Translation of the human papillomavirus type 16 E7 oncoprotein from bicistronic mRNA is independent of splicing events within the E6 open reading frame

Abstract

In this study we investigated the translational capacities of bicistronic and spliced mRNAs originating from the E6 and E7 regions of the high-risk genital human papillomavirus type 16 (HPV-16) and the low-risk HPV-11. For HPV-16 it was found, unexpectedly, that E7 protein could be translated from full-length bicistronic E6-E7 mRNAs. E6*I and E6*II splicing events were not required for E7 synthesis, nor did splicing increase the efficiency of E7 translation significantly. In cells, E7 synthesis from all known naturally occurring mRNA structures was very inefficient compared with that from synthetic monocistronic controls, suggesting that HPV-16 employs translational mechanisms to restrict E7 protein levels. For HPV-11, only RNAs initiated at the P 264 promoter, located within the E6 open reading frame, were capable of providing an efficient template for E7 synthesis. P 264-initiated mRNAs were as efficient in vivo as monocistronic controls, suggesting that the low-risk HPV-11 does not limit E7 synthesis by translational mechanisms. A detailed analysis of HPV-16 templates by using site-directed mutagenesis showed that the majority of ribosomes which ultimately translate E7 have not reinitiated after translating some or all of the upstream open reading frames. The data support a model in which the failure of 40S ribosomal initiation complexes to recognize the E6 AUG renders them capable of proceeding efficiently to translate E7.

Extended effects of human papillomavirus 16 E6-specific short hairpin RNA on cervical carcinoma cells

“…In our study, the E6A shRNA is the most effective one among the four E6 shRNAs, this is likely that E6A shRNA targets upstream of the splicing donor site of E6 mRNA and can degrade both the full‐length E6 and the truncated E6*, whereas the other three E6 shRNAs target the splicing donor site and can only disrupt the full‐length E6 and leave the truncated E6* intact. Both the full‐length E6–E7 bicistronic mRNA and the spliced structures E6* are capable of acting as templates for E7 translation (31) . Therefore, degradation of the E6 and E6* by E6A shRNA must cause the disruption of E7 at the same time.…”

Paclitaxel combined with siRNA targeting HPV16 oncogenes improves cytotoxicity for cervical carcinoma

“…There are conflicting reports as to whether the HPV16 E7 protein is translated more efficiently from the spliced E6* I transcript 29 or with equal efficiency from spliced and full-length transcripts. 30 Short interfering RNA (siRNA) technology can generate specific silencing of target mRNA in mammalian cells. 31 To silence HPV E6/E7 oncogenes, two strategies have been adopted.…”

A promoter within the E6 ORF of human papillomavirus type 16 contributes to the expression of the E7 oncoprotein from a monocistronic mRNA

“…Splicing within the E6 ORF was previously believed to facilitate efficient translation of E7 (Smotkin et al, 1989). However, Stacey et al (1995Stacey et al ( , 2000 showed that the three mRNAs E6*IE7, E6*IIE7 and the unspliced E6E7 produced equal amounts of E7, whereas a synthetic monocistronic construct initiating at nt 553 was 10-fold more efficient. In our study, we have found that the monocistronic mRNA initiated from P542 leads to the highest level of E7 protein, whereas both the spliced and unspliced polycistronic mRNAs lead to less E7 protein.…”

“…The presence of an upstream ORF generally inhibits expression from a second downstream ORF. The expression of E7 has been shown to be substantially lower from the polycistronic mRNA than from an E7 monocistronic control construct (Tan et al, 1994a;Stacey et al, 1995). This can be explained by a leaky scanning mechanism, where ribosomes initiate scanning at the 59 end, but somehow bypass the E6 start codon and continue down to the start codon for E7 (Stacey et al, 2000).…”