The E1 Protein of Human Papillomavirus Type 16 Is Dispensable for Maintenance Replication of the Viral Genome

Abstract

dPapillomavirus genomes are thought to be amplified to about 100 copies per cell soon after infection, maintained constant at this level in basal cells, and amplified for viral production upon keratinocyte differentiation. To determine the requirement for E1 in viral DNA replication at different stages, an E1-defective mutant of the human papillomavirus 16 (HPV16) genome featuring a translation termination mutation in the E1 gene was used. The ability of the mutant HPV16 genome to replicate as nuclear episomes was monitored with or without exogenous expression of E1. Unlike the wild-type genome, the E1-defective HPV16 genome became established in human keratinocytes only as episomes in the presence of exogenous E1 expression. Once established, it could replicate with the same efficiency as the wild-type genome, even after the exogenous E1 was removed. However, upon calcium-induced keratinocyte differentiation, once again amplification was dependent on exogenous E1. These results demonstrate that the E1 protein is dispensable for maintenance replication but not for initial and productive replication of HPV16. P apillomaviruses (PVs) are small, double-stranded DNA viruses that infect stratified squamous epithelium. Human PVs (HPVs) are very important causative agents for various lesions, ranging from verrucas to cancer. Among them, a subset of HPVs, the so-called high-risk types such as type 16 and 18, are associated with more than 90% of all cervical carcinomas as primary etiological factors (45). PVs establish long-term persistent infections in squamous epithelium, and the viral life cycle is tightly linked with the differentiation state of the host keratinocytes (7).PV genome is replicated and amplified in three different stages: establishment, maintenance, and productive stages of the life cycle. In the establishment stage, soon after infection of the basal layer keratinocytes, a single or a few initial copies of the viral genome amplify and establish residence as multicopy circular extrachromosomal elements (episomes) in the nucleus. In the maintenance stage, the viral genomes in each affected cell replicate approximately once in a cell cycle in proliferating basal layer keratinocytes. Then, in the productive stage, they are exponentially amplified in terminally differentiating keratinocytes and packaged into progeny virions. It is important to understand the molecular mechanisms underlying this triphasic model for development of new therapies against HPV-infected lesions, such as cervical intraepithelial neoplasias, which can progress to cervical cancer.The regulation of viral DNA replication is thought to differ in these three distinct stages of the viral life cycle. Studies of lesions experimentally induced by rabbit oral papillomavirus (ROPV) infection showed that the genome copy number of ROPV is low in the basal layer and increases up to four orders of magnitude during the terminal differentiation of host keratinocytes (21). This corresponds to more than 13 rounds of continuous replication of the viral ...

Human Papillomavirus Episome Stability Is Reduced by Aphidicolin and Controlled by DNA Damage Response Pathways

“…In light of the present results, it is conceivable that aphidicolin uncouples pol␣ and E1, resulting in a stalled, unstable replication fork. However, multiple examples of E1-independent maintenance and replication of HPV episomes have been reported (44)(45)(46)(47), making it possible only to speculate on a role for E1 in the episome loss reported here. For this reason, we are interested in examining the status of the viral genome under conditions of instability.…”

Activation of NF-κB by Human Papillomavirus 16 E1 Limits E1-Dependent Viral Replication through Degradation of E1

“…Papillomaviruses undergo three phases of genome replication, the initial amplification, maintenance replication, and productive amplification (38). While the underlying mechanisms for the differentiation-dependent productive amplification are becoming gradually clear, virtually nothing is known about the molecular mechanisms for transition from the initial amplification to maintenance replication within the basal compartment.…”

“…pxCANCre was obtained from the Riken DNA bank (RDB10748; Riken Bioresource center, Tsukuba, Japan). Lentivirus vec-tors, CSII-CMV-tetON-ADV and CSII-TRE-Tight-HA16E1, were described previously (38). The codon-optimized HPV16 E2 cDNA (GenScript, Piscataway, NJ) was used to construct CSII-TRE-Tight-HA16E2, in which the N-terminal hemagglutinin (HA)-tagged HPV16 E2 was inserted under a tetracycline-responsive promoter.…”

“…Total genomic DNA for Southern blotting was isolated after several passages and at least one freezing and thawing from transfection as described previously (38). For quantitative realtime PCR (qPCR), total genomic DNA was isolated using a Wizard SV genomic purification system according to the manufacturer's protocol (catalog no.…”

“…When necessary, an insoluble fraction was lysed in an SDS sample buffer (50 mM Tris-HCl, 2% SDS, and 10% glycerol). The equivalent protein amounts of cell lysates were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blot analysis was conducted as described previously (38). The primary antibodies used in this study were as follows: mouse monoclonal antibodies (MAbs) HA (16B12; Covance, Princeton, NJ), FLAG (M2; Sigma-Aldrich, St. Louis, MO, USA), vinculin (hVIN-1; Sigma-Aldrich), pATM(S1981) Indirect immunofluorescence analysis.…”