Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

Human papillomavirus (HPV) infections pose serious medical problem. To date, there are no HPV-specific antivirals available due to poor understanding of the molecular mechanisms of virus infection cycle. The infection cycle of HPV involves initial amplification of the viral genomes and maintenance of the viral genomes with a constant copy number, followed by another round of viral genome amplification and new viral particle formation. The viral protein E1 is critical for the initial amplification of the viral genome. However, E1 involvement in other phases of the viral life cycle has remained controversial. In the present study, we show that at least two different replication modes of the HPV18 genome are undertaken simultaneously during the maintenance and vegetative amplification phases, i.e., replication of the majority of the HPV18 genome proceeds under the control of the host cell replication machinery without E1 function, whereas a minority of the genome replicates in an E1-dependent manner. ABSTRACT The life cycle of human papillomaviruses (HPVs) comprises three distinct phases of DNA replication: initial amplification, maintenance of the genome copy number at a constant level, and vegetative amplification. The viral helicase E1 is one of the factors required for the initiation of HPV genome replication. However, the functions of the E1 protein during other phases of the viral life cycle are largely uncharacterized. Here, we studied the role of the HPV18 E1 helicase in three phases of viral genome replication by downregulating E1 expression using RNA interference or inducing degradation of the E1 protein via inhibition of casein kinase 2α expression or catalytic activity. We generated a novel modified HPV18 genome expressing Nanoluc and tagged E1 and E2 proteins and created several stable HPV18-positive cell lines. We showed that, in contrast to initial amplification of the HPV18 genome, other phases of viral genome replication involve also an E1-independent mechanism. We characterize two distinct populations of HPV18 replicons existing during the maintenance and vegetative amplification phases. We show that a subset of these replicons, including viral genome monomers, replicate in an E1-dependent manner, while some oligomeric forms of the HPV18 genome replicate independently of E1 function. IMPORTANCE Human papillomavirus (HPV) infections pose serious medical problem. To date, there are no HPV-specific antivirals available due to poor understanding of the molecular mechanisms of virus infection cycle. The infection cycle of HPV involves initial amplification of the viral genomes and maintenance of the viral genomes with a constant copy number, followed by another round of viral genome amplification and new viral particle formation. The viral protein E1 is critical for the initial amplification of the viral genome. However, E1 involvement in other phases of the viral life cycle has remained controversial. In the present study, we show that at least two different replication modes of the HPV18 genome are undertaken simultaneously during the maintenance and vegetative amplification phases, i.e., replication of the majority of the HPV18 genome proceeds under the control of the host cell replication machinery without E1 function, whereas a minority of the genome replicates in an E1-dependent manner.