A finely tuned interplay between calcium binding, ionic strength and pH modulates conformational and oligomerization equilibria in the Respiratory Syncytial Virus Matrix (M) protein.

As in most enveloped RNA viruses, the Respiratory Syncytial Virus Matrix (RSV-M) protein plays key roles in viral assembly and uncoating. It also plays non-structural roles related to transcription modulation through nucleo-cytoplasmic shuttling and nucleic acid binding ability. We dissected the structural and conformational changes underlying the switch between multiple functionalities, identifying Ca2+ binding as a key factor. To this end, we tackled the analysis of M's conformational stability and equilibria. While in silico calculations predict two potential calcium binding sites per protomer, purified RSV-M dimer contains only one strongly bound calcium ion per protomer. Incubation of RSV-M in the presence of excess Ca2+ leads to an increase in the thermal stability, confirming additional Ca2+ binding sites. Moreover, mild denaturant concentrations trigger the formation of higher order oligomers which are otherwise prevented under Ca2+ saturation conditions, in line with the stabilizing effect of the additional low affinity binding site. On the other hand, Ca2+ removal by chelation at pH 7.0 causes a substantial decrease in the thermal stability leading to the formation of amorphous, spherical-like aggregates, as assessed by TEM. Even though the Ca2+ content modulates RSV-M oligomerization propensity, it does affect its weak RNA binding ability. RSV-M undergoes a substantial conformational change at pHs 4.0 to 5.0 that results in the exposure of hydrophobic surfaces, an increase beta sheet content but burial of tryptophan residues. While low ionic strength promotes dimer dissociation at pH 4.0, physiological concentrations of NaCl lead to the formation of soluble oligomers smaller than 400 kDa at pH 4.0 or insoluble aggregates with tubular morphology at pH 5.0, supporting a fine tuning by pH. Furthermore, the dissociation constants estimated for the low- and high affinity calcium binding sites are 13 µM and 58 nM, respectively, suggesting an intracellular calcium sensing mechanism of RSV-M upon infection. We uncover a finely tuned interplay between calcium binding, ionic strength, and pH changes compatible with the different cellular compartments where M plays key roles, revealing diverse conformational equilibria, oligomerization, and high order structures, required to stabilize the virion particle by a layer of molecules positioned between the membrane and the nucleocapsid.

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.

Positive response to imatinib mesylate therapy for childhood chronic myeloid leukemia

Chronic myeloid leukemia (CML) is rare in the pediatric population, accounting for 2-3 percent of childhood leukemia cases, with an annual incidence of one case per million children. The low toxicity profile of imatinib mesylate has led to its approval as a front-line therapy in children for whom interferon treatment has failed or who have relapsed after allogeneic transplantation. We describe the positive responses of 2 children (case 1 - from a 7-year-old male since May 2005; case 2 - from a 5-year-old female since June 2006) with Philadelphia-positive chromosome CML treated with imatinib (300 mg/day, orally) for up to 28 months, as evaluated by morphological, cytogenetic, and molecular approaches. Our patients are alive, are in the chronic phase, and are in continuous morphological complete remission.

Positive response to imatinib mesylate therapy for childhood chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is rare in the pediatric population, accounting for 2-3% of childhood leukemia cases, with an annual incidence of one case per million children. The low toxicity profile of imatinib mesylate has led to its approval as a front-line therapy in children for whom interferon treatment has failed or who have relapsed after allogeneic transplantation. We describe the positive responses of 2 children (case 1 - from a 7-year-old male since May 2005; case 2 - from a 5-year-old female since June 2006) with Philadelphia-positive chromosome CML treated with imatinib (300 mg/day, orally) for up to 28 months, as evaluated by morphological, cytogenetic, and molecular approaches. Our patients are alive, are in the chronic phase, and are in continuous morphological complete remission.

Expression of metalloproteinases and their tissue inhibitors in gingiva affected by hereditary gingival fibromatosis: analysis of three cases within a family.

BACKGROUND AND OBJECTIVE: Hereditary gingival fibromatosis (HGF) is a benign disorder manifested by fibrous enlargement of keratinized gingiva. Evidence exists concerning the role of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in mediating normal and pathological processes, including HGF. Nevertheless, there are few and contradictory results on the analysis of MMPs and TIMPs transcripts in this pathology. MATERIAL AND METHODS: We studied the expression of the transcripts encoding MMP-1, -2 and -9 and TIMP-1 and -2 in gingival biopsies, obtained from three cases of HGF within a family, by semi-quantitative reverse transcriptase-polymerase chain reaction analysis. Samples were also processed for gelatin zymography. RESULTS: Except for MMP-9, most transcripts presented a higher level of expression in biopsies from HGF patients compared with control subjects. Accordingly, MMP-9 gelatinase activity was detected at low and similar levels among samples. Moreover, MMP-2 enzymatic activity was not detected at all. CONCLUSION: The mRNA expression of MMP-1 and -2 and TIMP-1 and -2 does not explain the gingival overgrowth presented in these cases. In addition, it is suggested that the gene expression of those molecules in the course of HGF is regulated at the translational or post-translational level.