Amanda Roa

Publications

• 5.15

  Impact points

  RING Domain Mutations Uncouple TRIM5α Restriction of HIV-1 From Inhibition of Reverse Transcription and Acceleration of Uncoating

  Amanda Roa, Fumiaki Hayashi, Yang Yang, Maritza Lienlaf, Jing Zhou, Jiong Shi, Satoru Watanabe, Takanori Kigawa, Shigeyuki Yokoyama, Chris Aiken, Felipe Diaz-Griffero

  Journal of Virology. 01/2011;

  Rhesus TRIM5α(TRIM5αrh) is a cytosolic protein that potently restricts HIV-1 at an early post-entry stage, prior to reverse transcription. The ability of TRIM5αrh to block HIV-1 infection has been correlated with a decrease of pelletable HIV-1 capsid during infection. To genetically dissect the abil... [more] Rhesus TRIM5α(TRIM5αrh) is a cytosolic protein that potently restricts HIV-1 at an early post-entry stage, prior to reverse transcription. The ability of TRIM5αrh to block HIV-1 infection has been correlated with a decrease of pelletable HIV-1 capsid during infection. To genetically dissect the ability of TRIM5α to block reverse transcription, we studied a set of TRIM5αrh RING domain mutants that potently restrict HIV-1 but allow the occurrence of reverse transcription. These TRIM5αrh RING variants blocked HIV-1 infection after reverse transcription but prior to integration, as suggested by routing of nuclear viral DNA to circularization in the form of 2-LTR circles. The folding of RING domain variants was similar to the wild type, as evaluated by Nuclear Magnetic Resonance. RING domain changes that allowed the occurrence of reverse transcription were impaired in their ability to decrease the amount of pelletable capsid when compared with wild-type TRIM5α. Similar effects of this particular group of mutations were observed with human TRIM5α inhibition of N-MLV. Interestingly, TRIM5αrh RING domain variants also prevented the degradation of TRIM5αrh that occurs following cell entry of HIV-1. These data correlated the block of reverse transcription with the ability of TRIM5αto accelerate uncoating. Collectively, these results suggest that TRIM5αrh blocks HIV-1 reverse transcription by inducing premature viral uncoating in target cells.

• Evaluating properties of several cheese-ripening fungi for potential biotechnological applications

  R. Chávez, Amanda Roa, K. Navarrete, J. Trebotich, Y. Espinosa, I. Vaca.

  Mycoscience. 01/2010; 51:84-87.

  Strains of Penicillium camemberti and Penicillium roqueforti were tested for properties that could be important for future biotechnological applications of these fungi. Penicillium camemberti CECT 2267 and P. roqueforti NRRL 849 showed the most promising performances in terms of growth, protoplast p... [more] Strains of Penicillium camemberti and Penicillium roqueforti were tested for properties that could be important for future biotechnological applications of these fungi. Penicillium camemberti CECT 2267 and P. roqueforti NRRL 849 showed the most promising performances in terms of growth, protoplast production, and protoplast regeneration abilities. Transformation of these strains with a plasmid containing gene encoding phleomycin resistance showed that they also have a high transformation frequency. In addition, both strains showed low extracellular proteolytic activity. Thus, these strains have all the characteristics to make them suitable for future genetic improvement, recombinant protein production, and other potential biotechnological applications.
• 3.94

  Impact points

  Membrane topology and identification of functional amino acid residues in the Wzx O-antigen translocase from Escherichia coli O157:H4

  Cristina L. Marolda, Bo Li, 1 Michael Lung, Lucy Yang, Amanda Roa Rosales, Miguel A. Valvano.

  Journal of Bacteriology. 01/2010; 192:6160-6170.

  Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of function... [more] Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the Escherichia coli O157 Wzx (WzxEcO157) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletion-fusions to green fluorescent protein and alkaline phosphatase. We conclude that WzxEcO157 consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PP-linked saccharide substrate across the membrane.
• 1.18

  Impact points

  Molecular characterization of the niaD and pyrG genes from Penicillium camemberti, and its uses as transformation markers

  K. Navarrete, Amanda Roa, I. Vaca, Y.Espinoza, C. Navarro, R. Chávez

  Cellular and molecular biology. 01/2009; 14:692-702..

  Molecular characterization of the niaD and pyrG genes from Penicillium camemberti, and its uses as transformation markers. , 14(4):