The primer for reverse transcription in human immunodeficiency virus type 1 (HIV-1), human tRNALys 3, is selectively packaged into the virion along with tRNALys 1, 2. Human lysyl-tRNA synthetase (LysRS), the only cellular factor known to interact specifically with all three tRNALys isoacceptors, is also selectively packaged into HIV-1. Previous work has defined a tRNALys packaging complex that includes the tRNALys isoacceptors, LysRS, Gag, GagPol, and viral RNA. Numerous studies support the hypothesis that during tRNA Lys packaging, a Gag/GagPol complex interacts with a tRNALys /LysRS complex, with the capsid (CA) domain of Gag interacting specifically with LysRS, and GagPol interacting with both Gag and tRNALys.
In this work, we have identified critical residues along one face of the dimerization helix 7 (H7) of LysRS involved in packaging of LysRS into virions. Mutation of these residues affects binding to Gag in vitro , as well as the oligomerization state and aminoacylation activity of the synthetase. Taken together with previous work, these data support the conclusion that the LysRS H7–CA interaction interface represents a novel antiviral target. With this target in mind, a support-bound cyclic peptide (CP) library containing randomized amino acid sequences and different ring sizes was synthesized and screened against CA and the monomeric form of the CA C-terminal domain (WM-CA CTD). Out of 3 x 105 CPs screened, 21 hits were obtained and 6 CPs were chosen for detailed in vitro analysis. Two CPs, CP2 and CP4 showed strong binding (Kd ~ 500 nM) to both CA and WM-CA CTD in vitro. Scrambled variants of CP2 and CP4 and point mutants at each of the randomized positions eliminated binding, suggesting a sequence-specific mode of interaction. CP2 and CP4 also inhibited LysRS/CA interaction in vitro with an IC50 value of ~ 1 μM. Furthermore, nuclear magnetic resonance (NMR), mutational studies along with in silico analysis revealed that CP2 and CP4 bind to a site proximal to helix 4 (h4) of the CA-CTD, which is the known site of LysRS interaction. Preliminary studies to understand the packaging of host 7SL RNA in HIV-1 have also been carried out. Preliminary binding studies with N-terminally truncated LysRS showed that 7SL RNA bound with high affinity and specificity. The binding affinity obtained was comparable to that of cognate tRNALys, thereby suggesting structural homology between the packaged RNAs. These studies may provide insights into the mechanism/function of 7SL RNA in HIV-1.
In summary, HIV-1 has evolved several intricate mechanisms for the packaging of host molecules. Targeting these interactions for anti-viral development may provide an opportunity to block the viral replication.
|Commitee:||Hadad, Christopher M., Ibba, Michael, Ottesen, Jennifer|
|School:||The Ohio State University|
|School Location:||United States -- Ohio|
|Source:||DAI-B 78/11(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry, Biophysics|
|Keywords:||HIV-1 capsid, Host-viral interactions, Lysyl-tRNA synthetase|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be