Characterization of a Panel of Insertion Mutants in Human Cytomegalovirus Glycoprotein B

Abstract

Glycoprotein B (gB; gpUL55) of human cytomegalovirus (HCMV) plays a critical role in virus entry and cell-to-cell spread of infection. To define the structure-function relationships in gB, a panel of linker-insertion mutations was generated throughout the coding region. This strategy yielded a panel of 22 mutants with four amino acid insertions and 3 large truncation mutants. Assessment of the mutant proteins' biosynthetic properties and folding patterns analyzed in context with predicted secondary features revealed novel insights into gB's structure and trafficking properties. All of the insertion mutants were able to assemble into oligomers, suggesting that oligomerization is tolerant of small insertions and/or that multiple regions of the protein may be involved. Computer algorithm predictions of gB's secondary structure indicate that the furin-recognized cleavage site falls within an exposed loop. This loop may be particularly sensitive to structural alterations, since insertions upstream and downstream of the cleavage site rendered the mutant proteins cleavage defective. In addition, a strong correlation existed between terminal folding and cleavage of gB. Interestingly, terminal folding was not correlated with delivery to the cell surface but may influence the rate of transport to the cell surface. Nine mutants, containing insertions in both the extracellular and intracellular portions of gB, retained wild-type structural properties. This panel of characterized gB mutants, the first of this type for an HCMV protein, will be a useful tool in dissecting the role of gB during HCMV infection.Human cytomegalovirus (HCMV), a member of the Herpesviridae, is a ubiquitous pathogen that causes significant morbidity and mortality in immunocompromised individuals (1). The frequency and severity of HCMV diseases and the paucity of desirable therapeutic options indicate the need to identify and understand the structures and functions of key potential antiviral targets. An ideal target for either prevention or therapy would be to block entry and/or cell-to-cell spread of HCMV into and between cells. To rationally develop drugs, a detailed understanding of the viral and cellular components necessary for virus entry is needed. While HCMV entry is not completely understood, significant progress has been made in recent years identifying the major viral components involved in entry (reviewed in reference 13). Entry of HCMV into host cells requires a complex series of sequential interactions between multiple viral and cellular molecules (7,13,28). Viral entry can be divided into two distinct stages: attachment and penetration. Virus binding to heparan sulfate proteoglycans (HSPGs) is the initial step in the entry pathway (15,27). Virus bound to cell surface HSPG is rapidly converted to a more stable binding state likely mediated by interaction of the virion with a second cellular receptor (15). Once stably attached, the virion penetrates into the cell by direct fusion of the virus envelope and the cellular plasma membrane (14)...

Antigenic Domain 1 Is Required for Oligomerization of Human Cytomegalovirus Glycoprotein B

“…Similar findings have been reported by Singh et al, who described linker insertion mutants of gB with mutations outside of AD-1 that lead to loss of oligomerization and cleavage (44). Interestingly, these investigators also noted that a linker insertion mutant within AD-1 (aa 627) leads to loss of reactivity by an AD-1-specific MAb and loss of oligomer formation (44). This finding independently demonstrated that AD-1 formation was required for oligomer formation and transport of gB from the ER.…”

“…We would argue that the gB Cys mutations at positions 508 and 550 resulted in misfolded molecules that failed to exit the ER and did not oligomerize, based on their lack of reactivity with MAb 27-39. Similar findings have been reported by Singh et al, who described linker insertion mutants of gB with mutations outside of AD-1 that lead to loss of oligomerization and cleavage (44). Interestingly, these investigators also noted that a linker insertion mutant within AD-1 (aa 627) leads to loss of reactivity by an AD-1-specific MAb and loss of oligomer formation (44).…”

Identification of Functional Domains in Herpes Simplex Virus 2 Glycoprotein B

“…The more global approach of linker-insertion mutagenesis was used with HSV-1 gB (5,24,29) and with HCMV gB (36). For the HCMV protein, several mutants that retained correct folding were identified, but no functional assay was available to test their activity (36).…”

“…For the HCMV protein, several mutants that retained correct folding were identified, but no functional assay was available to test their activity (36). For the HSV-1 protein, all mutants except one either retained functional activity or were misfolded, so only one candidate functional domain was identified.…”

Disulfide Bond Configuration of Human Cytomegalovirus Glycoprotein B

“…An in-frame, four-amino-acid insertion mutant six amino acids downstream of C250 (mutant I-256) resulted in the loss of recognition by monoclonal antibody 27-39, which recognizes a folded and conformation specific epitope, indicating that this region may be important in the final folded form of gB (28). The I-256 insertion mutant also showed a defect in proteolytic processing by furin despite the fact that the furin cleavage site is over 200 amino acids downstream of the insertion.…”