Extreme genetic fragility of the HIV-1 capsid

Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

The isolation and characterisation of temperature-dependent Ricin A chain molecules in Saccharomyces cerevisiae.

Ricin is a heterodimeric plant protein that is potently toxic to mammalian cells. Toxicity results from the catalytic depurination of eukaryotic ribosomes by ricin A chain (RTA) that follows toxin endocytosis to, and translocation across, the endoplasmic reticulum (ER) membrane. To ultimately identify proteins required for these later steps in the entry process, it will be useful to express the catalytic subunit within the ER of yeast cells in a manner that initially permits cell growth. A subsequent switch in conditions to provoke innate toxin action would permit only those strains containing defects in genes normally essential for toxin retro-translocation, refolding or degradation to survive. As a route to such a screen, several RTA mutants with reduced catalytic activity have previously been isolated. Here we report the use of Saccharomyces cerevisiae to isolate temperaturedependent mutants of endoplasmic reticulum-targeted RTA. Two such toxin mutants with opposing phenotypes were isolated. One mutant RTA (RTAF108L/L151P) allowed the yeast cells that express it to grow at 37°C while the same cells did not grow at 23ºC. Both mutations were required for temperature-dependent growth. The second toxin mutant (RTAE177D) allowed cells to grow at 23°C but not at 37°C. Interestingly, RTAE177D has been previously reported to have reduced catalytic activity, but this is the first demonstration of a temperature-sensitive phenotype. To provide a more detailed characterisation of these mutants we have investigated their N-glycosylation, stability, catalytic activity and, where appropriate, a three dimensional structure. The potential utility of these mutants is discussed

The alphaviruses: gene expression, replication, and evolution

The alphaviruses are a genus of 26 enveloped viruses that cause disease in humans and domestic animals. Mosquitoes or other hematophagous arthropods serve as vectors for these viruses. The complete sequences of the +/- 11.7-kb plus-strand RNA genomes of eight alphaviruses have been determined, and partial sequences are known for several others; this has made possible evolutionary comparisons between different alphaviruses as well as comparisons of this group of viruses with other animal and plant viruses. Full-length cDNA clones from which infectious RNA can be recovered have been constructed for four alphaviruses; these clones have facilitated many molecular genetic studies as well as the development of these viruses as expression vectors. From these and studies involving biochemical approaches, many details of the replication cycle of the alphaviruses are known. The interactions of the viruses with host cells and host organisms have been exclusively studied, and the molecular basis of virulence and recovery from viral infection have been addressed in a large number of recent papers. The structure of the viruses has been determined to about 2.5 nm, making them the best-characterized enveloped virus to date. Because of the wealth of data that has appeared, these viruses represent a well-characterized system that tell us much about the evolution of RNA viruses, their replication, and their interactions with their hosts. This review summarizes our current knowledge of this group of viruses

A direct interaction between fascin and microtubules contributes to adhesion dynamics and cell migration

Fascin is an actin-binding and bundling protein that is highly upregulated in most epithelial cancers. Fascin promotes cell migration and adhesion dynamics in vitro and tumour cell metastasis in vivo. However, potential non-actin bundling roles for fascin remain unknown. Here we show for the first time that fascin can directly interact with the microtubule cytoskeleton and that this does not depend upon fascin-actin bundling. Microtubule binding contributes to fascin-dependent control of focal adhesion dynamics and cell migration speed. We also show that fascin forms a complex with focal adhesion kinase (FAK) and Src, and that this signalling pathway lies downstream of fascin-microtubule association in the control of adhesion stability. These findings shed light on new non actin-dependent roles for fascin and may have implications for the design of therapies to target fascin in metastatic disease

Binding of small interfering RNA molecules is crucial for RNA interference suppressor activity of rice hoja blanca virus NS3 in plants

The NS3 protein of rice hoja blanca tenuivirus represents a viral suppressor of RNAi that sequesters small interfering (si)RNAs in vitro. To determine whether this siRNA binding property is the critical determinant for the suppressor activity of NS3, an alanine point mutational analysis was performed and the resulting mutant proteins were tested for both siRNA binding ability and RNAi suppressor activity in plants. Alanine substitutions of lysine residues at position 173-175 resulted in mutant proteins that lost both their affinity for siRNAs and their RNAi suppressor activity in planta. This indicates that siRNA binding of NS3 is indeed essential for the suppressor function of NS3 and that residues at position 173-175 are involved in the siRNA binding and suppressor activit

Towards Symbolic Model-Based Mutation Testing: Combining Reachability and Refinement Checking

Model-based mutation testing uses altered test models to derive test cases that are able to reveal whether a modelled fault has been implemented. This requires conformance checking between the original and the mutated model. This paper presents an approach for symbolic conformance checking of action systems, which are well-suited to specify reactive systems. We also consider nondeterminism in our models. Hence, we do not check for equivalence, but for refinement. We encode the transition relation as well as the conformance relation as a constraint satisfaction problem and use a constraint solver in our reachability and refinement checking algorithms. Explicit conformance checking techniques often face state space explosion. First experimental evaluations show that our approach has potential to outperform explicit conformance checkers.Comment: In Proceedings MBT 2012, arXiv:1202.582

Automated metamorphic testing on the analyses of feature models

Copyright © 2010 Elsevier B.V. All rights reserved.Context: A feature model (FM) represents the valid combinations of features in a domain. The automated extraction of information from FMs is a complex task that involves numerous analysis operations, techniques and tools. Current testing methods in this context are manual and rely on the ability of the tester to decide whether the output of an analysis is correct. However, this is acknowledged to be time-consuming, error-prone and in most cases infeasible due to the combinatorial complexity of the analyses, this is known as the oracle problem.Objective: In this paper, we propose using metamorphic testing to automate the generation of test data for feature model analysis tools overcoming the oracle problem. An automated test data generator is presented and evaluated to show the feasibility of our approach.Method: We present a set of relations (so-called metamorphic relations) between input FMs and the set of products they represent. Based on these relations and given a FM and its known set of products, a set of neighbouring FMs together with their corresponding set of products are automatically generated and used for testing multiple analyses. Complex FMs representing millions of products can be efficiently created by applying this process iteratively.Results: Our evaluation results using mutation testing and real faults reveal that most faults can be automatically detected within a few seconds. Two defects were found in FaMa and another two in SPLOT, two real tools for the automated analysis of feature models. Also, we show how our generator outperforms a related manual suite for the automated analysis of feature models and how this suite can be used to guide the automated generation of test cases obtaining important gains in efficiency.Conclusion: Our results show that the application of metamorphic testing in the domain of automated analysis of feature models is efficient and effective in detecting most faults in a few seconds without the need for a human oracle.This work has been partially supported by the European Commission(FEDER)and Spanish Government under CICYT project SETI(TIN2009-07366)and the Andalusian Government project ISABEL(TIC-2533)

Oncogenic RET Kinase domain mutations perturb the autophosphorylation trajectory by enhancing substrate presentation in trans

To decipher the molecular basis for RET kinase activation and oncogenic deregulation, we defined the temporal sequence of RET autophosphorylation by label-free quantitative mass spectrometry. Early autophosphorylation sites map to regions flanking the kinase domain core, while sites within the activation loop only form at later time points. Comparison with oncogenic RET kinase revealed that late autophosphorylation sites become phosphorylated much earlier than wild-type RET, which is due to a combination of an enhanced enzymatic activity, increased ATP affinity, and surprisingly, by providing a better intermolecular substrate. Structural analysis of oncogenic M918T and wild-type RET kinase domains reveal a cis-inhibitory mechanism involving tethering contacts between the glycine-rich loop, activation loop, and αC-helix. Tether mutations only affected substrate presentation but perturbed the autophosphorylation trajectory similar to oncogenic mutations. This study reveals an unappreciated role for oncogenic RET kinase mutations in promoting intermolecular autophosphorylation by enhancing substrate presentation