Keratinocyte Differentiation-Dependent Human Papillomavirus Gene Regulation

Abstract

Human papillomaviruses (HPVs) cause diseases ranging from benign warts to invasive cancers. HPVs infect epithelial cells and their replication cycle is tightly linked with the differentiation process of the infected keratinocyte. The normal replication cycle involves an early and a late phase. The early phase encompasses viral entry and initial genome replication, stimulation of cell division and inhibition of apoptosis in the infected cell. Late events in the HPV life cycle include viral genome amplification, virion formation, and release into the environment from the surface of the epithelium. The main proteins required at the late stage of infection for viral genome amplification include E1, E2, E4 and E5. The late proteins L1 and L2 are structural proteins that form the viral capsid. Regulation of these late events involves both cellular and viral proteins. The late viral mRNAs are expressed from a specific late promoter but final late mRNA levels in the infected cell are controlled by splicing, polyadenylation, nuclear export and RNA stability. Viral late protein expression is also controlled at the level of translation. This review will discuss current knowledge of how HPV late gene expression is regulated.

Keywords: RNA processing; epithelial differentiation; gene regulation; human papillomavirus; infection.

Figure 1

Eosin-stained HPV16-infected cervical epithelium (nuclei are stained purple). Key events in the viral replication cycle are noted on the left hand side. The approximate region of expression of viral proteins is shown on the right hand side. It is expected that E1 follows the pattern of E2 while E5 follows the pattern of E4. However, lack of suitable antibodies against these proteins have precluded confirmation of their expression pattern.

Figure 2

Regulatory elements on the HPV16 genome. (A) Details of the cis-acting elements, and the proteins that bind, located in the HPV16 long control region (LCR), otherwise called the upstream regulatory region (URR). The 3′ end of the L1 open reading frame is shown in pink. The 5′ end of the E6 open reading frame is shown in olive. The approximate positions of the proximal promoter and transcriptional enhancer are shown with double headed arrows. The origin of replication (ori) is marked with a blue oval. The late polyadenylation site is indicated with a downward facing arrow and p(A)_L. The late regulatory element at the end of the L1 open reading frame is indicated with a red upward arrow and an orange box. The light blue circle indicates U1 snRNP, light green circle; SRSF1, dark red shape; U2AF, dark blue triangle; CUG-BP1 (see Table 1). The curved double headed black arrow indicates interactions between these factors and the polyadenylation complex (not shown). The viral transcription/replication factor E2 is shown as green ovals. Transcription factors are shown as rectangles. NF1; purple, Oct1; red, AP1; beige, Sp1; gray, YY1; orange. This is not an exhaustive list of transcription factors that bind the LCR. The curved double headed gray arrow indicates interactions between the enhancer and proximal promoter; (B) illustration of the LCR and early region of the HPV16 genome. Early open reading frames are shown as coloured boxes. The location on the genome of characterised promoters (P) is shown with arrows and associated numbers.

Figure 3

Splicing and polyadenylation of HPV16 late RNAs. (A) Diagram of the HPV16 genome. Coloured boxes indicate viral open reading frames. Promoters are indicated with arrows and (P). The positions of the early (pA_E) and late (pA_L) polyadenylation sites and indicated with downward arrows; (B) diagram of the main splicing events for generation of HPV16 late mRNAs. Boxes indicate open reading frames. Dotted lines indicate introns that are spliced out. (*), alternative splice acceptor sites can be used in the E6E7 gene region, including, for HPV16, sites at 409, 526 and 743. The putative coding potential of each mRNA is indicated to the right-hand side. This is a limited list of possible late mRNAs. A fuller list can be viewed at https://pave.niaid.nih.gov/#explore/transcript_maps.

Figure 4

Cartoon of HPV gene regulation in the nucleus and cytoplasm. Transcription of the HPV genome (blue circle) is carried out by RNA polymerase II (pink oval). The carboxyl terminal domain of the polymerase (pink thin rectangle) acts to integrate transcription and RNA processing by binding splicing factors (SF; e.g., SRSFs and hnRNPs, orange ovals) and polyadenylation factors (PF, olive rectangle). These are then able to bind and process the nascent RNA molecule as it emerges from the RNA polymerase exit channel. A. Fully processed mRNAs are able to be exported from the nucleus to the cytoplasm through nuclear pore complexes (NPC, blue tubes) to access the ribosomes (beige ovoids) for translation (E.). Aberrantly formed mRNAs are targeted for decay. B. mRNAs that aberrantly retain introns. C. mRNAs that are not properly polyadenylated, or capped. D. RNAs that bind protein complexes (olive shapes) that inhibit nuclear export. F. RNA decay factors (DF, purple circle), especially those that bind 3′ untranslated region motifs, can control levels of mRNAs in the cytoplasm linked to translation. This is not an exhaustive list of possible control mechanisms.