Limits...
Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: putative CD81 binding region 1 is not involved in CD81 binding.

Rothwangl KB, Manicassamy B, Uprichard SL, Rong L - Virol. J. (2008)

Bottom Line: Based on these characteristics, mutants either displayed wt characteristics (high infectivity [> or = 50% of wt HCVpp], CD81 binding, E1E2 expression, association, and incorporation into viral particles and proper conformation) or segregated into 4 distinct low infectivity (< or = 50% of wt HCVpp) mutant phenotypes: (I) CD81 binding deficient (despite wt E1E2 expression, incorporation and association and proper conformation); (II) CD81 binding competent, but lack of E1 detection on the viral particle, (despite adequate E1E2 expression in producer cell lysates and proper conformation); (III) CD81 binding competent, with adequate E1E2 expression, incorporation, association, and proper E2 conformation (i.e. no defect identified to explain the reduced infectivity observed); (IV) CD81 binding deficient due to disruption of E2 mutant protein conformation.Although most alanine substitutions within the putative CD81 binding region 1 (amino acids 474-492) displayed greatly reduced HCVpp infectivity, they retained soluble CD81 binding, proper E2 conformation, E1E2 association and incorporation into HCVpp suggesting that region 1 of E2 does not mediate binding to CD81.This region is highly conserved across genotypes, underlining its importance in mediating viral entry.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA. Katharin@uic.edu

ABSTRACT

Background: Hepatitis C virus (HCV) encodes two transmembrane glycoproteins E1 and E2 which form a heterodimer. E1 is believed to mediate fusion while E2 has been shown to bind cellular receptors including CD81. In this study, alanine substitutions in E2 were generated within putative CD81 binding regions to define residues critical for viral entry. The effect of each mutation was tested by challenging susceptible cell lines with mutant HCV E1E2 pseudotyped viruses generated using a lentiviral system (HCVpp). In addition to assaying infectivity, producer cell expression and HCVpp incorporation of HCV E1 and E2 proteins, CD81 binding profiles, and E1E2 association of mutants were examined.

Results: Based on these characteristics, mutants either displayed wt characteristics (high infectivity [> or = 50% of wt HCVpp], CD81 binding, E1E2 expression, association, and incorporation into viral particles and proper conformation) or segregated into 4 distinct low infectivity (< or = 50% of wt HCVpp) mutant phenotypes: (I) CD81 binding deficient (despite wt E1E2 expression, incorporation and association and proper conformation); (II) CD81 binding competent, but lack of E1 detection on the viral particle, (despite adequate E1E2 expression in producer cell lysates and proper conformation); (III) CD81 binding competent, with adequate E1E2 expression, incorporation, association, and proper E2 conformation (i.e. no defect identified to explain the reduced infectivity observed); (IV) CD81 binding deficient due to disruption of E2 mutant protein conformation.

Conclusion: Although most alanine substitutions within the putative CD81 binding region 1 (amino acids 474-492) displayed greatly reduced HCVpp infectivity, they retained soluble CD81 binding, proper E2 conformation, E1E2 association and incorporation into HCVpp suggesting that region 1 of E2 does not mediate binding to CD81. In contrast, conformationally correct E2 mutants (Y527 and W529) within the second putative CD81 binding region (amino acids 522-551) disrupted binding of E2 to CD81-GST, suggesting that region 2 is critical to CD81 binding. Likewise, all conformationally intact mutants within the third putative CD81 binding region (amino acids 612-619), except L615A, were important for E2 binding to CD81-GST. This region is highly conserved across genotypes, underlining its importance in mediating viral entry.

Show MeSH

Related in: MedlinePlus

Determining association of E1E2 mutants. 293T cells were transfected with HCV E1E2 wt, mutant, or E1 alone glycoprotein expression plasmids. Cells were lysed and cleared cell lysate was incubated with anti (α)-E2 antibody. Immune complexes were separated by SDS-PAGE and analyzed by Western Blot for E1 to determine if the E2 and E1 glycoproteins had formed dimers. Image is a composite.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2277408&req=5

Figure 5: Determining association of E1E2 mutants. 293T cells were transfected with HCV E1E2 wt, mutant, or E1 alone glycoprotein expression plasmids. Cells were lysed and cleared cell lysate was incubated with anti (α)-E2 antibody. Immune complexes were separated by SDS-PAGE and analyzed by Western Blot for E1 to determine if the E2 and E1 glycoproteins had formed dimers. Image is a composite.

Mentions: Having identified several E2 mutations that exhibit severely reduced infectivity while retaining the ability to bind CD81, we next investigated whether any of the alanine substitutions in E2 disrupted E1E2 association. It is known that E1 and E2 must properly dimerize in order to mediate HCV infectivity [2,43,46-49]. This is a relevant consideration for these mutations as E2 dimerization domains have been mapped to the transmembrane domains, a WHY motif at positions 487–489, amino acids 415–500 as well as amino acids L675, S678, L689 and L692 [48,50-53]. For this analysis, 293T cells were transiently transfected with the HCV glycoprotein constructs then lysed 48 h later. E2 protein was pulled down with polyclonal HCV E2 antibody and immunocomplexes were analyzed by Western Blot for the presence of E1 using a monoclonal antibody. To control for E2 antibody specificity, 293T cells were also transfected with E1 alone. For several mutants, most notably W549A, F550A and R614A, a greater amount of E2 was detected compared to wt (Fig. 5). Notably however, despite varying levels of E2 pulled down, E1 was detected in association with all the E2 mutants.


Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: putative CD81 binding region 1 is not involved in CD81 binding.

Rothwangl KB, Manicassamy B, Uprichard SL, Rong L - Virol. J. (2008)

Determining association of E1E2 mutants. 293T cells were transfected with HCV E1E2 wt, mutant, or E1 alone glycoprotein expression plasmids. Cells were lysed and cleared cell lysate was incubated with anti (α)-E2 antibody. Immune complexes were separated by SDS-PAGE and analyzed by Western Blot for E1 to determine if the E2 and E1 glycoproteins had formed dimers. Image is a composite.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2277408&req=5

Figure 5: Determining association of E1E2 mutants. 293T cells were transfected with HCV E1E2 wt, mutant, or E1 alone glycoprotein expression plasmids. Cells were lysed and cleared cell lysate was incubated with anti (α)-E2 antibody. Immune complexes were separated by SDS-PAGE and analyzed by Western Blot for E1 to determine if the E2 and E1 glycoproteins had formed dimers. Image is a composite.
Mentions: Having identified several E2 mutations that exhibit severely reduced infectivity while retaining the ability to bind CD81, we next investigated whether any of the alanine substitutions in E2 disrupted E1E2 association. It is known that E1 and E2 must properly dimerize in order to mediate HCV infectivity [2,43,46-49]. This is a relevant consideration for these mutations as E2 dimerization domains have been mapped to the transmembrane domains, a WHY motif at positions 487–489, amino acids 415–500 as well as amino acids L675, S678, L689 and L692 [48,50-53]. For this analysis, 293T cells were transiently transfected with the HCV glycoprotein constructs then lysed 48 h later. E2 protein was pulled down with polyclonal HCV E2 antibody and immunocomplexes were analyzed by Western Blot for the presence of E1 using a monoclonal antibody. To control for E2 antibody specificity, 293T cells were also transfected with E1 alone. For several mutants, most notably W549A, F550A and R614A, a greater amount of E2 was detected compared to wt (Fig. 5). Notably however, despite varying levels of E2 pulled down, E1 was detected in association with all the E2 mutants.

Bottom Line: Based on these characteristics, mutants either displayed wt characteristics (high infectivity [> or = 50% of wt HCVpp], CD81 binding, E1E2 expression, association, and incorporation into viral particles and proper conformation) or segregated into 4 distinct low infectivity (< or = 50% of wt HCVpp) mutant phenotypes: (I) CD81 binding deficient (despite wt E1E2 expression, incorporation and association and proper conformation); (II) CD81 binding competent, but lack of E1 detection on the viral particle, (despite adequate E1E2 expression in producer cell lysates and proper conformation); (III) CD81 binding competent, with adequate E1E2 expression, incorporation, association, and proper E2 conformation (i.e. no defect identified to explain the reduced infectivity observed); (IV) CD81 binding deficient due to disruption of E2 mutant protein conformation.Although most alanine substitutions within the putative CD81 binding region 1 (amino acids 474-492) displayed greatly reduced HCVpp infectivity, they retained soluble CD81 binding, proper E2 conformation, E1E2 association and incorporation into HCVpp suggesting that region 1 of E2 does not mediate binding to CD81.This region is highly conserved across genotypes, underlining its importance in mediating viral entry.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA. Katharin@uic.edu

ABSTRACT

Background: Hepatitis C virus (HCV) encodes two transmembrane glycoproteins E1 and E2 which form a heterodimer. E1 is believed to mediate fusion while E2 has been shown to bind cellular receptors including CD81. In this study, alanine substitutions in E2 were generated within putative CD81 binding regions to define residues critical for viral entry. The effect of each mutation was tested by challenging susceptible cell lines with mutant HCV E1E2 pseudotyped viruses generated using a lentiviral system (HCVpp). In addition to assaying infectivity, producer cell expression and HCVpp incorporation of HCV E1 and E2 proteins, CD81 binding profiles, and E1E2 association of mutants were examined.

Results: Based on these characteristics, mutants either displayed wt characteristics (high infectivity [> or = 50% of wt HCVpp], CD81 binding, E1E2 expression, association, and incorporation into viral particles and proper conformation) or segregated into 4 distinct low infectivity (< or = 50% of wt HCVpp) mutant phenotypes: (I) CD81 binding deficient (despite wt E1E2 expression, incorporation and association and proper conformation); (II) CD81 binding competent, but lack of E1 detection on the viral particle, (despite adequate E1E2 expression in producer cell lysates and proper conformation); (III) CD81 binding competent, with adequate E1E2 expression, incorporation, association, and proper E2 conformation (i.e. no defect identified to explain the reduced infectivity observed); (IV) CD81 binding deficient due to disruption of E2 mutant protein conformation.

Conclusion: Although most alanine substitutions within the putative CD81 binding region 1 (amino acids 474-492) displayed greatly reduced HCVpp infectivity, they retained soluble CD81 binding, proper E2 conformation, E1E2 association and incorporation into HCVpp suggesting that region 1 of E2 does not mediate binding to CD81. In contrast, conformationally correct E2 mutants (Y527 and W529) within the second putative CD81 binding region (amino acids 522-551) disrupted binding of E2 to CD81-GST, suggesting that region 2 is critical to CD81 binding. Likewise, all conformationally intact mutants within the third putative CD81 binding region (amino acids 612-619), except L615A, were important for E2 binding to CD81-GST. This region is highly conserved across genotypes, underlining its importance in mediating viral entry.

Show MeSH
Related in: MedlinePlus