp64

gp64 is the major envelope glycoprotein in the budded form of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). gp64 is essential for AcMNPV infection, as it mediates penetration of budded virus into host cells via the endocytic pathway. In this study, we used site-directed mutagenesis to map the positions of the N-linked glycans on AcMNPV gp64, characterize their structures, and evaluate their influence on gp64 function. We found that four of the five consensus N-glycosylation sites in gp64 are used, and we mapped the positions of those sites to amino acids 198, 355, 385, and 426 in the polypeptide chain. Endoglycosidase H sensitivity assays showed that N-linked glycans located at different positions are processed to various degrees. Lectin blotting analyses showed that each N-linked glycan on gp64 contains α-linked mannose, all but one contains α-linked fucose, and none contains detectable β-linked galactose or α2,6-linked sialic acid. The amounts of infectious progeny produced by AcMNPV mutants lacking one, two, or three N-linked glycans on gp64 were about 10- to 100-fold lower than wild-type levels. This reduction did not correlate with reductions in the expression, transport, or inherent fusogenic activity of the mutant gp64s or in the gp64 content of mutant budded virus particles. However, all of the mutant viruses bound more slowly than the wild type. Therefore, elimination of one or more N-glycosylation sites in AcMNPV gp64 impairs binding of budded virus to the cell, which explains why viruses containing these mutant forms of gp64 produce less infectious progeny.     

Common peptide epitopes in glycophorin and the endothelial sialoglycoprotein gp60.

Polyclonal anti-serum made against murine glycophorin gp3 (alpha gp) recognizes the endothelial albumin binding glycoprotein, gp60. In this study, we investigated the nature (peptide vs. carbohydrate) of the common epitope. First, a new technique was developed to remove oligosaccharides from glycoproteins that were first immobilized on filters and then subjected to beta-elimination. When greater than 90% of the glycans of gp60 were removed, alpha gp still recognized gp60 without apparent loss of affinity. Second, we used brefeldin A to accumulate unglycosylated glycophorin precursors in order to affinity-purify peptide-specific alpha gp immuno-globulins; these antibodies recognized gp60. Finally, alpha gp recognized from in vitro translations a 48 kDa putative polypeptide precursor of gp60. These different approaches indicate that gp60 and gp3 have at least one common epitope in their peptide backbones.     

Primary structure and processing of the Candida tsukubaensis alpha-glucosidase. Homology with the rabbit intestinal sucrase-isomaltase complex and human lysosomal alpha-glucosidase.

The nucleotide sequence of a 4.39-kb DNA fragment encoding the alpha-glucosidase gene of Candida tsukubaensis is reported. The cloned gene contains a major open reading frame (ORF 1) which encodes the alpha-glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa. N-terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the alpha-glucosidase precursor is proteolytically processed by removal of an N-terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63-65 kDa and 50-52 kDa, respectively. Both subunits are secreted by the heterologous host S. cerevisiae in a glycosylated form. Coincident with its efficient expression in the heterologous host, the C. tsukubaensis alpha-glucosidase gene contains many of the canonical features of highly expressed S. cerevisiae genes. There is considerable sequence similarity between C. tsukubaensis alpha-glucosidase, the rabbit sucrase-isomaltase complex (proSI) and human lysosomal acid alpha-glucosidase. The cloned DNA fragment from C. tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids. The function and identity of the polypeptide encoded by ORF 2 is not known.     

对虾白斑综合征病毒的细胞因子受体基因的分析与表达

在对虾白斑综合征病毒(White spot syndrome virus,WSSV)的基因组中发现一个具有细胞因子受体特征的开放阅读框,该阅读框全长2022个核苷酸,编码674个氨基酸,蛋白质理论分子量为76kDa。该基因含有真核生物细胞因子gp130受体特征序列。为了研究该基因的功能,采用PCR方法从病毒基因组中扩增出基因片段,克隆到pGEM-T Easy载体中,经BamH I和Sal I双酶切后插入pET28b表达载体中。重组质粒转化到大肠杆菌BL21中,IPTG诱导后,经SDS-PAGE电泳表明在。76kDa处有目的蛋白表达。用冰浴超声波对诱导后的菌液进行处理以获得初步纯化的蛋白,作为抗原人工免疫实验兔子以获得含特异性抗体的抗血清。该基因的表达成功,为其功能的进一步深入研究奠定了基础。     

Ubiquitous 9-O-acetylation of sialoglycoproteins restricted to the Golgi complex

9-O-Acetylation of sialic acid is known as a cell type-specific modification of secretory and plasma membrane glycoconjugates of higher vertebrates with important functions in modulating cell-cell recognition. Using a recombinant probe derived from influenza C virus hemagglutinin, we discovered 9-O-acetylated protein in the Golgi complex of various cell lines, most of which did not display 9-O-acetylated sialic acid on the cell surface. All cell lines expressed a sulfated glycoprotein of 50 kDa (sgp50) carrying 9-O-acetylated sialic acids, which was used as a model substrate. Like gp40, the major receptor for influenza C virus of Madin-Darby canine kidney I cells, sgp50 is 9-O-acetylated on O-linked glycans. However, gp40 was not 9-O-acetylated when expressed in Madin-Darby canine kidney II or COS-7 cells. The results demonstrate the existence of two 9-O-acetylation machineries for O-glycosylated proteins with distinct substrate specificities. The widespread occurrence of 9-O-acetylated protein in the Golgi furthermore suggests an additional intracellular role for this modification.