HIV-1 RT-dependent DNAzyme expression inhibits HIV-1 replication without the emergence of escape viruses

DNAzymes are easier to prepare and less sensitive to chemical and enzymatic degradation than ribozymes; however, a DNA enzyme expression system has not yet been developed. In this study, we exploited the mechanism of HIV-1 reverse transcription (RT) in a DNA enzyme expression system. We constructed HIV-1 RT-dependent lentiviral DNAzyme expression vectors including the HIV-1 primer binding site, the DNA enzyme, and either a native tRNA (Lys-3), tRMDtRL, or one of two truncated tRNAs (Lys-3), tRMDΔARMtRL or tRMD3′-endtRL. Lentiviral vector-mediated DNAzyme expression showed high levels of inhibition of HIV-1 replication in SupT1 cells. We also demonstrated the usefulness of this approach in a long-term assay, in which we found that the DNAzymes prevented escape from inhibition of HIV. These results suggest that HIV-1 RT-dependent lentiviral vector-derived DNAzymes prevent the emergence of escape mutations

In silico modeling indicates the development of HIV-1 resistance to multiple shRNA gene therapy differs to standard antiretroviral therapy

<p>Abstract</p> <p>Background</p> <p>Gene therapy has the potential to counter problems that still hamper standard HIV antiretroviral therapy, such as toxicity, patient adherence and the development of resistance. RNA interference can suppress HIV replication as a gene therapeutic via expressed short hairpin RNAs (shRNAs). It is now clear that multiple shRNAs will likely be required to suppress infection and prevent the emergence of resistant virus.</p> <p>Results</p> <p>We have developed the first biologically relevant stochastic model in which multiple shRNAs are introduced into CD34+ hematopoietic stem cells. This model has been used to track the production of gene-containing CD4+ T cells, the degree of HIV infection, and the development of HIV resistance in lymphoid tissue for 13 years. In this model, we found that at least four active shRNAs were required to suppress HIV infection/replication effectively and prevent the development of resistance. The inhibition of incoming virus was shown to be critical for effective treatment. The low potential for resistance development that we found is largely due to a pool of replicating wild-type HIV that is maintained in non-gene containing CD4+ T cells. This wild-type HIV effectively out-competes emerging viral strains, maintaining the viral <it>status quo</it>.</p> <p>Conclusions</p> <p>The presence of a group of cells that lack the gene therapeutic and is available for infection by wild-type virus appears to mitigate the development of resistance observed with systemic antiretroviral therapy.</p

Frater a mentoring innovative Project for freshmen

The degree in Pharmacy of the University of Castilla-La Mancha is included in the branch of Health Sciences and its main objective is to train specialists in all aspects of drugs and medicines, in accordance with European Directives relating to the pharmacology profession. The degree requires five years of full time study, and there are two semesters per year. The first two years are foundation years in which students study anatomy, biology, chemistry, basic pharmaceutical science and are introduced to pharmacy. The remaining three years are devoted to higher levels of biomedical and pharmaceutical sciences, together with specialized clinical pharmacy studies. During the fourth and fifth year, considerable time is spent in clinical placements in community and hospital pharmacies, where valuable practical experience is gained. A first-year student either coming from high-school or the vocational training system find many difficulties in our degree related to the high level of demand together with the multidisciplinary typology of the different subjects they have to take during the year. The teaching staff of this degree hasdesigned an innovative project with the aim of helping first-year students to achieve their full potential. Particularly, the program is based on a mentoring relationship between freshmen and senior. The mentoring partnership formulated is a structured one-to-one relationship that focuses on the needs of the mentored participant.The mentoring program in the Faculty of Pharmacy of the University of Castilla-La Mancha is called ‘FRATER’ and it takes shaped on four stages: selection, formation, execution, and evaluation stage. Selection Stage (before starting the classes): specialized psychologists look for students who show the ability to relate to mentees, motivate, listen, and assist them to make plans and carry them through. The mentors also identify a mentees hidden talents and skills, communicate hope and optimism, make time available and help mentees to become more connected to school, maintaining confidentiality and trust within appropriate limits. The selection is carried out with the help of testtaking strategies and personal interviews.Training Stage (first month of the first year degree): three-year students are trained to be mentors. Mentors have to know all university regulations, be able to share knowledge, proper communication, improve their emotional intelligence, and make a good management time wisely. Specialized psychologists and the teachers participating in the program are in charge of all this training. Execution Stage (during the year): the mentors develop their mentoring job by means of several meetings along the year. The mentors have to record every event that takes place during the meetings by filling out subsequent reports Evaluation Stage (at the end of the year): the evaluation of the mentoring program will be achieved by the coordination team of the mentoring program through different satisfaction tests and personal interviews with mentors and mentees.Herein, in this short communication we would like to report the results obtained from ‘FRATER’ and sum up the benefits that mentor and mentee can obtain from such academic experienc

Polymerase Activity, Protein-Protein Interaction, and Cellular Localization of the Usutu Virus NS5 Protein

Usutu virus (USUV) has become increasingly relevant in recent years, with large outbreaks that sporadically have affected humans being reported in wildlife. Similarly to the rest of flaviviruses, USUV contains a positive-sense single-stranded RNA genome which is replicated by the activity of nonstructural protein 5 (NS5). USUV NS5 shows high sequence identity with the remaining viruses in this genus.</jats:p

An ad hoc final degree project to help students to get the second cycle educational level (MECES 3)

A final degree project (TFG by its Spanish acronym) has been implemented in a newly created degree of Pharmacy at the University of Castilla-La Mancha (Spain). The new subject aims to carry out activities to ensure that students can get the second cycle educational level (MECES 3). According to each students records, pharmacy students select a specific TFG from a pull previously designed by professors of the School of Pharmacy. Each professor proposes a TFG with a specific title accompanied by a short description of the main objectives. Once each student selects a specific TFG, the professor becomes the mentor of the student leading him to reach the aims proposed in the TFG and, in parallel, the MECES level 3 competencies. This student-mentor framework is possible thanks to TFG procedures which include the TFG report guidelines, the TFG progress report, and the Supervisor evaluation report, among others, and evaluation criteria developed by an ad hoc committee. To get a better insight into the potential of the TFG as a tool to aid students, a survey was conducted to gather the student’s feelings and experiences. Interestingly, the questionnaire revealed that most of the students (more than 90%) think that they have acquired a high achievement on each one of the seven MECES level 3 competencies thanks to the collaborative student-mentor work

Tempo and Mode of Plant RNA Virus Escape from RNA Interference-Mediated Resistance ▿

A biotechnological application of artificial microRNAs (amiRs) is the generation of plants that are resistant to virus infection. This resistance has proven to be highly effective and sequence specific. However, before these transgenic plants can be deployed in the field, it is important to evaluate the likelihood of the emergence of resistance-breaking mutants. Two issues are of particular interest: (i) whether such mutants can arise in nontransgenic plants that may act as reservoirs and (ii) whether a suboptimal expression level of the transgene, resulting in subinhibitory concentrations of the amiR, would favor the emergence of escape mutants. To address the first issue, we experimentally evolved independent lineages of Turnip mosaic virus (TuMV) (family Potyviridae) in fully susceptible wild-type Arabidopsis thaliana plants and then simulated the spillover of the evolving virus to fully resistant A. thaliana transgenic plants. To address the second issue, the evolution phase took place with transgenic plants that expressed the amiR at subinhibitory concentrations. Our results show that TuMV populations replicating in susceptible hosts accumulated resistance-breaking alleles that resulted in the overcoming of the resistance of fully resistant plants. The rate at which resistance was broken was 7 times higher for TuMV populations that experienced subinhibitory concentrations of the antiviral amiR. A molecular characterization of escape alleles showed that they all contained at least one nucleotide substitution in the target sequence, generally a transition of the G-to-A and C-to-U types, with many instances of convergent molecular evolution. To better understand the viral population dynamics taking place within each host, as well as to evaluate relevant population genetic parameters, we performed in silico simulations of the experiments. Together, our results contribute to the rational management of amiR-based antiviral resistance in plants

Emergence of a Complex Relationship between HIV-1 and the microRNA Pathway

Recent experimental evidences support the existence of an increasingly complex and multifaceted interaction between viruses and the microRNA-guided RNA silencing machinery of human cells. The discovery of small interfering RNAs (siRNAs), which are designed to mediate cleavage of specific messenger RNAs (mRNAs), prompted virologists to establish therapeutic strategies based on siRNAs with the aim to suppress replication of several viruses, including human immunodeficiency virus type 1 (HIV-1). It has been appreciated only recently that viral RNAs can also be processed endogenously by the microRNA-generating enzyme Dicer or recognized by cellular miRNAs, in processes that could be viewed as an adapted antiviral defense mechanism. Known to repress mRNA translation through recognition of specific binding sites usually located in their 3′ untranslated region, miRNAs of host or viral origin may exert regulatory effects towards host and/or viral genes and influence viral replication and/or the host response to viral infection. This article summarizes our current state of knowledge on the relationship between HIV-1 and miRNA-guided RNA silencing, and discusses the different aspects of their interaction