Biology subjects

DeStefano, J. J.

Publications and source records attributed to DeStefano, J. J..

5 recordsLinked to original sources

bioRxiv2021

Cryo-EM Structure of the Pol Polyprotein Provides Insights into HIV Maturation

Many retroviral proteins are initially translated from unspliced full-length RNA as polyprotein precursors that are subsequently processed by the viral protease (PR) to yield the mature forms. In HIV-1, the enzymes, PR, reverse transcriptase (RT), and integrase (IN), are produced as part of the Gag-Pol polyprotein. While structures of the mature proteins have aided our understanding of catalytic mechanisms and the design of antiretroviral drugs, knowledge of the architecture and functional implications of the immature forms prior to PR-mediated cleavage is limited. We developed a system to produce and purify the HIV-1 Pol polyprotein intermediate precursor and determined its high-resolution cryo-EM structure. The RT portion of the polyprotein has an architecture similar to the mature RT p66/p51 heterodimer, and dimerization of the RT portion draws together two PR monomers to activate proteolytic processing. HIV-1 thus may leverage the dimerization interfaces in Pol to regulate the assembly and maturation of the polyprotein precursors.

biophysics↗

bioRxiv2021

Physiological Magnesium Concentrations Increase Fidelity of Diverse Reverse Transcriptases from HIV-1, HIV-2, and Foamy Virus, but not MuLV or AMV

Reverse transcriptases (RTs) are typically assayed in vitro using optimized Mg2+ concentrations ([~]5-10 mM) several-fold higher than physiological cellular free Mg2+ ([~]0.5 mM). Analysis of fidelity using lacZ-based -complementation assays showed that tested HIV RTs, including HIV-1 from subtype B (HXB2-derived), HIV-2, subtype A/E, and several drug-resistant HXB2 derivatives all showed significantly higher fidelity using physiological Mg2+. This also occurred with prototype foamy virus (PFV) RT. In contrast, Moloney murine leukemia virus (MuLV) and avian myeloblastosis virus (AMV) RTs demonstrated equivalent fidelity in both low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated {approx} equal fidelity, except for PFV RT which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to accurately determine mutation rates and profiles was used to examine the types of mutations made by HIV-1 (subtype B, wild type) in low (0.5 mM) and high (6 mM) Mg2+ with DNA or RNA that coded for lacZ. Unlike the -complementation assay, which is dependent on LacZ activity, the NGS assay scores mutations at all positions and of every type. Consistent with -complementation assays, a [~]4-fold increase in mutations was observed in high Mg2+. These findings help explain why HIV RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g., MuLV and AMV), yet cellular fidelity for these viruses is comparable. Establishing in vitro conditions that accurately represent RTs activity in cells is pivotal to determining the contribution of RT and other factors to the mutation profile observed with HIV.

biochemistry↗

bioRxiv2021

Xeno-nucleic Acid (XNA) 2'-Fluoro-Arabino Nucleic Acid (FANA) Aptamers to the Receptor Binding Domain of SARS-CoV-2 S Protein Block ACE2 Binding

The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor binding domain (RBD) on the viral S protein with angiotensin converting enzyme 2 (ACE2) on the surface of human host cells. Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2-fluoroarabinonucleic acid (FANA). The best selected ~ 79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equilibrium dissociation constants (KD,app) of ~ 10-20 nM and a binding half-life for the RBD of 53 {+/-} 18 minutes. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per weight basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.

microbiology↗

bioRxiv2020

HIV reverse transcriptase pre-steady-state kinetic analysis of chain terminators and translocation inhibitors reveals interactions between magnesium and nucleotide 3'-OH

Deoxythymidine triphosphate analogs with various 3' sugar groups (-OH (dTTP), -H, -N3, -NH2, -F, -O-CH3, no group (2',3'-didehydro-2',3'-dideoxythymidine triphosphate (d4TTP)), and those retaining the 3'-OH but with 4' additions (4'-C-methyl, 4'-C-ethyl) or sugar ring modifications (D-carba dTTP) were evaluated using pre-steady-state kinetics in low (0.5 mM) and high (6 mM) Mg2+ with HIV reverse transcriptase (RT). Analogs showed diminished incorporation rates (k) compared to dTTP ranging from about 2-fold (3'-H, -N3, and d4TTP with high Mg2+) to >10-fold (3'-NH2 and 3'-F with low Mg2+), while 3'-O-CH3 dTTP incorporated much slower than other analogs. Illustrating the importance of interactions between Mg2+ and the 3'-OH, k using 5 M dTTP and 0.5 mM Mg2+ was only modestly slower (1.6-fold) than with 6 mM Mg2+, while analogs with 3' alterations incorporated 2.8-5.1-fold slower in 0.5 mM Mg2+. In contrast, 4'-C-methyl and D-carb dTTP, which retain the 3'-OH, were not significantly affected by Mg2+. Consistent with these results, analogs with 3' modifications were better inhibitors in 6 mM vs. 0.5 mM Mg2+. Equilibrium dissociation constant (Kd) and kpol determinations for dTTP and analogs lacking a 3'-OH indicated that low Mg2+ caused a several-fold greater reduction in kpol with the analogs but had little effect on Kd, results consistent with a role for 3'-OH/Mg2+ interactions in catalysis rather than nucleotide binding. Overall, results emphasize the importance previously unreported interactions between Mg2+ and the 3'-OH of the incoming nucleotide and suggest inhibitors with 3'-OH groups may have advantages in the low free Mg2+ in physiological settings.

biochemistry↗

bioRxiv2020

Crystal structure of prototype foamy virus (PFV) protease-reverse transcriptase fusion (PR-RT) reveals conformational plasticity: implications for function

In most cases, proteolytic processing of the retroviral Pol portion of the Gag-Pol polyprotein precursor produces protease (PR), reverse transcriptase (RT), and integrase (IN). However, foamy viruses (FVs), express Pol separately from Gag and, when Pol is processed, only the IN domain is released. Here, we report a 2.9 [A] resolution crystal structure of the mature PR-RT from prototype FV (PFV) that carries out both proteolytic processing and reverse transcription. PFV PR-RT is monomeric and the architecture of PFV PR is similar to one of the subunits of HIV-1 PR, which is a dimer. There is a C-terminal extension of PFV PR (101-145) that consists of two helices which are adjacent to the base of the RT palm subdomain, and anchors PR to RT. The polymerase domain of PFV RT consists of fingers, palm, thumb, and connection subdomains whose spatial arrangements are similar to the p51 subunit of HIV-1 RT. The RNase H and polymerase domains of PFV RT are connected by flexible linkers. Significant spatial and conformational (sub)domain rearrangements are therefore required for nucleic acid binding. The structure of PFV PR-RT provides insights into the conformational maturation of retroviral Pol polyproteins. IMPORTANCERetroviruses such as HIV and other human and animal pathogens have evolved to maximize the protein-coding capacity of their genomes. Retroviruses synthesize large polyprotein precursors that are subsequently cleaved to produce mature functional enzymes and structural proteins from the same mRNA template. These polyproteins play an important role in the viral life cycle. We report the monomeric structure of the prototype foamy virus protease-reverse transcriptase fusion (PR-RT) which serves as both a protease and reverse transcriptase. The monomeric PR domain is folded in a similar way as a single subunit of dimeric HIV-1 PR. PR is anchored close to the fingers subdomain of the RT by a long C-terminal extension (CTE) that folds into two helices that pack against the palm subdomain of the RT. The RT is folded similarly to the p51 subunit of HIV-1 RT with the RNase H subdomain packed against the thumb and opposite the connection and palm subdomains. This structure offers insight into the structural arrangement of the domains and subdomains of immature retroviral polyproteins and provides support for the concept of developing drugs that target interfaces between immature enzymes that are present only in retroviral polyprotein precursors.

biophysics↗