The E1∧E4 Protein of Human Papillomavirus Type 16 Associates with a Putative RNA Helicase through Sequences in Its C Terminus

Abstract

Human papillomavirus type 16 (HPV16) infects cervical epithelium and is associated with the majority of cervical cancers. The E1 ∧ E4 protein of HPV16 but not those of HPV1 or HPV6 was found to associate with a novel member of the DEAD box protein family of RNA helicases through sequences in its C terminus. This protein, termed E4-DBP (E4-DEAD box protein), has a molecular weight of 66,000 (66K) and can shuttle between the nucleus and the cytoplasm. It binds to RNA in vitro, including the major HPV16 late transcript (E1 ∧ E4.L1), and has an RNA-independent ATPase activity which can be partially inhibited by E1 ∧ E4. E4-DBP was detectable in the cytoplasm of cells expressing HPV16 E1 ∧ E4 (in vivo and in vitro) and could be immunoprecipitated as an E1 ∧ E4 complex from cervical epithelial cell lines. In cell lines lacking cytoplasmic intermediate filaments, loss of the leucine cluster-cytoplasmic anchor region of HPV16 E1 ∧ E4 resulted in both proteins colocalizing exclusively to the nucleoli. Two additional HPV16 E1 ∧ E4-binding proteins, of 80K and 50K, were identified in pull-down experiments but were not recognized by antibodies to E4-DBP or the conserved DEAD box motif. Sequence analysis of E4-DBP revealed homology in its E4-binding region with three Escherichia coli DEAD box proteins involved in the regulation of mRNA stability and degradation (RhlB, SrmB, and DeaD) and with the Rrp3 protein of Saccharomyces cerevisiae, which is involved in ribosome biogenesis. The synthesis of HPV16 coat proteins occurs after E1 ∧ E4 expression and genome amplification and is regulated at the level of mRNA stability and translation. Identification of E4-DBP as an HPV16 E1 ∧ E4-associated protein indicates a possible role for E1 ∧ E4 in virus synthesis.

E1^E4-mediated keratin phosphorylation and ubiquitylation: a mechanism for keratin depletion in HPV16-infected epithelium

“…16E1^E4 was expressed either by transfection using the pMV11.16E1^E4 plasmid (Davy et al, 2002) or infection using the rAd16E1^E4 plasmid (Doorbar et al, 2000). LLKLL was expressed by infection using the rAd16E1^E4LLKLL plasmid (Wang et al, 2004).…”

“…Nuclei were stained using DAPI (4-6-diamidino-2-phenylindole) (Sigma). For immunoblotting, 16E1^E4 was detected using TVG402 (Doorbar et al, 2000) and GAPDH using MAB374 (Chemicon). The ubiquitin antibodies Sc-8017 (Santa Cruz) and Z0458 (DAKO) were used for immunoblotting and immunofluorescence microscopy, respectively.…”

E1 ∧ E4 Protein of Human Papillomavirus Type 16 Associates with Mitochondria

“…They are made in terminally differentiated squamae that are keratin-rich, and it has been suggested that E4 may contribute to virus release by perturbing the cytokeratin network and increasing cell fragility. In addition to interacting with cytokeratins, E4 also binds to a DEAD-box protein (12). The function of this association is not yet clear, largely because the function of this DEAD-box protein is poorly defined.…”

Mecanismo De Infección Y Transformación Neoplásica Producido Por Virus Papiloma Humano en El Epitelio Cervical

“…Se postula que durante esta etapa se aseguraría que el ADN viral se distribuya difusamente por las células basales proliferantes y que al mantener un número reducido de copias se impediría la activación de la respuesta inmune (17). Las células basales proliferantes migran a los estratos parabasal y espinoso, amplificándose la expresión de genes virales tempranos a través de la región no codificante (URR), los cuales permiten producir ADN a cientos de copias por célula (18); ésta etapa en el ciclo viral es conocida como la fase vegetante, proliferante o productiva (19). En este proceso de replicación del ADN viral participan un grupo de proteínas, cuyas características principales se mencionan a continuación.…”