Mutations in HPV18 E1^E4 Impact Virus Capsid Assembly, Infectivity Competence, and Maturation

Abstract

The most highly expressed protein during the productive phase of the human papillomavirus (HPV) life cycle is E1^E4. Its full role during infection remains to be established. HPV E1^E4 is expressed during both the early and late stages of the virus life cycle and contributes to viral genome amplification. In an attempt to further outline the functions of E1^E4, and determine whether it plays a role in viral capsid assembly and viral infectivity, we examined wild-type E1^E4 as well as four E1^E4 truncation mutants. Our study revealed that HPV18 genomes containing the shortest truncated form of E1^E4, the 17/18 mutant, produced viral titers that were similar to wild-type virus and significantly higher compared to virions containing the three longer E1^E4 mutants. Additionally, the infectivity of virus containing the shortest E1^E4 mutation was equivalent to wild-type and significantly higher than the other three mutants. In contrast, infectivity was completely abrogated for virus containing the longer E1^E4 mutants, regardless of virion maturity. Taken together, our results indicate for the first time that HPV18 E1^E4 impacts capsid assembly and viral infectivity as well as virus maturation.

Keywords: E1^E4; HPV18; Human Papillomavirus (HPV); infection; viral titer; virion maturation.

Figure 1

Mutations in the human papillomavirus type-18 (HPV18) E1^E4 protein sequence. Outlined in red are the sites where translation termination stop codons were inserted at amino acids 17/18, 34, 54, and 63/67. The codon modifications are indicated below the wild-type (WT) sequence.

Figure 2

Mutations in the HPV18 E1^E4 protein do not affect organotypic raft growth. HPV18 WT and HPV18 mutant E1^E4 organotypic raft cultures grown for 10 (top row) and 20 (bottom row) days. Tissue sections were cut and stained with hematoxylin and eosin (H&E). Images are 10× magnification. Tissue sections are representative of rafts grown from at least two different cell lines produced for each virus type.

Figure 3

HPV18 virus titer, but not genomic replication or L1 synthesis is affected by mutations in E1^E4. Organotypic rafts harvested at either day 10 or day 20 of tissue growth were homogenized and both (A) total viral genomes and (B) viral titer were quantified. The total number of viral genomes were quantitated via a qPCR assay amplifying the E2 open reading frame and were measured against a standard curve of known concentrations of HPV genomes. To measure viral titer, homogenized samples were treated with benzonase to remove any non-protected HPV genomes. The quantity of protected viral genomes was then analyzed via qPCR against a standard curve of known concentrations of HPV genomes; (C) Western blot analysis of HPV18 L1 (equal loading as determined by Bradford Assay) from 10-day and 20-day WT and HPV18 E1^E4 mutant rafts. The blot was probed with the HPV18 L1 specific H18.7E antibody. The arrow points to HPV18 L1; (D) Immunofluorescent (IF) staining of 10—(bottom section) and 20—(top section) day raft tissue with HPV18 L1 (top rows, red) and E1^E4 protein (bottom rows, green). IF images are at 10× magnification. (A,B) Are averages from at least three individual experiments utilizing at least 2 different sets of rafts/virus preparations. Bars represent standard deviation. An asterisk (*) denotes significance by students t-test. Statistical significance was defined as p ≤ 0.05. (C) is representative of at least two different western blots (WBs) with samples from at least 2 different rafts/virus preparations. IF staining is representative of at least two different raft tissues from two different HPV18 WT and mutant cell lines.

Figure 3

HPV18 virus titer, but not genomic replication or L1 synthesis is affected by mutations in E1^E4. Organotypic rafts harvested at either day 10 or day 20 of tissue growth were homogenized and both (A) total viral genomes and (B) viral titer were quantified. The total number of viral genomes were quantitated via a qPCR assay amplifying the E2 open reading frame and were measured against a standard curve of known concentrations of HPV genomes. To measure viral titer, homogenized samples were treated with benzonase to remove any non-protected HPV genomes. The quantity of protected viral genomes was then analyzed via qPCR against a standard curve of known concentrations of HPV genomes; (C) Western blot analysis of HPV18 L1 (equal loading as determined by Bradford Assay) from 10-day and 20-day WT and HPV18 E1^E4 mutant rafts. The blot was probed with the HPV18 L1 specific H18.7E antibody. The arrow points to HPV18 L1; (D) Immunofluorescent (IF) staining of 10—(bottom section) and 20—(top section) day raft tissue with HPV18 L1 (top rows, red) and E1^E4 protein (bottom rows, green). IF images are at 10× magnification. (A,B) Are averages from at least three individual experiments utilizing at least 2 different sets of rafts/virus preparations. Bars represent standard deviation. An asterisk (*) denotes significance by students t-test. Statistical significance was defined as p ≤ 0.05. (C) is representative of at least two different western blots (WBs) with samples from at least 2 different rafts/virus preparations. IF staining is representative of at least two different raft tissues from two different HPV18 WT and mutant cell lines.

Figure 4

Mutations in HPV18 E1^E4 affect viral infectivity. HaCaT cells were infected with WT or mutant HPV18 virus, harvested at either day 10 or day 20 of tissue growth, at a multiplicity of infection (MOI) of 10. Forty-eight hours post infection (h.p.i.) mRNA was harvested and levels of the E1^E4 splice transcript were determined by RT-qPCR. (A) To measure the relative difference in infectivity between 10-day WT HPV18 and the 10-day mutant HPV18 viruses, the level of infection of 10-day WT HPV18 is set equal to one; (B) To measure the relative difference in infectivity between 20-day WT HPV18 and the 20-day mutant HPV18 viruses, the level of infection of 20-day WT HPV18 is set equal to one; (C) To compare the relative difference between the infection of 10-day virus and 20-day virus for WT HPV18 and each of the HPV18 mutants, each 10-day infection level was set equal to one. (A–C) Are representative of at least three individual infections utilizing at least two different virus preparations. Bars represent standard deviation. An asterisk (*) denotes significance by students t-test. Statistical significance was defined at p ≤ 0.05.

Figure 5

Mutations in HPV18 E1^E4 affects viral stability. HaCaT cells were infected with virus from tissue grown for (A) 10 days or (B) 20 days. Virus was incubated either alone or in the presence of the HPV18 L1 specific antibody, H18.J4. Forty-eight h.p.i., mRNA was harvested and RT-qPCR was utilized to quantify the E1^E4 splice transcript as a measure of infectivity. Stability of 10-day and 20-day virus for (A) WT and (B) the HPV18 17/18 E1^E4 mutant was determined by ultracentrifugation. Equal volumes of fractions were used to measure total genome copies per fraction via a qPCR assay amplifying the E2 open reading frame against a standard curve of known HPV18 genome concentrations. (A,B) Are representative of three individual experiments utilizing at least 2 different virus preparations. The bars represent standard deviation. An asterisk (*) denotes significance by students t-test. Statistical significance was defined as p ≤ 0.05. (C,D) Represent fractionation profiles of an individual experiment, with all repeats having a similar trend.