APOBEC3 (A3) proteins are restriction factors that provide innate immunity against HIV-1 in the absence of viral infectivity factors (Vif). These single-stranded DNA- (ssDNA) and RNA-binding proteins are active deoxycytidine deaminases. Their ability to restrict of HIV replication is primarily linked to C ? U deamination of the minus-strand of DNA copy of viral RNA, resulting in a G ? A mutation in the viral DNA genome and functional inactivation of the virus. Despite the importance of A3G in HIV restriction, knowledge regarding the molecular mechanisms that underlie the interactions of A3 with nucleic acids is very limited. The objective of this application is to unravel mechanisms of A3-ssDNA interactions and the features of A3G-ssRNA that define A3G?s incorporation in virions. Our ultimate goal is to translate this knowledge toward the prevention and treatment of HIV. Our central hypothesis is that the two-domain property of A3G and A3F proteins is critical for maintaining all aspects of A3 antiviral activity and encapsidation in virions. Our rationale is that understanding the fundamental mechanisms of A3-ssDNA and A3-RNA interactions will guide the development of approaches to control and inhibit virus replication. To accomplish this goal and test our hypothesis, we will use a set of the nanoimaging and probing tools and develop a nanoarray approach that allows us to assemble A3 proteins in oligomers with defined sizes. Guided by strong preliminary data, we will test our major hypothesis through the following three specific aims:
Specific Aim 1 : Reveal the interplay between the catalytic and DNA-binding domains of A3 proteins. Hypothesis: The non-catalytic DNA-binding domain defines the complex assembly and modulates the deamination activity of A3 proteins.
Specific Aim 2 : Identify the role of A3G and A3F oligomerization in interactions with DNA. Hypothesis: Oligomerization increases deamination activity and stability of A3 proteins in complexes with ssDNA.
Specific Aim 3 : Characterize the interaction of A3G and A3F with RNA. Hypothesis: Oligomerization of A3 proteins is a critical for interaction with viral RNA and encapsidation in virions. These studies are expected to lead to the development of new and innovative preventative strategies and treatments for HIV. The proposed study is innovative in that it presents a novel approach to study A3-DNA and RNA complexes and develops a set of new nanotechnology methods with broad biomedical applications. The proposed research is significant in that the findings will lay the foundation for further improving the innate immunity property of A3. Additionally, A3 oligomers can avoid Vif-dependent degradation. Thus, the availability of A3 oligomers of select sizes assembled as nanoarrays opens prospects for testing these nanoassemblies as a means of HIV prevention.

Public Health Relevance

APOBEC3 (A3) proteins can provide innate immunity against HIV. The work proposed is relevant to public health because unraveling structural and dynamic properties of APOBEC3-viral nucleic acid complexes will increase understanding of the molecular mechanisms of HIV restriction with the existing innate mechanism of immunity of A3 proteins. Elucidating the principle of A3 restriction of HIV can generate novel treatment preventative approaches. The proposed research will provide knowledge on the stabilities and assembly process of APOBEC3 with nucleic acids, as well as information on how to control the stability of the A3-nucleic acid complex. Thus, the proposed research is relevant to the NIH?s mission to develop fundamental knowledge that will improve preventive and therapeutic treatments for diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM118006-04
Application #
9625638
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Sakalian, Michael
Project Start
2016-03-02
Project End
2021-01-31
Budget Start
2019-02-01
Budget End
2021-01-31
Support Year
4
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Nebraska Medical Center
Department
Other Basic Sciences
Type
Schools of Pharmacy
DUNS #
168559177
City
Omaha
State
NE
Country
United States
Zip Code
68198
Lv, Zhengjian; Banerjee, Siddhartha; Zagorski, Karen et al. (2018) Supported Lipid Bilayers for Atomic Force Microscopy Studies. Methods Mol Biol 1814:129-143
Lyubchenko, Yuri L (2018) Direct AFM Visualization of the Nanoscale Dynamics of Biomolecular Complexes. J Phys D Appl Phys 51:
Maity, Sibaprasad; Viazovkina, Ekaterina; Gall, Alexander et al. (2018) Polymer Nanoarray Approach for the Characterization of Biomolecular Interactions. Methods Mol Biol 1814:63-74
Pan, Yangang; Zagorski, Karen; Shlyakhtenko, Luda S et al. (2018) The Enzymatic Activity of APOBE3G Multimers. Sci Rep 8:17953
Maity, Sibaprasad; Pramanik, Apurba; Lyubchenko, Yuri L (2018) Probing Intermolecular Interactions within the Amyloid ? Trimer Using a Tethered Polymer Nanoarray. Bioconjug Chem 29:2755-2762
Sun, Zhiqiang; Hashemi, Mohtadin; Warren, Galina et al. (2018) Dynamics of the Interaction of RecG Protein with Stalled Replication Forks. Biochemistry 57:1967-1976
Zhang, Yuliang; Hashemi, Mohtadin; Lv, Zhengjian et al. (2018) High-speed atomic force microscopy reveals structural dynamics of ?-synuclein monomers and dimers. J Chem Phys 148:123322
Banerjee, Siddhartha; Hashemi, Mohtadin; Lv, Zhengjian et al. (2017) A novel pathway for amyloids self-assembly in aggregates at nanomolar concentration mediated by the interaction with surfaces. Sci Rep 7:45592
Pan, Yangang; Sun, Zhiqiang; Maiti, Atanu et al. (2017) Nanoscale Characterization of Interaction of APOBEC3G with RNA. Biochemistry 56:1473-1481
Banerjee, Siddhartha; Sun, Zhiqiang; Hayden, Eric Y et al. (2017) Nanoscale Dynamics of Amyloid ?-42 Oligomers As Revealed by High-Speed Atomic Force Microscopy. ACS Nano 11:12202-12209

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