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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.

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Binding of mutant HCV E1E2 glycoproteins to soluble CD81. (A) 293T cells transfected with HCV E1E2 wt or mutant expression vectors were lysed 24 h post-transfection. Cleared cell lysate was incubated with soluble CD81-GST fusion protein. Binding to CD81 was determined by Western Blot analysis of E2 and the GST tag. As a negative control, GST protein without soluble CD81 was incubated with HCV wt. Image is a composite. (B) 293T cells transfected with HCV E1E2 wt or specific mutant expression vectors were lysed 24 h post-transfection. Cleared cell lysate was incubated with AR3A (C1) conformational antibody to assess conformation of mutations. Immunoprecipitated proteins were detected by subsequent Western Blot analysis of E2. Image is a composite.
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Figure 4: Binding of mutant HCV E1E2 glycoproteins to soluble CD81. (A) 293T cells transfected with HCV E1E2 wt or mutant expression vectors were lysed 24 h post-transfection. Cleared cell lysate was incubated with soluble CD81-GST fusion protein. Binding to CD81 was determined by Western Blot analysis of E2 and the GST tag. As a negative control, GST protein without soluble CD81 was incubated with HCV wt. Image is a composite. (B) 293T cells transfected with HCV E1E2 wt or specific mutant expression vectors were lysed 24 h post-transfection. Cleared cell lysate was incubated with AR3A (C1) conformational antibody to assess conformation of mutations. Immunoprecipitated proteins were detected by subsequent Western Blot analysis of E2. Image is a composite.

Mentions: To confirm adequate expression of the various E2 alanine substitutions, mutant E2 protein levels in the 293T producer cell lysates transiently transfected with HIV-luc backbone and the HCV E1E2 glycoprotein plasmids were examined by Western Blot analysis. Actin levels were used as a control for protein loading. When probed with anti-E2 antibody, a band of ~70kDa was detected in the cell lysate of wt and all mutant glycoprotein transfected cells, corresponding to the size of the HCV E2 protein (Fig. 3A). Overall, cell lysate levels of E2 were reduced for the mutants in the putative CD81 binding regions 2 and 3, compared to region 1 (Fig. 3A). To determine if this reduced intracellular expression has an effect on E1E2 incorporation onto the viral particle, virions were pelleted through a 20% sucrose cushion and examined by Western Blot, probing for p24 capsid levels to control for pseudovirus particle loading. E2 extracted from the viral particle displayed a diffuse migration pattern with at least three distinct bands, most likely due to extensive N-linked glycosylation (Fig. 3B)[43]. These various forms of E2 were incorporated into particles regardless of the specific E2 mutation present and independent of the intracellular accumulation levels of the protein (Fig. 3A). Hence, the lower intracellular E2 levels detected for the mutants in regions 2 and 3 were not reflected in the amount of E2 incorporated into the viral particle. Levels of E1 detected on the various mutant viral particles however, varied greatly. Most dramatically, although E2 incorporation was not impaired, E1 was not detected in W487A or W549A mutant viral particles. This could either be due to the loss of the monoclonal antibody epitope the Western Blot was probed with or due to a lack of incorporation onto the viral particle. Based on these two E2 mutations coming down in the conformational antibody immunoprecipitation (Fig. 4B), we suspect E1 is present on HCVpp since both E1 and E2 need to be present for proper folding [44]. In any case, the level of E1 detected on the different mutant viral particles did not correlate with infectivity levels or correspond to a specific binding region. At the positions where greater levels of E1 were detected, the bands appeared as a couplet.


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)

Binding of mutant HCV E1E2 glycoproteins to soluble CD81. (A) 293T cells transfected with HCV E1E2 wt or mutant expression vectors were lysed 24 h post-transfection. Cleared cell lysate was incubated with soluble CD81-GST fusion protein. Binding to CD81 was determined by Western Blot analysis of E2 and the GST tag. As a negative control, GST protein without soluble CD81 was incubated with HCV wt. Image is a composite. (B) 293T cells transfected with HCV E1E2 wt or specific mutant expression vectors were lysed 24 h post-transfection. Cleared cell lysate was incubated with AR3A (C1) conformational antibody to assess conformation of mutations. Immunoprecipitated proteins were detected by subsequent Western Blot analysis of E2. Image is a composite.
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Figure 4: Binding of mutant HCV E1E2 glycoproteins to soluble CD81. (A) 293T cells transfected with HCV E1E2 wt or mutant expression vectors were lysed 24 h post-transfection. Cleared cell lysate was incubated with soluble CD81-GST fusion protein. Binding to CD81 was determined by Western Blot analysis of E2 and the GST tag. As a negative control, GST protein without soluble CD81 was incubated with HCV wt. Image is a composite. (B) 293T cells transfected with HCV E1E2 wt or specific mutant expression vectors were lysed 24 h post-transfection. Cleared cell lysate was incubated with AR3A (C1) conformational antibody to assess conformation of mutations. Immunoprecipitated proteins were detected by subsequent Western Blot analysis of E2. Image is a composite.
Mentions: To confirm adequate expression of the various E2 alanine substitutions, mutant E2 protein levels in the 293T producer cell lysates transiently transfected with HIV-luc backbone and the HCV E1E2 glycoprotein plasmids were examined by Western Blot analysis. Actin levels were used as a control for protein loading. When probed with anti-E2 antibody, a band of ~70kDa was detected in the cell lysate of wt and all mutant glycoprotein transfected cells, corresponding to the size of the HCV E2 protein (Fig. 3A). Overall, cell lysate levels of E2 were reduced for the mutants in the putative CD81 binding regions 2 and 3, compared to region 1 (Fig. 3A). To determine if this reduced intracellular expression has an effect on E1E2 incorporation onto the viral particle, virions were pelleted through a 20% sucrose cushion and examined by Western Blot, probing for p24 capsid levels to control for pseudovirus particle loading. E2 extracted from the viral particle displayed a diffuse migration pattern with at least three distinct bands, most likely due to extensive N-linked glycosylation (Fig. 3B)[43]. These various forms of E2 were incorporated into particles regardless of the specific E2 mutation present and independent of the intracellular accumulation levels of the protein (Fig. 3A). Hence, the lower intracellular E2 levels detected for the mutants in regions 2 and 3 were not reflected in the amount of E2 incorporated into the viral particle. Levels of E1 detected on the various mutant viral particles however, varied greatly. Most dramatically, although E2 incorporation was not impaired, E1 was not detected in W487A or W549A mutant viral particles. This could either be due to the loss of the monoclonal antibody epitope the Western Blot was probed with or due to a lack of incorporation onto the viral particle. Based on these two E2 mutations coming down in the conformational antibody immunoprecipitation (Fig. 4B), we suspect E1 is present on HCVpp since both E1 and E2 need to be present for proper folding [44]. In any case, the level of E1 detected on the different mutant viral particles did not correlate with infectivity levels or correspond to a specific binding region. At the positions where greater levels of E1 were detected, the bands appeared as a couplet.

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