Probing the Structural Topology of HIV-1 Virion Infectivity Factor (VIF): A Dissertation

Human Immunodeficiency Virus Type 1 (HIV-1), the virus that causes Acquired Immunodeficiency Syndrome (AIDS), attacks the immune system leaving patients susceptible to opportunistic infections that eventually cause death. Highly Active Antiretroviral Therapy, HAART, is the current drug strategy used to combat HIV. It is a combination therapy that includes HIV-1 Reverse Transcriptase and HIV-1 Protease inhibitors. Drug resistant strains arise that evade current HAART treatments; therefore novel drugs are needed. HIV-1 regulatory proteins such as Tat, Rev, Nef, Vpr, Vpu, and Vif are attractive new drug targets. Of particular interest is the HIV-1 Vif protein and its cellular binding partner APOBEC3G. In the absence of HIV-1 Vif, APOBEC3G, a cytidine deaminase, is able to mutate the viral cDNA and render the virus noninfectious. HIV-1 Vif binds to APOBEC3G and targets it for proteosomal degradation through an interaction with a Cullin-RING ligase complex. Blocking the HIV-1 Vif APOBEC3G interaction would allow APOBEC3G to perform its antiviral function. An attractive strategy to target the HIV-1 Vif APOBEC3G interaction would be a structure-based one. To apply structure-based drug design approaches to HIV-1 Vif and APOBEC3G, I attempted to collect high resolution structural data on HIV-1 Vif and APOBEC3G. My attempts were unsuccessful because the milligram quantities of soluble protein required were not obtained. Therefore, in Chapter III I used chemical cross-linking and mass spectrometry to probe the structural topology of HIV-1 Vif obtaining low resolution structural data. Chemical cross-linking formed HIV-1 Vif multimers including dimers, trimers, and tetramers. Analysis of the cross-linked monomer revealed that HIV-1 Vif’s N-terminal domain is a well-folded, compact, globular domain, where as the C-teriminal domain is predicted to be disordered. In addition, disorder prediction programs predicted the C-terminal domain of HIV-1 Vif to be disordered. Upon oligomerization the C-terminal domain undergoes a disorder-to-order transition that not only facilitates oligomerization but may facilitate other protein-protein interactions. In addition, HIV-1 Vif oligomerization bring Lys34 and Glu134 in close proximity to each other likely creating one molecular surface forming a “hot spot” of biological activity. In Chapter IV I confirmed my low resolution structural data via peptide competition experiments where I identified peptides that can be used as scaffolds for future drug design. HIV-1 Vif oligomerization is concentration dependent. The HIV-1 Vif peptides Vif(29-43) and Vif(125-139) were able to disrupt HIV-1 Vif oligomerization, which confirms the low resolution structural data. HIV-1 Vif peptides Vif(25-39) and Vif(29-43) reduced the amount of APOBEC3G immobilized on the Protein A beads, reduced the amount of HIV-1 Vif interacting with APOBEC3G, or degraded APOBEC3G itself. These peptides could be used as scaffolds to design novel drugs that disrupt the function of HIV-1 Vif and or APOBEC3G. Therefore, low resolution structural data and peptide competition experiments were successful in identifying structurally important domains in HIV-1 Vif. They also provided insight into a possible mechanism for HIV-1 Vif function where a disorder-to-order transition facilitates HIV-1 Vif’s ability to interact with a diverse set of macromolecules. These data advance our structural understanding of HIV-1 Vif and they will facilitate future highresolution studies and novel drug designs

Cross-sectional Ct distributions from qPCR tests can provide an early warning signal for the spread of COVID-19 in communities

BackgroundSARS-CoV-2 PCR testing data has been widely used for COVID-19 surveillance. Existing COVID-19 forecasting models mainly rely on case counts obtained from qPCR results, even though the binary PCR results provide a limited picture of the pandemic trajectory. Most forecasting models have failed to accurately predict the COVID-19 waves before they occur. Recently a model utilizing cross-sectional population cycle threshold (Ct—the number of cycles required for the fluorescent signal to cross the background threshold) values obtained from PCR tests (Ct-based model) was developed to overcome the limitations of using only binary PCR results. In this study, we aimed to improve on COVID-19 forecasting models using features derived from the Ct-based model, to detect epidemic waves earlier than case-based trajectories.MethodsPCR data was collected weekly at Northeastern University (NU) between August 2020 and January 2022. Campus and county epidemic trajectories were generated from case counts. A novel forecasting approach was developed by enhancing a recent deep learning model with Ct-based features and applied in Suffolk County and NU campus. For this, cross-sectional Ct values from PCR data were used to generate Ct-based epidemic trajectories, including effective reproductive rate (Rt) and incidence. The improvement in forecasting performance was compared using absolute errors and residual squared errors with respect to actual observed cases at the 7-day and 14-day forecasting horizons. The model was also tested prospectively over the period January 2022 to April 2022.ResultsRt curves estimated from the Ct-based model indicated epidemic waves 12 to 14 days earlier than Rt curves from NU campus and Suffolk County cases, with a correlation of 0.57. Enhancing the forecasting models with Ct-based information significantly decreased absolute error (decrease of 49.4 and 221.5 for the 7 and 14-day forecasting horizons) and residual squared error (40.6 and 217.1 for the 7 and 14-day forecasting horizons) compared to the original model without Ct features.ConclusionCt-based epidemic trajectories can herald an earlier signal for impending epidemic waves in the community and forecast transmission peaks. Moreover, COVID-19 forecasting models can be enhanced using these Ct features to improve their forecasting accuracy. In this study, we make the case that public health agencies should publish Ct values along with the binary positive/negative PCR results. Early and accurate forecasting of epidemic waves can inform public health policies and countermeasures which can mitigate spread

Asenapine effects in animal models of psychosis and cognitive function

Asenapine, a novel psychopharmacologic agent in the development for schizophrenia and bipolar disorder, has high affinity for serotonergic, α-adrenergic, and dopaminergic receptors, suggesting potential for antipsychotic and cognitive-enhancing properties. The effects of asenapine in rat models of antipsychotic efficacy and cognition were examined and compared with those of olanzapine and risperidone. Amphetamine-stimulated locomotor activity (Amp-LMA; 1.0 or 3.0 mg/kg s.c.) and apomorphine-disrupted prepulse inhibition (Apo-PPI; 0.5 mg/kg s.c.) were used as tests for antipsychotic activity. Delayed non-match to place (DNMTP) and five-choice serial reaction (5-CSR) tasks were used to assess short-term spatial memory and attention, respectively. Asenapine doses varied across tasks: Amp-LMA (0.01–0.3 mg/kg s.c.), Apo-PPI (0.001–0.3 mg/kg s.c.), DNMTP (0.01–0.1 mg/kg s.c.), and 5-CSR (0.003–0.3 mg/kg s.c.). Asenapine was highly potent (active at 0.03 mg/kg) in the Amp-LMA and Apo-PPI assays. DNMTP or 5-CSR performance was not improved by asenapine, olanzapine, or risperidone. All agents (P < 0.01) reduced DNMTP accuracy at short delays; post hoc analyses revealed that only 0.1 mg/kg asenapine and 0.3 mg/kg risperidone differed from vehicle. All active agents (asenapine, 0.3 mg/kg; olanzapine, 0.03–0.3 mg/kg; and risperidone, 0.01–0.1 mg/kg) significantly impaired 5-CSR accuracy (P < 0.05). Asenapine has potent antidopaminergic properties that are predictive of antipsychotic efficacy. Asenapine, like risperidone and olanzapine, did not improve cognition in normal rats. Rather, at doses greater than those required for antipsychotic activity, asenapine impaired cognitive performance due to disturbance of motor function, an effect also observed with olanzapine and risperidone

Evaluating protein cross-linking as a therapeutic strategy to stabilize SOD1 variants in a mouse model of familial ALS

Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment. We hypothesized that cyclic thiosulfinate cross-linkers, which selectively target a rare, 2 cysteine-containing motif, can stabilize fALS-causing SOD1 variants in vivo. We created a library of chemically diverse cyclic thiosulfinates and determined structure-cross-linking-activity relationships. A pre-lead compound, “S-XL6,” was selected based upon its cross-linking rate and drug-like properties. Co-crystallographic structure clearly establishes the binding of S-XL6 at Cys 111 bridging the monomers and stabilizing the SOD1 dimer. Biophysical studies reveal that the degree of stabilization afforded by S-XL6 (up to 24°C) is unprecedented for fALS, and to our knowledge, for any protein target of any kinetic stabilizer. Gene silencing and protein degrading therapeutic approaches require careful dose titration to balance the benefit of diminished fALS SOD1 expression with the toxic loss-of-enzymatic function. We show that S-XL6 does not share this liability because it rescues the activity of fALS SOD1 variants. No pharmacological agent has been proven to bind to SOD1 in vivo. Here, using a fALS mouse model, we demonstrate oral bioavailability; rapid engagement of SOD1G93A by S-XL6 that increases SOD1G93A’s in vivo half-life; and that S-XL6 crosses the blood–brain barrier. S-XL6 demonstrated a degree of selectivity by avoiding off-target binding to plasma proteins. Taken together, our results indicate that cyclic thiosulfinate-mediated SOD1 stabilization should receive further attention as a potential therapeutic approach for fALS

Mass spectrometry analysis of HIV-1 Vif reveals an increase in ordered structure upon oligomerization in regions necessary for viral infectivity

HIV-1 Vif, an accessory protein in the viral genome, performs an important role in viral pathogenesis by facilitating the degradation of APOBEC3G, an endogenous cellular inhibitor of HIV-1 replication. In this study, intrinsically disordered regions are predicted in HIV-1 Vif using sequence-based algorithms. Intrinsic disorder may explain why traditional structure determination of HIV-1 Vif has been elusive, making structure-based drug design impossible. To characterize HIV-1 Vif\u27s structural topology and to map the domains involved in oligomerization we used chemical cross-linking, proteolysis, and mass spectrometry. Cross-linking showed evidence of monomer, dimer, and trimer species via denaturing gel analysis and an additional tetramer via western blot analysis. We identified 47 unique linear peptides and 24 (13 intramolecular; 11 intermolecular) noncontiguous, cross-linked peptides, among the noncross-linked monomer, cross-linked monomer, cross-linked dimer, and cross-linked trimer samples. Almost complete peptide coverage of the N-terminus is observed in all samples analyzed, however reduced peptide coverage in the C-terminal region is observed in the dimer and trimer samples. These differences in peptide coverage or protections between dimer and trimer indicate specific differences in packing between the two oligomeric forms. Intramolecular cross-links within the monomer suggest that the N-terminus is likely folded into a compact domain, while the C-terminus remains intrinsically disordered. Upon oligomerization, as evidenced by the intermolecular cross-links, the C-terminus of one Vif protein becomes ordered by wrapping back on the N-terminal domain of another. In addition, the majority of the intramolecular cross-links map to regions that have been previously reported to be necessary for viral infectivity. Thus, this data suggests HIV-1 Vif is in a dynamic equilibrium between the various oligomers potentially allowing it to interact with other binding partners

Mass spectrometry tools for analysis of intermolecular interactions

The small quantities of protein required for mass spectrometry (MS) make it a powerful tool to detect binding (protein-protein, protein-small molecule, etc.) of proteins that are difficult to express in large quantities, as is the case for many intrinsically disordered proteins. Chemical cross-linking, proteolysis, and MS analysis, combined, are a powerful tool for the identification of binding domains. Here, we present a traditional approach to determine protein-protein interaction binding sites using heavy water ((18)O) as a label. This technique is relatively inexpensive and can be performed on any mass spectrometer without specialized software

Heavy Sugar and Heavy Water Create Tunable Intact Protein Mass Increases for Quantitative Mass Spectrometry in Any Feed and Organism

Stable isotope labeling techniques for quantitative top-down proteomics face unique challenges. These include unpredictable mass shifts following isotope labeling, which impedes analysis of unknown proteins and complex mixtures and exponentially greater susceptibility to incomplete isotope incorporation, manifesting as broadening of labeled intact protein peaks. Like popular bottom-up isotope labeling techniques, most top-down labeling methods are restricted to defined media/feed as well as amino acid auxotrophic organisms. We present a labeling method optimized for top-down proteomics that overcomes these challenges. We demonstrated this method through the spiking of ¹³C-sugar or ²H-water into standard laboratory feedstocks, resulting in tunable intact protein mass increases (TIPMI). After mixing of labeled and unlabeled samples, direct comparison of light and heavy peaks allowed for the relative quantitation of intact proteins in three popular model organisms, including prokaryotic and eukaryotic microorganisms and an animal. This internal standard method proved to be more accurate than label-free quantitation in our hands. Advantages over top-down SILAC include working equally well in nutrient-rich media, conceivably expanding applicability to any organism and all classes of biomolecules, not requiring high-resolving power MS for quantitation and being relatively inexpensive

A Novel Rolling Circle Amplification-Based Detection of SARS-CoV-2 with Multi-Region Padlock Hybridization

SARS-CoV-2 has remained a global health burden, primarily due to the continuous evolution of different mutant strains. These mutations present challenges to the detection of the virus, as the target genes of qPCR, the standard diagnostic method, may possess sequence alterations. In this study, we develop an isothermal one-step detection method using rolling circle amplification (RCA) for SARS-CoV-2. This novel strategy utilizes a multi-padlock (MP-RCA) approach to detect viral-RNA via a simplified procedure with the reliable detection of mutated strains over other procedures. We designed 40 padlock-based probes to target different sequences across the SARS-CoV-2 genome. We established an optimal one-step isothermal reaction protocol utilizing a fluorescent output detected via a plate reader to test a variety of padlock combinations. This method was tested on RNA samples collected from nasal swabs and validated via PCR. S-gene target failure (SGTF)-mutated strains of SARS-CoV-2 were included. We demonstrated that the sensitivity of our assay was linearly proportional to the number of padlock probes used. With the 40-padlock combination the MP-RCA assay was able to correctly detect 45 out 55 positive samples (81.8% efficiency). This included 10 samples with SGTF mutations which we were able to detect as positive with 100% efficiency. We found that the MP-RCA approach improves the sensitivity of the MP-RCA assay, and critically, allows for the detection of SARS-CoV-2 variants with SGTF. Our method offers the simplicity of the reaction and requires basic equipment compared to standard qPCR. This method provides an alternative approach to overcome the challenges of detecting SARS-CoV-2 and other rapidly mutating viruses