Browsing by Author "Summers, Michael F."
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Item Assigning NMR spectra of RNA, peptides and small organic molecules using molecular network visualization software(Springer Netherlands, 2019-07-19) Marchant, Jan; Summers, Michael F.; Johnson, Bruce A.NMR assignment typically involves analysis of peaks across multiple NMR spectra. Chemical shifts of peaks are measured before being assigned to atoms using a variety of methods. These approaches quickly become complicated by overlap, ambiguity, and the complexity of correlating assignments among multiple spectra. Here we propose an alternative approach in which a network of linked peak-boxes is generated at the predicted positions of peaks across all spectra. These peak-boxes correlate known relationships and can be matched to the observed spectra. The method is illustrated with RNA, but a variety of molecular types should be readily tractable with this approach.Item Clonally expanded HIV-1 proviruses with 5’-Leader defects can give rise to nonsuppressible residual viremia(American Society for Clinical Investigation, 2023-01-05) White, Jennifer A.; Wu, Fengting; Yasin, Saif; Moskovljevic, Milica; Zheng, Mei Y.; Tadzong, Ndeh F.; Patel, Heer B.; Quiambao, Jeanelle Mae C.; Summers, Michael F.; et alBACKGROUND. Antiretroviral therapy (ART) halts HIV-1 replication, decreasing viremia to below the detection limit of clinical assays. However, some individuals experience persistent nonsuppressible viremia (NSV) originating from CD4⁺ T cell clones carrying infectious proviruses. Defective proviruses represent over 90% of all proviruses persisting during ART and can express viral genes, but whether they can cause NSV and complicate ART management is unknown. METHODS. We carried an in-depth characterization of proviruses causing NSV in 4 study participants with optimal adherence and no drug resistance. We investigated the impact of the observed defects on 5’-Leader RNA properties, virus infectivity, and gene expression. Integration-site specific assays were used to track these proviruses over time and among cell subsets. RESULTS. Clones carrying proviruses with 5’-Leader defects can cause persistent NSV up to ~10³ copies/mL. These proviruses had small, often identical deletions or point mutations involving the major splicing donor site (MSD) and showed partially reduced RNA dimerization and nucleocapsid binding. Nevertheless, they were inducible and produced non-infectious virions containing viral RNA but lacking Envelope. CONCLUSION. These findings show that proviruses with 5’-Leader defects in CD4⁺ T cell clones can give rise to NSV, affecting clinical care. Sequencing of the 5’-Leader can help understanding failure to completely suppress viremia. FUNDING. Office of the NIH Director and National Institute of Dental & Craniofacial Research, NIH; Howard Hughes Medical Institute; Johns Hopkins University Center for AIDS Research; National Institute for Allergy and Infectious Diseases, NIH, to the PAVE, BEAT-HIV and DARE Martin Delaney collaboratories.Item Identification of the initial nucleocapsid recognition element in the HIV-1 RNA packaging signal(Proceedings of the National Academy of Sciences, 2020-07-09) Ding, Pengfei; Kharytonchyk, Siarhei; Waller, Alexis; Mbaekwe, Ugonna; Basappa, Sapna; Kuo, Nansen; Frank, Heather M.; Quasney, Christina; Kidane, Aaron; Swanson, Canessa; Van, Verna; Sarkar, Mitali; Cannistraci, Emily; Chaudhary, Ridhi; Flores, Hana; Telesnitsky, Alice; Summers, Michael F.Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5ʹ-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. Here, we show that the dimeric 5′-leader contains more than two dozen NC binding sites with affinities ranging from 40 nM to 1.4 μM, and that all high-affinity sites (Kd ≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, ΨCES). The four initial binding sites with highest affinity reside near two symmetrically equivalent three-way junction structures. Unlike the other high-affinity sites, which bind NC with exothermic energetics, binding to these sites occurs endothermically due to concomitant unwinding of a weakly base-paired [UUUU]:[GGAG] helical element. Mutations that stabilize base pairing within this element eliminate NC binding to this site and severely impair RNA packaging into virus-like particles. NMR studies reveal that a recently discovered small-molecule inhibitor of HIV-1 RNA packaging that appears to function by stabilizing the structure of the leader binds directly to the [UUUU]:[GGAG] helix. Our findings suggest a sequential NC binding mechanism for Gag-genome assembly and identify a potential RNA Achilles’ heel to which HIV therapeutics may be targeted.Item NMR Studies of Retroviral Genome Packaging(MDPI, 2020-09-30) Boyd, Patricia S.; Brown, Janae B.; Brown, Joshua D.; Catazaro, Jonathan; Chaudry, Issac; Ding, Pengfei; Dong, Xinmei; Marchant, Jan; O’Hern, Colin T.; Singh, Karndeep; Swanson, Canessa; Summers, Michael F.; Yasin, SaifNearly all retroviruses selectively package two copies of their unspliced RNA genomes from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Over the past four decades, combinations of genetic experiments, phylogenetic analyses, nucleotide accessibility mapping, in silico RNA structure predictions, and biophysical experiments were employed to understand how retroviral genomes are selected for packaging. Genetic studies provided early clues regarding the protein and RNA elements required for packaging, and nucleotide accessibility mapping experiments provided insights into the secondary structures of functionally important elements in the genome. Three-dimensional structural determinants of packaging were primarily derived by nuclear magnetic resonance (NMR) spectroscopy. A key advantage of NMR, relative to other methods for determining biomolecular structure (such as X-ray crystallography), is that it is well suited for studies of conformationally dynamic and heterogeneous systems—a hallmark of the retrovirus packaging machinery. Here, we review advances in understanding of the structures, dynamics, and interactions of the proteins and RNA elements involved in retroviral genome selection and packaging that are facilitated by NMR.Item Replicating Meyerhoff for inclusive excellence in STEM(American Association for the Advancement of Science, 2019-04-26) Sto. Domingo, Mariano R.; Sharp, Starlette; Freeman, Amy; Freeman Jr., Thomas; Freeman, Thomas, Jr.; Harmon, Keith; Wiggs, Mitsue; Sathy, Viji; Panter, Abigail T.; Oseguera, Leticia; Sun, Shuyan; Williams, Mary Elizabeth; Templeton, Joseph; Folt, Carol L.; Barron, Eric J.; Hrabowski III, Freeman A.; Maton, Kenneth I.; Crimmins, Michael; Fisher, Charles R.; Summers, Michael F.