Diagnostic consequences of cytomegalovirus glycoprotein B polymorphisms.
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ABSTRACT:
SUBMITTER: Novak Z
PROVIDER: S-EPMC3147759 | biostudies-literature | 2011 Aug
REPOSITORIES: biostudies-literature
Publications
Diagnostic consequences of cytomegalovirus glycoprotein B polymorphisms.
Novak Zdenek Z Chowdhury Nazma N Ross Shannon A SA Pati Sunil K SK Fowler Karen K Boppana Suresh B SB
Journal of clinical microbiology 20110608 8
Failure of a cytomegalovirus (CMV) real-time PCR assay targeting glycoprotein B (gB) was investigated. A multiplex assay targeting gB and immediate-early 2 (IE2) genes showed discordant results (gB negative and IE positive or a >10-fold-higher viral load with IE primers) in saliva from 14.6% of CMV-infected newborns. Sequencing revealed 3 patterns of gB variations. ...[more]
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Project description:BackgroundMultiple strains infection of human cytomegalovirus (HCMV) was found to be correlated with increased viral load in immunodeficient patients. However, the pathogenic mechanism underlying this correlation remains unclear. To evaluate genetic polymorphisms of HCMV glycoprotein and their potential role in its viral load, HCMV glycoprotein B, N, and O (gB, gN and gO) genotypes was studied in the population of HCMV infected acquired immune deficiency syndrome (AIDS) patients. The association between glycoprotein polymorphisms and HCMV viral load was analyzed.MethodsThe genetic polymorphisms of glycoprotein from sera of 60 HCMV infected AIDS patients was investigated by multiplex nested PCR and sequencing. HCMV viral load was evaluated by quantitative PCR.ResultsgB1, gO1a, and gN4a were the predominant glycoprotein genotypes in HCMV infected AIDS patients and composed 86.96%, 78.8%, and 49.2%, respectively. Only gN4a genotype infection significantly increased viral load (P = 0.048). 71% (43/60) of HCMV infected AIDS patients were found to carry multiple HCMV strains infection. A novel potential linkage of gO1a/gN4a was identified from multiple HCMV infected patients. It was the most frequent occurrence, accounted for 51.5% in 33 patients with gO and gN genotypes infection. Furthermore, the gO1a/gN4a linkage was correlated to an increased viral load (P = 0.020).ConclusionThe gN4a correlates to higher level HCMV load in AIDS patients. Interestingly, a novel gO1a/gN4a linkage is identified from the patients with multiple HCMV strains infection and is also associated with an increased viral load. Therefore, the pathogenic mechanism underlying glycoprotein polymorphisms and interaction of variants should be analyzed further.
| S-EPMC5415198 | biostudies-literature
Project description:Human cytomegalovirus glycoprotein B (gB) represents a target for diagnosis and treatment in view of the role it plays in virus entry and spread. Nevertheless, to our knowledge, rare detection of a gB antigen has been reported in transplant patients and limited information is available about diagnostic gB monoclonal antibodies (mAbs). Our aim was to develop gB mAbs with diagnostic potential. Hydrophilic gB peptides (ST: amino acids 27-40, SH: amino acids 81-94) of favorable immunogenicity were synthesized and used to immunize BALB/c mice. Two mAbs, named ZJU-FH6 and ZJU-FE6, were generated by the hybridoma technique and limited serial dilution and then characterized by indirect ELISA, Western blotting, immunoprecipitation, and immunohistochemical staining. The mAbs displayed high titers of specific binding affinities for the ST and SH synthetic peptides at an mAb dilution of 1:60,000 and 1:240,000, respectively. Western blotting and immunoprecipitation indicated that these mAbs recognized both denatured and native gB of the Towne and AD169 strains. The mAbs, when used as the primary antibody, showed positive staining in cells infected with both Towne and AD169 strains. The mAbs were then tested on patients submitted to allogeneic hematopoietic stem cell transplantation. The gB antigen positivity rates of the patients tested using ZJU-FH6 and ZJU-FE6 were 62.0 and 63.0%, respectively. The gB antigen showed a significant correlation with the level of pp65 antigen in peripheral blood leukocytes. In conclusion, two potential diagnostic gB mAbs were developed and were shown to be capable of recognizing gB in peripheral blood leukocytes in a reliable manner.
| S-EPMC3854279 | biostudies-literature
Project description:Congenital Cytomegalovirus (CMV) infection is an important medical problem that has yet no current solution. A clinical trial of CMV glycoprotein B (gB) vaccine in young women showed promising efficacy. Improved understanding of the basis for prevention of CMV infection is essential for developing improved vaccines.We genotyped 142 women previously vaccinated with three doses of CMV gB for single nucleotide polymorphisms (SNPs) in TLR 1-4, 6, 7, 9, and 10, and their associated intracellular signaling genes. SNPs in the platelet-derived growth factor receptor (PDGFRA) and integrins were also selected based on their role in binding gB. Specific SNPs in TLR7 and IKBKE (inhibitor of nuclear factor kappa-B kinase subunit epsilon) were associated with antibody responses to gB vaccine. Homozygous carriers of the minor allele at four SNPs in TLR7 showed higher vaccination-induced antibody responses to gB compared to heterozygotes or homozygotes for the common allele. SNP rs1953090 in IKBKE was associated with changes in antibody level from second to third dose of vaccine; homozygotes for the minor allele exhibited lower antibody responses while homozygotes for the major allele showed increased responses over time.These data contribute to our understanding of the immunogenetic mechanisms underlying variations in the immune response to CMV vaccine.
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Project description:The core, conserved function of the herpesvirus gH/gL is to promote gB-mediated membrane fusion during entry, although the mechanism is poorly understood. The human cytomegalovirus (HCMV) gH/gL can exist as either the gH/gL/gO trimer or the gH/gL/UL128/UL130/UL131 (gH/gL/UL128-131) pentamer. One model suggests that gH/gL/gO provides the core fusion role during entry into all cells within the broad tropism of HCMV, whereas gH/gL/UL128-131 acts at an earlier stage, by a distinct receptor-binding mechanism to enhance infection of select cell types, such as epithelial cells, endothelial cells, and monocytes/macrophages. To further study the distinct functions of these complexes, mutants with individual charged cluster-to-alanine (CCTA) mutations of gH and gL were combined to generate a library of 80 mutant gH/gL heterodimers. The majority of the mutant gH/gL complexes were unable to facilitate gB-mediated membrane fusion in transient-expression cell-cell fusion experiments. In contrast, these mutants supported the formation of gH/gL/UL128-131 complexes that could block HCMV infection in receptor interference experiments. These results suggest that receptor interactions with gH/gL/UL128-131 involve surfaces contained on the UL128-131 proteins but not on gH/gL. gH/gL/UL128-131 receptor interference could be blocked with anti-gH antibodies, suggesting that interference is a cell surface phenomenon and that anti-gH antibodies can block gH/gL/UL128-131 in a manner that is distinct from that for gH/gL/gO.Interest in the gH/gL complexes of HCMV (especially gH/gL/UL128-131) as vaccine targets has far outpaced our understanding of the mechanism by which they facilitate entry and contribute to broad cellular tropism. For Epstein-Barr virus (EBV), gH/gL and gH/gL/gp42 are both capable of promoting gB fusion for entry into epithelial cells and B cells, respectively. In contrast, HCMV gH/gL/gO appears to be the sole fusion cofactor that promotes gB fusion activity, whereas gH/gL/UL128-131 expands cell tropism through a distinct yet unknown mechanism. This study suggests that the surfaces of HCMV gH/gL are critical for promoting gB fusion but are dispensable for gH/gL/UL128-131 receptor interaction. This underscores the importance of gH/gL/gO in HCMV entry into all cell types and reaffirms the complex as a candidate target for vaccine development. The two functionally distinct forms of gH/gL present in HCMV make for a useful model with which to study the fundamental mechanisms by which herpesvirus gH/gL regulates gB fusion.
| S-EPMC4810707 | biostudies-literature
Project description:Human cytomegalovirus (HCMV), a dsDNA, enveloped virus, is a ubiquitous pathogen that establishes lifelong latent infections and caused disease in persons with compromised immune systems, e.g., organ transplant recipients or AIDS patients. HCMV is also a leading cause of congenital viral infections in newborns. Entry of HCMV into cells requires the conserved glycoprotein B (gB), thought to function as a fusogen and reported to bind signaling receptors. gB also elicits a strong immune response in humans and induces the production of neutralizing antibodies although most anti-gB Abs are non-neutralizing. Here, we report the crystal structure of the HCMV gB ectodomain determined to 3.6-Å resolution, which is the first atomic-level structure of any betaherpesvirus glycoprotein. The structure of HCMV gB resembles the postfusion structures of HSV-1 and EBV homologs, establishing it as a new member of the class III viral fusogens. Despite structural similarities, each gB has a unique domain arrangement, demonstrating structural plasticity of gB that may accommodate virus-specific functional requirements. The structure illustrates how extensive glycosylation of the gB ectodomain influences antibody recognition. Antigenic sites that elicit neutralizing antibodies are more heavily glycosylated than those that elicit non-neutralizing antibodies, which suggest that HCMV gB uses glycans to shield neutralizing epitopes while exposing non-neutralizing epitopes. This glycosylation pattern may have evolved to direct the immune response towards generation of non-neutralizing antibodies thus helping HCMV to avoid clearance. HCMV gB structure provides a starting point for elucidation of its antigenic and immunogenic properties and aid in the design of recombinant vaccines and monoclonal antibody therapies.
| S-EPMC4617298 | biostudies-literature
Project description:Glycoprotein B (gB) is the most highly conserved of the envelope glycoproteins of human herpesviruses. The gB protein of human cytomegalovirus (CMV) serves multiple roles in the life cycle of the virus. To investigate structural properties of gB that give rise to its function, we sought to determine the disulfide bond arrangement of gB. To this end, a recombinant form of gB (gB-S) comprising the entire ectodomain of the glycoprotein (amino acids 1 to 750) was constructed and expressed in insect cells. Proteolytic fragmentation and mass spectrometry were performed using purified gB-S, and the five disulfide bonds that link 10 of the 11 highly conserved cysteine residues of gB were mapped. These bonds are C94-C550, C111-C506, C246-C250, C344-C391, and C573-C610. This configuration closely parallels the disulfide bond configuration of herpes simplex type 2 (HSV-2) gB (N. Norais, D. Tang, S. Kaur, S. H. Chamberlain, F. R. Masiarz, R. L. Burke, and F. Markus, J. Virol. 70:7379-7387, 1996). However, despite the high degree of conservation of cysteine residues between CMV gB and HSV-2 gB, the disulfide bond arrangements of the two homologs are not identical. We detected a disulfide bond between the conserved cysteine residue 246 and the nonconserved cysteine residue 250 of CMV gB. We hypothesize that this disulfide bond stabilizes a tight loop in the amino-terminal fragment of CMV gB that does not exist in HSV-2 gB. We predicted that the cysteine residue not found in a disulfide bond of CMV gB, cysteine residue 185, would play a role in dimerization, but a cysteine substitution mutant in cysteine residue 185 showed no apparent defect in the ability to form dimers. These results indicate that gB oligomerization involves additional interactions other than a single disulfide bond. This work represents the second reported disulfide bond structure for a herpesvirus gB homolog, and the discovery that the two structures are not identical underscores the importance of empirically determining structures even for highly conserved proteins.
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Project description:IntroductionCytomegalovirus (CMV) predisposes to several clinical complications and is a major cause of morbidity and mortality in immunocompromised patients, including patients with hematological malignancies (HM). The present study was carried out to determine the distribution of CMV glycoprotein B, N, and O (gB, gN, and gO) genotypes and their potential effect on its viral load and on clinical outcomes in a cohort of Tunisian non-hematopoietic stem cell transplant (HSCT) patients with HM undergoing chemotherapy.MethodsCMV viral load was evaluated by real-time quantitative PCR. The gB, gN, and gO genotypes of the CMV strains were analyzed by multiplex nested PCR and sequencing.ResultsThis prospective study involved 60 clinical isolates obtained from 60 non-HSCT patients with HM undergoing chemotherapy. Mixed CMV gB, gN, and gO genotypes were the predominant glycoprotein genotypes in 31%, 41.4%, and 46.4% of patients, respectively. Mixed gB genotypes were associated with higher initial levels of CMV load (p = 0.001), increased rate of fever (0.025), and co-infection with other herpesviruses (HHVs) (p = 0.024) more frequently than in single gB genotype. Mixed gN genotypes were more associated with severe lymphopenia (ALC < 500/µL) (p = 0.01) and increased risk of death (p = 0.042) than single gN genotype. Single gO2b genotype had also a more unfavorable outcome (p = 0.009) than the other single gO genotype. Mixed gO genotypes were associated with female gender (p = 0.015), acute leukemia disease (p = 0.036), initial high level of CMV viral load (at least 1000 copies/mL) (p = 0.029), skin rash (p = 0.01) more frequently than in single gO genotype. The gO1a/gN3b linkage was associated with an increased initial viral load (p = 0.012).ConclusionInfection with mixed CMV genotypes was common and multiple gB, gN, and gO genotypes were associated with clinical manifestation and higher viral load.
| S-EPMC8322362 | biostudies-literature
Project description:A cytomegalovirus (CMV) was isolated from its natural host, Peromyscus maniculatus, and was designated Peromyscus CMV (PCMV). A recombinant PCMV was constructed that contained Sin Nombre virus glycoprotein G1 (SNV-G1) fused in frame to the enhanced green fluorescent protein (EGFP) gene inserted into a site homologous to the human CMV UL33 (P33) gene. The recombinant CMV was used for expression and immunization of deer mice against SNV-G1. The results of the study indicate that P. maniculatus could be infected with as few as 10 virus particles of recombinant virus. Challenge of P. maniculatus with either recombinant or wild-type PCMV produced no overt pathology in infected animals. P. maniculatus immunized with recombinant virus developed an antibody response to SNV and EGFP. When rechallenged with recombinant virus, animals exhibited an anamnestic response against SNV. Interestingly, a preexisting immune response against PCMV did not prevent reinfection with recombinant PCMV.
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Project description:Herpes viruses persist in the infected host and are transmitted between hosts in the presence of a fully functional humoral immune response, suggesting that they can evade neutralization by antiviral antibodies. Human cytomegalovirus (HCMV) encodes a number of polymorphic highly glycosylated virion glycoproteins (g), including the essential envelope glycoprotein, gN. We have tested the hypothesis that glycosylation of gN contributes to resistance of the virus to neutralizing antibodies. Recombinant viruses carrying deletions in serine/threonine rich sequences within the glycosylated surface domain of gN were constructed in the genetic background of HCMV strain AD169. The deletions had no influence on the formation of the gM/gN complex and in vitro replication of the respective viruses compared to the parent virus. The gN-truncated viruses were significantly more susceptible to neutralization by a gN-specific monoclonal antibody and in addition by a number of gB- and gH-specific monoclonal antibodies. Sera from individuals previously infected with HCMV also more efficiently neutralized gN-truncated viruses. Immunization of mice with viruses that expressed the truncated forms of gN resulted in significantly higher serum neutralizing antibody titers against the homologous strain that was accompanied by increased antibody titers against known neutralizing epitopes on gB and gH. Importantly, neutralization activity of sera from animals immunized with gN-truncated virus did not exhibit enhanced neutralizing activity against the parental wild type virus carrying the fully glycosylated wild type gN. Our results indicate that the extensive glycosylation of gN could represent a potentially important mechanism by which HCMV neutralization by a number of different antibody reactivities can be inhibited.
| S-EPMC3486915 | biostudies-literature
Project description:Envelopment of a herpesvirus particle is a complex process of which much is still to be learned. We previously identified the glycoprotein gpUL132 of human cytomegalovirus (HCMV) as an envelope component of the virion. In its carboxy-terminal portion, gpUL132 contains at least four motifs for sorting of transmembrane proteins to endosomes; among them are one dileucine-based signal and three tyrosine-based signals of the YXXØ and NPXY (where X stands for any amino acid, and Ø stands for any bulky hydrophobic amino acid) types. To investigate the role of each of these trafficking signals in intracellular localization and viral replication, we constructed a panel of expression plasmids and recombinant viruses in which the signals were rendered nonfunctional by mutagenesis. In transfected cells wild-type gpUL132 was mainly associated with the trans-Golgi network. Consecutive mutation of the trafficking signals resulted in increasing fractions of the protein localized at the cell surface, with gpUL132 mutated in all four trafficking motifs predominantly associated with the plasma membrane. Concomitant with increased surface expression, endocytosis of mutant gpUL132 was reduced, with a gpUL132 expressing all four motifs in mutated form being almost completely impaired in endocytosis. The replication of recombinant viruses harboring mutations in single trafficking motifs was comparable to replication of wild-type virus. In contrast, viruses containing mutations in three or four of the trafficking signals showed pronounced deficits in replication with a reduction of approximately 100-fold. Moreover, recombinant viruses expressing gpUL132 with three or four trafficking motifs mutated failed to incorporate the mutant protein into the virus particle. These results demonstrate a role of endocytosis of an HCMV envelope glycoprotein for incorporation into the virion and optimal virus replication.
| S-EPMC2898235 | biostudies-literature