Construction of a Full Transcription Map of Human Papillomavirus Type 18 during Productive Viral Infection

Abstract

Human papillomavirus type 18 (HPV18) is the second most common oncogenic HPV genotype, responsible for ϳ15% of cervical cancers worldwide. In this study, we constructed a full HPV18 transcription map using HPV18-infected raft tissues derived from primary human vaginal or foreskin keratinocytes. By using 5 rapid amplification of cDNA ends (RACE), we mapped two HPV18 transcription start sites (TSS) for early transcripts at nucleotide (nt) 55 and nt 102 and the HPV18 late TSS frequently at nt 811, 765, or 829 within the E7 open reading frame (ORF) of the virus genome. HPV18 polyadenylation cleavage sites for early and late transcripts were mapped to nt 4270 and mainly to nt 7299 or 7307, respectively, by using 3 RACE. Although all early transcripts were cleaved exclusively at a single cleavage site, HPV18 late transcripts displayed the heterogeneity of 3 ends, with multiple minor cleavage sites for late RNA polyadenylation. HPV18 splice sites/splice junctions for both early and late transcripts were identified by 5 RACE and primer walking techniques. Five 5 splice sites (donor sites) and six 3 splice sites (acceptor sites) that are highly conserved in other papillomaviruses were identified in the HPV18 genome. HPV18 L1 mRNA translates a L1 protein of 507 amino acids (aa), smaller than the 568 aa residues previously predicted. Collectively, a full HPV18 transcription map constructed from this report will lead us to further understand HPV18 gene expression and virus oncogenesis.Cervical cancer is a leading cause of death for women in the developing world, with about 493,000 new cases and nearly 273,000 deaths each year (www.who.int/hpvcenter/en/). Oncogenic human papillomavirus (HPV) infection is widely recognized as a principal cause of cervical, penile, and anal cancers (62). Among over 120 genotypes with human origin (3), infection with HPV16 and HPV18, the two most common oncogenic HPV types, leads to the development of ϳ70% of all cervical and other anogenital cancers (55, 62). HPV18 alone accounts for more than 15% of all cervical cancer cases worldwide (14). Persistent HPV16 infection is responsible for development of both cervical squamous cell carcinoma and adenocarcinoma. In contrast, persistent HPV18 infection is a preferential risk factor for the development of cervical adenocarcinoma (7, 10).HPVs are a group of small DNA viruses which have a high degree of conservation with respect to genome structure and organization as well as gene expression (44, 91). The circular, double-stranded viral genome is approximately 8 kb in size and contains eight open reading frames (ORFs) which are all transcribed from the same strand. In general, the viral genome can be divided into three major regions: an early region, a late region, and a long control region (LCR) or a noncoding region (NCR). The early region is positioned in the 5Ј half of the virus genome and encodes six common ORFs (E1, E2, E4, E5, E6, and E7) with regulatory functions in viral replication, gene expression, and pathogenesis. The late region lies dow...

HPV-18 E6 mutants reveal p53 modulation of viral DNA amplification in organotypic cultures

“…5 H and I and Table 1, species g) had two splices from nucleotide 929 to nucleotide 3434 and then from nucleotide 3696 to nucleotide 5613 at the L1 initiation codon. It is identical to a previous report (30) and predicted to encode both E1^E4 and L1. Band 2 ( Fig.…”

“…The relatively low abundance of this RNA is in accordance with L2 being a minor capsid protein. In this regard, we hypothesize that the more abundant singly spliced transcript (nucleotides 929^3434), which spans E1^E4, E5, and the entire L region (29,30), is not a message for L2, because L2 would be the third ORF, an unfavorable position for protein translation. We suggest that this long transcript is the splicing intermediate for the twice-spliced (nucleotides 929^3434 to 3696^5613) mature bicistronic mRNA encoding E1E4 and L1 (Table 1, species g).…”

“…Using E1F1/E4R or E1F1/ E5R primer set, a major band and a minor band (Fig. 5 B and C, bands 1 and 2) were detected; they correspond to the E1^E4, E5 transcript with a splice at nucleotides 929^3434 (Table 1, species a) and the E5 transcript with the splice at nucleotides 929^3465 (Table 1, species b), respectively (29,30). A product with the splice nucleotides 929^3506 was not observed.…”

“…5 H and I and Table 1, species H) had two splices (nucleotides 929^3465 to 3696^5613), connecting the short E1 ORF to a closed frame within the E2/E4 overlap region; it could encode L1 only (30). An early E5 RNA with the same or comparable first splice only has been reported for HPV-18 and -11 (30,31). A twice-spliced band 3 had a first splice (nucleotides 929^3506) not previously reported ( Fig.…”

Human Papillomavirus Type 58 Genome Variations and RNA Expression in Cervical Lesions

“…7, HPV58 early transcripts are most likely to start at nt 100 or 102 from a viral early promoter containing a TATA box at nt 72 in the virus genome and are polyadenylated from a nt 4230A cleavage site using an early poly(A) signal at nt 4209. Similar to HPV16 and HPV18 early transcripts (27,28), HPV58 early transcripts also consist of three exons and two introns, which can be alternatively spliced. Splicing from nt 232 to 510 in the E6 ORF leads to an E6*I ORF, which is truncated compared to the E6 ORF.…”