A conformational switch balances viral RNA accessibility and protection in a nucleocapsid ring model.

Virus from the Mononegavirales order share common features ranging from virion structure arrangement to mechanisms of replication and transcription. One of them is the way the nucleoprotein (N) wraps and protects the RNA genome from degradation by forming a highly ordered helical nucleocapsid. However, crystal structures from numerous Mononegavirales reveal that binding to the nucleoprotein results in occluded nucleotides that hinder base pairing necessary for transcription and replication. This hints at the existence of alternative conformations of the N protein that would impact on the protein-RNA interface, allowing for transient exposure of the nucleotides without complete RNA release. Moreover, the regulation between the alternative conformations should be finely tuned. Recombinant expression of N from the respiratory syncytial virus form regular N/RNA common among all Mononegavirales, and these constitute an ideal minimal unit for investigating the mechanisms through which these structures protect RNA so efficiently while allowing for partial accessibility during transcription and replication. Neither pH nor high ionic strength could dissociate the RNA but led to irreversible aggregation of the nucleoprotein. Low concentrations of guanidine chloride dissociated the RNA moiety but leading to irreversible aggregation of the protein moiety. On the other hand, high concentrations of urea and long incubation periods were required to remove bound RNA. Both denaturants eventually led to unfolding but converged in the formation of an RNA-free ß-enriched intermediate species that remained decameric even at high denaturant concentrations. Although the N-RNA rings interact with the phosphoprotein P, the scaffold of the RNA polymerase complex, this interaction did not lead to RNA dissociation from the rings in vitro. Thus, we have uncovered complex equilibria involving changes in secondary structure of N and RNA loosening, processes that must take place in the context of RNA transcription and replication, whose detailed mechanisms and cellular and viral participants need to be established.

Expressions of E2 and E7-HPV16 proteins in pre-malignant and malignant lesions of the uterine cervix.

Continuous production of the E7 protein from different types of high risk human papilloma virus (HPV) is required for progression of malignancy. We developed antibodies against HPV type 16 E7 and E2 proteins to evaluate their utility as markers for diagnosis during early stages of cervical cancer. Forty biopsies from uterine cervices were diagnosed as low grade intraepithelial lesion (LSIL), high grade intraepithelial lesion (HSIL), squamous carcinoma (SC), in situ adenocarcinoma (ISA) and invasive adenocarcinoma (AC), all of which were infected with HPV 16. Immunohistochemistry was used to investigate the expressions of E7 and E2 (both from HPV 16) and p16. P16 was expressed in eight of 12 LSILs, in all HSILs, in 16 of 18 SC and in all ACs. E2 was expressed in six of 12 LSILs. E7 was positive in eight of 12 LSILs and in all HSIL and carcinomas. The expressions of E2 and E7 of HPV16 related to p16 expression confirmed the value of the viral oncogenic proteins as complementary to histology and support the carcinogenic model of the uterine cervix, because HPVDNA integration into cellular DNA implies the destruction of the gene encoding E2, which suppresses the expression of the E6 and E7 oncoproteins. E2 from HPV16 could be marker for LSILs, while E7 could be a marker for progression of LSILs to HSILs in patients infected by HPV16, because viral typing has little positive predictive value.