Cross-species retroviral infection as selective pressure in the evolution of the host resistance factor APOBEC3
Japan (IFMSA-Japan) - Kindai University, Osaka(Kansai area)
Department of Immunology
Prof. Masaaki Miyazawa, M.D., Ph.D.
Prof. Yoshiyuki Hakata, Ph.D.
4 weeks
Cities/Months Jan Feb Mar Apr May Jun Jul Augt Sep Oct Nov Dec
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Type of Research Project
- Basic science
What is the background of the project?
APOBEC3 is a DNA mutator enzyme that functions as a physiologically effective restriction factor to retroviral infections. High levels of APOBEC3 expression are associated with resistance to HIV in humans. We were the first in discovering functional genetic polymorphisms at the mouse APOBEC3 locus: strains of mice resistant to retroviral infections express high levels of APOBEC3 that lacks exon 5, while susceptible strains express low levels of APOBEC3 that includes exon5. Genetic analyses of field species indicated that rodent ancestors expressed exon 5- deficient APOBEC3, but one branch of descendants acquired the exon to reduce the protein levels. Currently resistant, exon 5-lacking species of wild mice possess fossil evidence of repeated infection with a specific clade of retroviruses that came from non-rodent species like monkeys and cats, while exon 5-epxressing species lack such evidence. This indicates that retroviruses that came from monkeys and cats and attacked rodent ancestors were the selective pressure to retain exon 5-deficient APOBEC3. On the other hand, present species of mice that express exon 5- containing APOBEC3 may have another selective factor for the acquisition of exon 5.
What is the aim of the project?
To prove at molecular levels that exon 5-deficient APOBEC3 is required to restrict “xenotropic retroviruses” that originate from non-rodent species, while exon 5- proficient APOBEC3 is not effective in restricting xenotroipic viruses.
What techniques and methods are used?
Molecular cloning and construction of expression vectors; DNA transfection and selection of stable transfectant cell lines; Transfection of infectious molecular clones of retroviruses; Detection of retroviral replication; Detection of cells infected with retroviruses with specific monoclonal antibodies.
What is the role of the student?
What are the tasks expected to be accomplished by the student?
The students perform, first with Prof. Hakata and his technician and then by themselves, DNA transfection of infectious molecular clones and prepare virus samples that contain exon 5-deficient or -proficient APOBEC3. The students infect target cells and measure the replication of retroviruses by detecting cells expressing the relevant viral protein under the guidance and supervision of Prof. Hakata. If the students have enough time and enthusiasm, they will prepare virus samples by ultracentrifugation and detect incorporated APOBEC3 by western blotting with Prof. Hakata and his technician. The students can always get necessary guidance and technical help from Prof. Hakata, his technician, and a Japanese medical student who is doing her research at the Department of Immunology.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Students will be given an introductory personal course that explains the background and aim of the research project from Prof. Miyazawa. Reprints of related scientific papers and experimental protocols will also be provided from Prof. Miyazawa and Hakata.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a presentation
- The student will prepare a scientific report
- The student will prepare an abstract
- The student’s name will be mentioned in a future publication
- The student will have the opportunity to present the results together with the supervisor at a conference
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Basic techniques of tissue culturing Subjects passed: preferably, cell biology and microbiology Previous experience with: preferably tissue culturing, but this is not an absolute requirement.
Are there any legal limitations in the student’s involvement
Handling of genetically modified organisms is regulated by a law in Japan, but we can provide the students a short course of explanation as soon as they arrive here and they will quickly receive a permit by filling out an application.
Type of students accepted
This project accepts:
- Medical students
- Li; J.; Y. Hakata; E. Takeda; Q. Liu; Y. Iwatani; C. A. Kozak; and M. Miyazawa. Two genetic determinants acquired late in Mus evolution regulate the inclusion of exon 5; which alters mouse APOBEC3 translation efficiency. PLoS Pathogens 8: e1002478; 2012. (