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Principal Investigator: PAUL T WINGFIELD
Organization: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
Fiscal Year: 2021
Award: $1,638,558
Funding agency: National Institute of Arthritis and Musculoskeletal and Skin Diseases
In uninfected cells, RNA is transcribed from DNA, processed, and then transported out of the nucleus and translated into protein. In cells infected with HIV-1, the viral RNA genomes must be exported out of the nucleus without being processed so they can be packaged into new viral particles. To do this the cell must bind its own RNA genome from among the host RNA in the nucleus. This is achieved using the HIV-1 Rev protein that recognizes a Rev response element (RRE) in the viral RNA. Once bound to RRE, Rev self-associates and binds other host proteins, forming a multiprotein-RNA complex that is exported from the nucleus. Our current studies are directed at describing the molecular details of this complex. In addition to contributing fundamental information on the mechanism of viral replication, these studies may highlight points of vulnerability that may be suitable targets for therapeutic intervention including Rev itself.
We have focused first on Rev RNA interactions. Using a shortened and non-polymerizing form of Rev that incorporates amino acid residues 1-93 (wild type Rev is 1-115) and is further stabilized with a single chain variable fragment (scFv) antibody, we have prepared complexes with various RNA preparations corresponding to regions of the RRE. Also we have used RNA aptamers, which are RNAs that fold into 3-dimensional conformations that bind to their targets (in this case Rev). Aptamers that bind with higher affinity than Rev-binding sites on RRE have potential anti-HIV activity. We have recently determined a high resolution structure by X-ray crystallography of Rev with a high affinity binding aptamer. In this structure, dimeric Rev bridges two discontinuous aptamers, suggesting when it binds to RRE the Rev dimer is binding two RNA sequences co-localized by the RNA conformation. Using the Rev 1-93 - scFv as a proven crystallization platform we are extending structural studies to solve interactions with other high affinity aptamers.
The antibody fragment (Fab) used for stabilizing Rev for structural studies was derived from a phage display antibody library. This chimeric antibody (human framework and rabbit variable domains), expressed in bacteria, was humanized and was effective by binding to Rev with a very high affinity, thereby preventing its oligomerization. In previous work we showed that this antibody had anti-HIV-1 activity. We also showed that cyclic peptides (up to 12 amino acids long) from the antibody variable regions (CDRs) could bind to Rev but we have not yet shown whether they also have anti-HIV-1 activity. In addition, we are attempting to co-crystallize the peptides with Rev in order to obtain high-resolution structures of the complexes, which may help design or model low-molecular weight mimics with improved (stronger) binding to Rev.
HIV-1 AIDS is associated with improper chromatid separation and aneuploidy. We have previously shown that Rev interacts strongly and stoichiometrically with tubulin to form double-ring-like complexes in vitro. We have also shown that treatment of tubulin with Cryptophycin-45 (cancer drug) forms highly stable single-rings composed of 8 tubulin heterodimers, and that these bind Rev stoichiometrically to also form double-rings. The first structure of Hela tubulin in complex with cryptophycin was determined at 3.45 Angstrom resolution using cryo-EM techniques. We have also have determined structure of Hela tubulin-cryptophycin rings in complex with HIV-Rev protein at 3.85 Angstrom resolution also using cryo-EM techniques. The structure shows tubulin double rings formed in the presence of Rev. These findings are of interest to both the cancer and tubulin research communities. The double-rings, formed when Rev is added will also further insights into Rev structure and function. A manuscript describing a novel mechanism for tubulin depolymerization by HIV-Rev is in preparation for submission September 2021.
SARS-cov-2 related studies (A-C)
A. APN01: There remains an urgent need for safe and effective therapeutics for Covid19. We hypothesized that aerosol administration of soluble human recombinant ACE2 receptor would neutralize SARS-CoV-2 in the airways, limiting infection in the lung and potentially also mitigate damage caused by deregulated signaling in the renin-angiotensin pathway. Prerequisite to a clinical test of this hypothesis, we evaluated both virus binding activity (with SARS-CoV-2 strain USA_WA1/2020) and enzymatic activity for cleavage of Ang II following aerosolization using a cGMP-grade form of recombinant human ACE2. Both activities were retained. Based on these results, a Phase I clinical trial in healthy volunteers is planned, with subsequent Phase II testing in individuals with SARS-CoV-2 infection. A manuscript titled Development of a novel aerosol intervention for COVID-19 disease: Tolerability of soluble ACE2 (APN01) administered via nebulizer, written in collaboration with Dr Robert Shoemaker (NCI) and others, is ready for submission to Science Translational Medicine. A companion manuscript reporting effective inhibition of recent SARS-CoV-2 variants of concern by APN01 is also in preparation.
B. MAP30 MAP30 is a multifunctional antiviral and antitumor protein derived from the seeds of bitter melon (Momordica charantia). MAP30 has previously been shown to possess several biological activities including inhibition of protein synthesis, DNA adenosine-glycosidase activity that leads to topological inactivation of viral DNAs, and inhibition of HIV-1 integrase. MAP30 has also been shown to inhibit ACE2, and to contain a peptide that when chemically synthesized inhibits ACE2 and reduces blood pressure in constitutively hypertensive rats. We have assessed MAP30 for inhibition of SARS-CoV-2. We have reproducibly observed ca. 40% reduction of virus-induced cytopathic effect (CPE) in Vero E6 monkey kidney cells in the low M range without concomitant toxicity. As MAP30 does not enter cells, this suggests inhibition of ACE2-mediated viral entry. However, in a viral replication assay in A549 human lung cells, a more appropriate cell type, we reproducibly observe 100% viral inhibition (IC50 = 0.27 M, CC50 = 4.17 M). Furthermore, appending an C-terminal TAT peptide (to mediate cell entry) increases the Hill coefficient from 1 to 2 without lowering the CC50, thereby broadening the effective range. In addition to the studies above, we have computationally designed a set of cyclic peptides, based on the structure of MAP30, and also on the SARS-cov-2 viral spike protein receptor binding domain. A total of 10 peptides have been commercially synthesized and will be tested in a Vero E6 CPE assay together with the recombinant ACE2 protein described above and a set of reference compounds that includes Remdesivir, Chloroquine, and Hydroxychloroquine. A manuscript titled Inhibition of the COVID-19 virus, SARS-cov-2 by rMAP30, a recombinant anti-viral and anti-tumor agent has been drafted in collaboration with Dr Robert Shoemaker (NCI).
C. One attractive target for therapeutic intervention is the main protease (Mpro), a dimeric enzyme necessary for SARS-cov-2 replication. Most work to date developing Mpro inhibitors has focused on the active site. Our work (collaboration D. Davis, NCI) has revealed a regulatory mechanism for Mpro activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits Mpro activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. This work has been accepted for publication in the journal mBio (July 2021 and is now on-line.
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