E6^E7, a Novel Splice Isoform Protein of Human Papillomavirus 16, Stabilizes Viral E6 and E7 Oncoproteins via HSP90 and GRP78

Abstract

Transcripts of human papillomavirus 16 (HPV16) E6 and E7 oncogenes undergo alternative RNA splicing to produce multiple splice isoforms. However, the importance of these splice isoforms is poorly understood. Here we report a critical role of E6^E7, a novel isoform containing the 41 N-terminal amino acid (aa) residues of E6 and the 38 C-terminal aa residues of E7, in the regulation of E6 and E7 stability. Through mass spectrometric analysis, we identified that HSP90 and GRP78, which are frequently upregulated in cervical cancer tissues, are two E6^E7-interacting proteins responsible for the stability and function of E6^E7, E6, and E7. Although GRP78 and HSP90 do not bind each other, GRP78, but not HSP90, interacts with E6 and E7. E6^E7 protein, in addition to self-binding, interacts with E6 and E7 in the presence of GRP78 and HSP90, leading to the stabilization of E6 and E7 by prolonging the half-life of each protein. Knocking down E6^E7 expression in HPV16-positive CaSki cells by a splice junction-specific small interfering RNA (siRNA) destabilizes E6 and E7 and prevents cell growth. The same is true for the cells with a GRP78 knockdown or in the presence of an HSP90 inhibitor. Moreover, mapping and alignment analyses for splicing elements in 36 alpha-HPVs (α-HPVs) suggest the possible expression of E6^E7 mostly by other oncogenic or possibly oncogenic α-HPVs (HPV18, -30, -31, -39, -42, -45, -56, -59, -70, and -73). HPV18 E6^E7 is detectable in HPV18-positive HeLa cells and HPV18-infected raft tissues. All together, our data indicate that viral E6^E7 and cellular GRP78 or HSP90 might be novel targets for cervical cancer therapy.

Transcriptome analysis of Mastomys natalensis papillomavirus in productive lesions after natural infection

“…Interestingly, though, we found a truncated E7 variant. The only previous E7 isoform described to our knowledge is an E6^E7 fusion which conserves the C-terminus of E7 (Ajiro & Zheng, 2015). On the contrary, the E7DC potentially encoded in transcripts J and K is a truncation variant which lacks the zincbinding terminal domain and could therefore render it biologically inactive.…”

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

“…Productive HPV18 infection in keratinocyte-derived raft tissues leads to the generation of more than 15 isoforms of viral early and late RNAs through alternative RNA splicing by selection of alternative splice sites ( Fig. 1) (24,26,27). With other, weak splice sites recently identified in HPV18-transfected U2OS cells (25), the updated HPV18 transcription map ( Fig.…”

E6 viral protein ratio correlates with outcomes in human papillomavirus related oropharyngeal cancer

“…Except for type 16, all other high risk HPVs have only a single splice acceptor. In addition to the full length E6 mRNA, the type 16 HPV E6 gene encodes E6*I and E6*II transcripts by alternative splicing 21 In cervical cancer cell lines, splicing may help with the translation of the E7 oncoprotein. 22 Our previous in vitro study of OSCC cell lines showed that the different isoforms of E6 affect the radiosensitivity by different degrees, with E6*I providing greatest radiosensitisation and E6*II having no effect.…”