Protein–DNA Interactions in Human Papillomavirus

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This entry is adapted from the peer-reviewed paper 10.3390/ijms24108493

Human papillomavirus (HPV) is a group of alpha papillomaviruses that cause various illnesses, including cancer. There are more than 160 types of HPV, with many being “high-risk” types that have been clinically linked to cervical and other types of cancer. “Low-risk” types of HPV cause less severe conditions, such as genital warts. Numerous studies have shed light on how HPV induces carcinogenesis. The HPV genome is a circular double-stranded DNA molecule that is approximately 8 kilobases in size. Replication of this genome is strictly regulated and requires two virus-encoded proteins, E1 and E2. E1 is a DNA helicase that is necessary for replisome assembly and replication of the HPV genome. On the other hand, E2 is responsible for initiating DNA replication and regulating the transcription of HPV-encoded genes, most importantly the E6 and E7 oncogenes.

HPV cervical cancer oropharyngeal cancer E2 protein

1. Introduction

Human papillomavirus (HPV) is one of the most common sexually transmitted infections, affecting millions worldwide annually. Human papillomaviruses belong to the papillomaviridae family of small, non-enveloped, double-stranded DNA viruses ^[1], which includes a large number of species-specific genotypes that predominantly infect the cutaneous and mucosal epithelium in various organisms ^[2]^[3]. The host’s immune system quickly clears most HPV infections within a couple of years. However, some HPV types cause persistent infections and become the causative agents of various genital and oropharyngeal cancers ^[4]. Currently, ~160 HPV strains, commonly called “types”, have been identified. These types are often classified into low- and high-risk types based on their propensity to induce cancer. Three vaccines against HPV are available, but no more than nine HPV types are covered by the latest vaccine: 6, 11, 16, 18, 31, 33, 45, 52, and 58. Of note, there are currently no treatments available for HPV infection. HPV accounts for approximately half a million deaths per year around the world. It is a silent viral epidemic.

2. The HPV Genome

The HPVs have double-stranded circular DNA genomes (~8 kb) containing eight open reading frames (ORFs) that encode viral proteins ^[5]. There is a noncoding region in the HPV genome called the long control region (LCR), which contains the origin of DNA replication and transcriptional regulatory elements (Figure 1A) ^[5]. The genome is divided into three main sections: the early region, containing the early genes; the late region, containing the late genes; and the long control region (LCR) ^[6]. The first six genes encode the early viral proteins and are expressed at the beginning of the viral life cycle. Of these viral proteins, E1 and E2 are the major replication proteins, E4 and E5 aid in genome amplification, and the E6 and E7 proteins are the oncoproteins (Figure 1). The late genes, L1 and L2, encode the L1 major and L2 minor capsid proteins. The L1 and L2 proteins together form the capsid. They are expressed in the later stages of the viral life cycle. The LCR is the only noncoding region of the genome. It contains the early viral promoters, enhancers, and the origin of viral DNA replication ^[7]^[8].

Figure 1. (A). A genetic map of the HPV genome. The HPV genome is ~8 kb in length and encodes eight major genes separated by their expression order during the life cycle. (B). List depicting the major functions of each of the HPV proteins.

The E2 protein can be considered the most pivotal protein in the HPV genome due to its role in the viral life cycle and oncogenesis. The HPV viral life cycle is tightly controlled by the E2 protein, which is also involved in transcriptional regulation, the expression of the E6 and E7 oncogenes, partitioning, and the maintenance of the viral genome, in addition to its role in the initiation of DNA replication ^[4].

3. Evolution and Diversity of HPV Types

Papillomaviruses are ancient DNA viruses spanning ~400 million years and have been identified in humans, nonhuman primates, bovines, and other animals such as dolphins ^[9]. HPVs have significantly diverged and evolved genetically and phenotypically over time. In the early 1900s, papillomaviruses were discovered in humans (HPV) and subsequently associated with cervical cancer by zur Hausen in 1975 ^[10].

There are five major known HPV genera: α (alpha), β (beta), γ (gamma), μ (mu), and υ (nu) ^[11]. Carcinogenic HPV types belong to the alpha or beta genus and infect the mucosal epithelium ^[12]^[13]. Recent studies have also shown the association of gamma HPV types with various oropharyngeal cancers ^[14]^[15].

The IARC working groups have classified many of the HPV types into three categories, low-risk, high-risk, and probable high-risk (Figure 2), leaving many HPV types unclassified. Low-risk HPV types commonly refer to the HPV types that cause anogenital warts and benign lesions. These types are generally non-lethal. High-risk HPV types commonly refer to the HPV types that have greater oncogenic potential and cause carcinomas ^[16]^[17]^[18]. Overall, 90% of cervical cancers are due to high-risk HPV infection. HPV16 and 18 are the most common high-risk HPV types and account for 70% of all cervical cancer cases ^[17].

Figure 2. Sequence variations of E2 binding sites correlate with cancer risks. E2 binding sites and detection of SNVs in the consensus sequence associated with clinically well-characterized HPV types. Variant nucleotides in the consensus sequence are indicated in red.

Yilmaz et al. discovered nucleotide variations in the E2 binding site consensus sequences (ACCGNNNNCGGT) of at least one of the three binding sites in all established high-risk HPV types (Figure 2) ^[19]. All HPV types classified as low-risk on the Papillomavirus database (PaVe) have intact consensus sequences. However, all high-risk or probable high-risk HPV types had at least one nucleotide variation in their consensus sequence (Figure 2). Using in vitro DNA binding assays with oligonucleotides containing normal and variant consensus sequences, this study established the attenuation of E2–DNA complex formation upon consensus sequence variations. Thus, the nucleotide variations affected HPV E2 binding to its binding sites, affecting the biological functions of E2 in HPV. This model can potentially be used to predict the oncogenic potential of all HPV types based on the DNA sequence of HPV.

As the Yilmaz classification of HPV types is based on the DNA sequence, any HPV type can be classified based on the consensus sequence mutation(s), without requiring clinical data on the potential to cause cancer, as shown in Figure 2 ^[19]. For example, HPV97, 102, and 114 are currently unclassified in terms of oncogenicity, but these HPV types contain variant E2 binding sites, making them high-risk.

4. Regulation of DNA Replication, Transcription, and Oncogenesis by E2 Protein

The HPV E2 protein is a DNA-binding protein with two conserved functional domains. The N-terminal has the transactivation domain, and the C-terminal has the DNA-binding domain. The two domains are connected by a flexible linker known as the “hinge region” (Figure 3A) ^[19]^[20].

Figure 3. (A). Schematic structure of the HPV16 E2 protein and its respective domains: transactivation, hinge, and DNA binding. (B). Ribbon representation of the HPV16 E2 transactivation domain crystal structure (PDB: 1DTO). (C). Ribbon representation of the crystal structure of HPV18 E2 DNA-binding domain as a dimer bound to E2 binding site 4 with a helix from each monomer interfaced with ACCG/CGGT motif (PDB: 1JJ4). (D). Homology model of the full-length HPV11 E2 protein monomer using Robetta Structure Prediction software from the University of Washington (25 April 2023).

The transactivation domain is the largest, with approximately 200 amino acids, and is required for its replication, transactivation, and segregation functions (Figure 3B). The hinge region is serine–arginine-rich and serves as a linker between the two domains. Finally, the DNA-binding domain interacts with sequence-specific binding sites within the long control region (LCR) and binds as a dimer (Figure 3C) ^[21]^[22]. To date, the crystal structure of the full-length HPV E2 protein has yet to be determined.

Various papillomaviruses have shorter, truncated E2 proteins, known as isoforms. In the bovine papillomavirus, truncated E2 isoforms were observed and determined to be repressors of transcription and replication ^[23]. In HPV, several types, such as HPV16, have isoforms predicted to have been generated through alternative splicing: E1^E2 and E8^E2. These E2 isoforms have a domain whose parts are either missing or misplaced. These alterations of E2 may result in similar protein functions but at a reduced capacity. For example, the E2/E8^E2 dimer has a truncated transactivation domain. While this might indicate regular E2 DNA binding, the recruitment of E1 might not occur ^[6]. Other studies have shown that, as in BPV, HPV16 E8^E2 can repress early transcription and replication ^[24]. All isoforms observed to date still maintain their replicational or transcriptional functions but not the partitioning of the viral genome. This could indicate that the partitioning function always requires a full-length E2 protein.

References

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