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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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Alanine substitutions within putative CD81 binding regions dramatically affect HCVpp entry. 293T cells were cotransfected with the HIV-luc packaging vector along with HCV E1E2 mutant expression plasmids. HCVpp was harvested at 24 h post-transfection and used to infect susceptible cell lines (A) Huh7 and (B) Hep3B. Infectivity was measured 72 h pi using a luciferase reporter assay. Infectivity of each mutant is expressed as a percentage of the infectivity observed for the wild-type (wt) H77 HCV E1E2. Values shown are the mean and standard error for a minimum of three assays.
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Figure 2: Alanine substitutions within putative CD81 binding regions dramatically affect HCVpp entry. 293T cells were cotransfected with the HIV-luc packaging vector along with HCV E1E2 mutant expression plasmids. HCVpp was harvested at 24 h post-transfection and used to infect susceptible cell lines (A) Huh7 and (B) Hep3B. Infectivity was measured 72 h pi using a luciferase reporter assay. Infectivity of each mutant is expressed as a percentage of the infectivity observed for the wild-type (wt) H77 HCV E1E2. Values shown are the mean and standard error for a minimum of three assays.

Mentions: To identify which of the charged, conserved amino acids in the three putative CD81 interaction sites of E2 are critical for infectivity, a panel of alanine substitutions was generated within the context of H77 E2 (Fig. 1 and Fig. 2). The substitutions are numbered based on their position within the polyprotein of the H77 clone and use the one letter amino acid code to denote the amino acid present at the site prior to alanine substitution. After sequence confirmation of the alanine substitutions, HCVpp infectivity of permissive Huh7 and Hep3B cells was assessed by inoculating cells with HIV virions pseudotyped with either wt E1E2 or the mutant E1E2 glycoproteins (Fig. 2). While Huh7 cells support robust HCV infection in vitro [29-32] and are thus obviously a relevant cell lines for this analysis, confirmatory screening was also performed in Hep3B cells, which have been shown to be permissive for HCVpp entry. Infectivity was determined as a measure of luciferase activity. In these experiments, VSVG/HIV virions were used as a positive control. As expected, infection of the cells by VSVG/HIV virus leads to a high level of luciferase activity (~107 RLU) (Fig. 2). Infection of the target cells by wt HCV E1E2/HIV virus resulted in luciferase levels at least 2 log above a negative control, EnvA/HIV [42]. In the first putative CD81 binding region, two mutations at positions 474 and 481 retained a significant degree of infectivity relative to that of wt HCVpp E1E2 control. D481A demonstrated quantitatively similar infectivity in both Huh7 (Fig 2A) and Hep3B (Fig. 2B) cells (64% and 71% respectively), compared to wt. While mutant Y474A retained a higher percent infectivity in the Huh7 cells (76%) compared to Hep3B cells (36%), in both cell lines Y474A exhibited one of the highest levels of infectivity among the mutants tested. In contrast to these 2 mutants, the remaining alanine substitutions within the first putative CD81 binding region reduced infectivity to 5% or less of wt in both Huh7 and Hep3B cells. In the second putative CD81 binding region, alanine substitution of conserved residues severely decreased infectivity in both cell lines. While the predominant phenotype of mutations in the putative CD81 binding region 2 was ablation of infection, the D533A and F550A mutants did retain some detectable level of infectivity in Huh7 cells, 12 and 19% respectively. However this minimal level of infection was not detected in the Hep3B cell assay, confirming the severity of the infectivity defect associated with changes in these residues. Finally, in the third putative CD81 binding region, all mutations, with the exception of L615A, impaired infectivity below 4%. As seen with the Y474A mutation in region 1, the exact amount of infectivity exhibited by L615A varied between cell lines with 44% of wt levels observed in Huh7 compared to 10% of wt levels observed in Hep3B cells; however, the trend of reduced infectivity was consistent.


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)

Alanine substitutions within putative CD81 binding regions dramatically affect HCVpp entry. 293T cells were cotransfected with the HIV-luc packaging vector along with HCV E1E2 mutant expression plasmids. HCVpp was harvested at 24 h post-transfection and used to infect susceptible cell lines (A) Huh7 and (B) Hep3B. Infectivity was measured 72 h pi using a luciferase reporter assay. Infectivity of each mutant is expressed as a percentage of the infectivity observed for the wild-type (wt) H77 HCV E1E2. Values shown are the mean and standard error for a minimum of three assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Alanine substitutions within putative CD81 binding regions dramatically affect HCVpp entry. 293T cells were cotransfected with the HIV-luc packaging vector along with HCV E1E2 mutant expression plasmids. HCVpp was harvested at 24 h post-transfection and used to infect susceptible cell lines (A) Huh7 and (B) Hep3B. Infectivity was measured 72 h pi using a luciferase reporter assay. Infectivity of each mutant is expressed as a percentage of the infectivity observed for the wild-type (wt) H77 HCV E1E2. Values shown are the mean and standard error for a minimum of three assays.
Mentions: To identify which of the charged, conserved amino acids in the three putative CD81 interaction sites of E2 are critical for infectivity, a panel of alanine substitutions was generated within the context of H77 E2 (Fig. 1 and Fig. 2). The substitutions are numbered based on their position within the polyprotein of the H77 clone and use the one letter amino acid code to denote the amino acid present at the site prior to alanine substitution. After sequence confirmation of the alanine substitutions, HCVpp infectivity of permissive Huh7 and Hep3B cells was assessed by inoculating cells with HIV virions pseudotyped with either wt E1E2 or the mutant E1E2 glycoproteins (Fig. 2). While Huh7 cells support robust HCV infection in vitro [29-32] and are thus obviously a relevant cell lines for this analysis, confirmatory screening was also performed in Hep3B cells, which have been shown to be permissive for HCVpp entry. Infectivity was determined as a measure of luciferase activity. In these experiments, VSVG/HIV virions were used as a positive control. As expected, infection of the cells by VSVG/HIV virus leads to a high level of luciferase activity (~107 RLU) (Fig. 2). Infection of the target cells by wt HCV E1E2/HIV virus resulted in luciferase levels at least 2 log above a negative control, EnvA/HIV [42]. In the first putative CD81 binding region, two mutations at positions 474 and 481 retained a significant degree of infectivity relative to that of wt HCVpp E1E2 control. D481A demonstrated quantitatively similar infectivity in both Huh7 (Fig 2A) and Hep3B (Fig. 2B) cells (64% and 71% respectively), compared to wt. While mutant Y474A retained a higher percent infectivity in the Huh7 cells (76%) compared to Hep3B cells (36%), in both cell lines Y474A exhibited one of the highest levels of infectivity among the mutants tested. In contrast to these 2 mutants, the remaining alanine substitutions within the first putative CD81 binding region reduced infectivity to 5% or less of wt in both Huh7 and Hep3B cells. In the second putative CD81 binding region, alanine substitution of conserved residues severely decreased infectivity in both cell lines. While the predominant phenotype of mutations in the putative CD81 binding region 2 was ablation of infection, the D533A and F550A mutants did retain some detectable level of infectivity in Huh7 cells, 12 and 19% respectively. However this minimal level of infection was not detected in the Hep3B cell assay, confirming the severity of the infectivity defect associated with changes in these residues. Finally, in the third putative CD81 binding region, all mutations, with the exception of L615A, impaired infectivity below 4%. As seen with the Y474A mutation in region 1, the exact amount of infectivity exhibited by L615A varied between cell lines with 44% of wt levels observed in Huh7 compared to 10% of wt levels observed in Hep3B cells; however, the trend of reduced infectivity was consistent.

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