Elucidation of HIV‐1 5' Leader Secondary Structure Through Novel Long Range Nuclear Magnetic Resonance (NMR) Techniques

Abstract

The human immunodeficiency virus (HIV‐1) is a global pandemic that has infected approximately 37 million individuals. The high mutation rate in the HIV‐1 genome renders current treatments that target regions of the replication cycle often insufficient. Thus, structural elucidation of the 5' leader within the HIV‐1 RNA is pertinent due to the high conservation of this packaging signal, making it paramount to viral replication and a promising target for antiretroviral therapy.Structural analysis of RNA constructs below 60 nucleotides has been effective by Nuclear Magnetic Resonance (NMR) spectroscopy. However, the size of the 5' leader – approximately 300 nucleotides – has stifled structural studies by NMR due to excessive signal broadening and long instrument acquisition times. To circumvent the latter complication, site‐specific lanthanide labeling of a reporter protein with a high binding affinity to a cognate RNA loop is proposed. It is well‐known that paramagnetic lanthanide ions induce measurable pseudocontact shifts (PCS) in NMR spectra. However, binding the metal ion tag directly to the RNA is deleterious. Consequently, the spliceosomal U1A reporter protein is utilized as an intermediary between the metal ion tag and the RNA. Prior to applying the technique to the 5' leader, a 46 nucleotide Moloney Murine Leukemia Virus (MMLV) derived RNA construct was used as a model for PCS observation and NMR experiment optimization. Through the use of Heteronuclear Multiple‐Quantum Correlation experiments, PCS data was obtained in conjunction with residual dipolar couplings, resolving the quaternary structure of U1A: MMLV. Furthermore, the experiment illustrated an increase in distance measurements from 5Å to 30Å in the presence of paramagnetic lanthanides. Our novel method is currently being applied toward the elucidation of the secondary structure of the 5' leader with the U1A binding loop strategically placed in regions of interest in an effort to enhance our understanding of HIV‐1 biology and to facilitate the design of novel therapeutics in the future.Support or Funding InformationThis investigation was sponsored by NIH/NIGMS MARC U*STAR T34 HHS 00026 National Research Service Award to UMBC. This research was also supported in part by a grant to UMBC from the Howard Hughes Medical Institute through the HHMI Adaptation Project. We would like to thank the MARC U*STAR program and UMBC Summer Biomedical Training Program for the strong support.