Fátima Pereira de Souza

Molecular models of NS3 protease variants of the Hepatitis C virus

BackgroundHepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed.ResultsThe atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures.ConclusionsThis project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure-based drug design of a new generation of NS3 protease variants inhibitors. All models in the database are publicly accessible via our interactive website, providing us with large amount of structural models for use in protein-ligand docking analysis.

Frequent respiratory pathogens of respiratory tract infections in children attending daycare centers

OBJECTIVESnTo identify and characterize respiratory viruses that infect children from daycare centers with symptoms of respiratory infection and to evaluate the association of clinical and epidemiological disease data with the identified virus.nnnMETHODSnWe conducted a study between 2003 and 2005 in 176 children with respiratory infection symptoms attending a municipal daycare center. Samples from nasopharyngeal secretion were tested by reverse transcriptase polymerase chain reaction and positive samples for picornavirus were sequenced.nnnRESULTSnAll 782 collected samples were analyzed and 31.8% were positive for at least one of the studied respiratory viruses. Respiratory infections were characterized by the presence of mild symptoms of the upper respiratory tract, the most common of which were runny nose and cough. In the 2 years of study, most cases of infection occurred in autumn and winter, but respiratory viruses were detected throughout all the study period.nnnCONCLUSIONSnRespiratory viruses and respiratory infections caused by them are part of the daily life of children attending daycare centers. Our results show the great impact that respiratory infections have on these children and suggest that more attention must be paid to viral pathogens.

Diversity and Adaptation of Human Respiratory Syncytial Virus Genotypes Circulating in Two Distinct Communities: Public Hospital and Day Care Center

HRSV is one of the most important pathogens causing acute respiratory tract diseases as bronchiolitis and pneumonia among infants. HRSV was isolated from two distinct communities, a public day care center and a public hospital in São José do Rio Preto – SP, Brazil. We obtained partial sequences from G gene that were used on phylogenetic and selection pressure analysis. HRSV accounted for 29% of respiratory infections in hospitalized children and 7.7% in day care center children. On phylogenetic analysis of 60 HRSV strains, 48 (80%) clustered within or adjacent to the GA1 genotype; GA5, NA1, NA2, BA-IV and SAB1 were also observed. SJRP GA1 strains presented variations among deduced amino acids composition and lost the potential O-glycosilation site at amino acid position 295, nevertheless this resulted in an insertion of two potential O-glycosilation sites at positions 296 and 297. Furthermore, a potential O-glycosilation site insertion, at position 293, was only observed for hospital strains. Using SLAC and MEME methods, only amino acid 274 was identified to be under positive selection. This is the first report on HRSV circulation and genotypes classification derived from a day care center community in Brazil.

Biophysical and Structural Characterization of the Recombinant Human eIF3L

The eukaryotic translation initiation factor 3, subunit L (eIF3L) is one of the subunits of the eIF3 complex, an accessory protein of the Polymerase I enzyme and may have an important role in the Flavivirus replication by interaction with a viral non-structural 5 protein. Considering the importance of eIF3L in a diversity of cellular functions, we have produced the recombinant full-length eIF3L protein in Escherichia coli and performed spectroscopic and in silico analyses to gain insights into its hydrodynamic behavior and structure. Dynamic light scattering showed that eIF3L behaves as monomer when it is not interacting with other molecular partners. Circular dichroism experiments showed a typical spectrum of α-helical protein for eIF3L, which is supported by sequence-based predictions of secondary structure and the 3D in silico model. The molecular docking with the K subunit of the eIF3 complex revealed a strong interaction. It was also predicted several potential interaction sites in eIF3L, indicating that the protein is likely capable of interacting with other molecules as experimentally shown in other functional studies. Moreover, bioinformatics analyses showed approximately 8 putative phosphorylation sites and one possible N-glycosylation site, suggesting its regulation by post-translational modifications. The production of the eIF3L protein in E. coli and structural information gained in this study can be instrumental for target-based drug design and inhibitors against Flavivirus replication and to shed light on the molecular mechanisms involved in the eukaryotic translation initiation.

Exploring the Binding Mechanism of Flavonoid Quercetin to Phospholipase A2: Fluorescence Spectroscopy and Computational Approach

The interaction of flavonoid Quercetin with Phospholipase n A2 isolated from snake venom Bothrops brazili (MTX-II) was n investigated by fluorescence spectroscopy and molecular n modeling. The fluorimetric titrations were conducted at n 288, 298 and 308 K and at pH 8.0. Stern-Volmer quenching n constant (KSV) and binding constant (Kb) were calculated n along with the corresponding thermodynamic parameters n ΔG, ΔH and ΔS at 288 and 298 K. From these analysis n evidences of complex formation in between MTX-II and QCT n are found. Besides that modified Stern-Volmer plot show n evidences for two types of intrinsic fluorophores with n different accessibilities at 308 K. The mean distance n between the donor (MTX-II) and acceptor (QCT) was n determined by fluorescence resonance energy transfer (FRET). The optimized structure of QCT was obtained by ab initio calculation, which geometry was performed in its n ground states by using DFT/B3LYP functional with 6-311+G n (d,p) basis set. The molecular docking analysis show that n QCT may be localized at two main clusters, the first is at the n dimer interface and the second at the active site like region. n The clusters positions and binding energies reinforce the experimental data.