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At least 199 records · Page 11

Quantum Chemical Computation of Omicron Mutations Near Cleavage Sites of the Spike Protein

The attachment of the spike protein in SARS-CoV-2 to host cells and the initiation of viral invasion are two critical processes in the viral infection and transmission in which the presence of unique furin (S1/S2) and TMPRSS2 (S2') cleavage sites play a pivotal role. We provide a detailed analysis of the impact of the BA.1 Omicron mutations vicinal to these cleavage sites using a novel computational method based on the amino acid–amino acid bond pair unit (AABPU), a specific protein structural unit as a proxy for quantifying the atomic interaction. Our study is focused mainly on the spike region between subdomain 2 (SD2) and the central helix (CH), which contains both S1/S2 and S2' cleavage sites. Based on ab initio quantum calculations, we have identified several key features related to the electronic structure and bonding of the Omicron mutations that significantly increase the size of the relevant AABPUs and the positive charge. These findings enable us to conjecture on the biological role of Omicron mutations and their specific effects on cleavage sites and identify the principles that can be of some value in analyzing new variants.

60 APPLIED LIFE SCIENCES↗

Identification of mobile genetic elements with geNomad

Identifying and characterizing mobile genetic elements in sequencing data is essential for understanding their diversity, ecology, biotechnological applications and impact on public health. Here we introduce geNomad, a classification and annotation framework that combines information from gene content and a deep neural network to identify sequences of plasmids and viruses. geNomad uses a dataset of more than 200,000 marker protein profiles to provide functional gene annotation and taxonomic assignment of viral genomes. Using a conditional random field model, geNomad also detects proviruses integrated into host genomes with high precision. In benchmarks, geNomad achieved high classification performance for diverse plasmids and viruses (Matthews correlation coefficient of 77.8% and 95.3%, respectively), substantially outperforming other tools. Leveraging geNomad’s speed and scalability, we processed over 2.7 trillion base pairs of sequencing data, leading to the discovery of millions of viruses and plasmids that are available through the IMG/VR and IMG/PR databases. geNomad is available at https://portal.nersc.gov/genomad.

59 BASIC BIOLOGICAL SCIENCES↗

Atypical Divergence of SARS-CoV-2 Orf8 from Orf7a within the Coronavirus Lineage Suggests Potential Stealthy Viral Strategies in Immune Evasion

Orf8, one of the most puzzling genes in the SARS lineage of coronaviruses, marks a unique and striking difference in genome organization between SARS-CoV-2 and SARS-CoV-1. Here, using sequence comparisons, we unequivocally reveal the distant sequence similarities between SARS-CoV-2 Orf8 with its SARS-CoV-1 counterparts and the X4-like genes of coronaviruses, including its highly divergent “paralog” gene Orf7a, whose product is a potential immune antagonist of known structure. Supervised sequence space walks unravel identity levels that drop below 10% and yet exhibit subtle conservation patterns in this novel superfamily, characterized by an immunoglobulin-like beta sandwich topology. We document the high accuracy of the sequence space walk process in detail and characterize the subgroups of the superfamily in sequence space by systematic annotation of gene and taxon groups. While SARS-CoV-1 Orf7a and Orf8 genes are most similar to bat virus sequences, their SARS-CoV-2 counterparts are closer to pangolin virus homologs, reflecting the fine structure of conservation patterns within the SARS-CoV-2 genomes. The divergence between Orf7a and Orf8 is exceptionally idiosyncratic, since Orf7a is more constrained, whereas Orf8 is subject to rampant change, a peculiar feature that may be related to hitherto-unknown viral infection strategies. Despite their common origin, the Orf7a and Orf8 protein families exhibit different modes of evolutionary trajectories within the coronavirus lineage, which might be partly attributable to their complex interactions with the mammalian host cell, reflected by a multitude of functional associations of Orf8 in SARS-CoV-2 compared to a very small number of interactions discovered for Orf7a.

60 APPLIED LIFE SCIENCES↗

AZD5582 plus SIV-specific antibodies reduce lymph node viral reservoirs in antiretroviral therapy-suppressed macaques

The main barrier to HIV cure is a persistent reservoir of latently infected CD4 + T cells harboring replication-competent provirus that fuels rebound viremia upon antiretroviral therapy (ART) interruption. A leading approach to target this reservoir involves agents that reactivate latent HIV proviruses followed by direct clearance of cells expressing induced viral antigens by immune effector cells and immunotherapeutics. We previously showed that AZD5582, an antagonist of inhibitor of apoptosis proteins and mimetic of the second mitochondrial-derived activator of caspases (IAPi/SMACm), induces systemic reversal of HIV/SIV latency but with no reduction in size of the viral reservoir. In this study, we investigated the effects of AZD5582 in combination with four SIV Env-specific Rhesus monoclonal antibodies (RhmAbs) ± N-803 (an IL-15 superagonist) in SIV-infected, ART-suppressed rhesus macaques. Here we confirm the efficacy of AZD5582 in inducing SIV reactivation, demonstrate enhancement of latency reversal when AZD5582 is used in combination with N-803 and show a reduction in total and replication-competent SIV-DNA in lymph-node-derived CD4 + T cells in macaques treated with AZD5582 + RhmAbs. Further exploration of this therapeutic approach may contribute to the goal of achieving an HIV cure.

60 APPLIED LIFE SCIENCES↗

Multidisciplinary studies with mutated HIV-1 capsid proteins reveal structural mechanisms of lattice stabilization

Abstract HIV-1 capsid (CA) stability is important for viral replication. E45A and P38A mutations enhance and reduce core stability, thus impairing infectivity. Second-site mutations R132T and T216I rescue infectivity. Capsid lattice stability was studied by solving seven crystal structures (in native background), including P38A, P38A/T216I, E45A, E45A/R132T CA, using molecular dynamics simulations of lattices, cryo-electron microscopy of assemblies, time-resolved imaging of uncoating, biophysical and biochemical characterization of assembly and stability. We report pronounced and subtle, short- and long-range rearrangements: (1) A38 destabilized hexamers by loosening interactions between flanking CA protomers in P38A but not P38A/T216I structures. (2) Two E45A structures showed unexpected stabilizing CA NTD -CA NTD inter-hexamer interactions, variable R18-ring pore sizes, and flipped N-terminal β-hairpin. (3) Altered conformations of E45A a α9-helices compared to WT, E45A/R132T, WT PF74 , WT Nup153 , and WT CPSF6 decreased PF74, CPSF6, and Nup153 binding, and was reversed in E45A/R132T. (4) An environmentally sensitive electrostatic repulsion between E45 and D51 affected lattice stability, flexibility, ion and water permeabilities, electrostatics, and recognition of host factors.

60 APPLIED LIFE SCIENCES↗

Cooperative multivalent receptor binding promotes exposure of the SARS-CoV-2 fusion machinery core

The molecular events that permit the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to bind and enter cells are important to understand for both fundamental and therapeutic reasons. Spike proteins consist of S1 and S2 domains, which recognize angiotensin-converting enzyme 2 (ACE2) receptors and contain the viral fusion machinery, respectively. Ostensibly, the binding of spike trimers to ACE2 receptors promotes dissociation of the S1 domains and exposure of the fusion machinery, although the molecular details of this process have yet to be observed. We report the development of bottom-up coarse-grained (CG) models consistent with cryo-electron tomography data, and the use of CG molecular dynamics simulations to investigate viral binding and S2 core exposure. We show that spike trimers cooperatively bind to multiple ACE2 dimers at virion-cell interfaces in a manner distinct from binding between soluble proteins, which processively induces S1 dissociation. We also simulate possible variant behavior using perturbed CG models, and find that ACE2-induced S1 dissociation is primarily sensitive to conformational state populations and the extent of S1/S2 cleavage, rather than ACE2 binding affinity. These simulations reveal an important concerted interaction between spike trimers and ACE2 dimers that primes the virus for membrane fusion and entry.

59 BASIC BIOLOGICAL SCIENCES↗

Minnesota peat viromes reveal terrestrial and aquatic niche partitioning for local and global viral populations

Abstract Background Peatlands are expected to experience sustained yet fluctuating higher temperatures due to climate change, leading to increased microbial activity and greenhouse gas emissions. Despite mounting evidence for viral contributions to these processes in peatlands underlain with permafrost, little is known about viruses in other peatlands. More generally, soil viral biogeography and its potential drivers are poorly understood at both local and global scales. Here, 87 metagenomes and five viral size-fraction metagenomes (viromes) from a boreal peatland in northern Minnesota (the SPRUCE whole-ecosystem warming experiment and surrounding bog) were analyzed for dsDNA viral community ecological patterns, and the recovered viral populations (vOTUs) were compared with our curated PIGEON database of 266,125 vOTUs from diverse ecosystems. Results Within the SPRUCE experiment, viral community composition was significantly correlated with peat depth, water content, and carbon chemistry, including CH 4 and CO 2 concentrations, but not with temperature during the first 2 years of warming treatments. Peat vOTUs with aquatic-like signatures (shared predicted protein content with marine and/or freshwater vOTUs) were significantly enriched in more waterlogged surface peat depths. Predicted host ranges for SPRUCE vOTUs were relatively narrow, generally within a single bacterial genus. Of the 4326 SPRUCE vOTUs, 164 were previously detected in other soils, mostly peatlands. None of the previously identified 202,371 marine and freshwater vOTUs in our PIGEON database were detected in SPRUCE peat, but 0.4% of 80,714 viral clusters (VCs, grouped by predicted protein content) were shared between soil and aquatic environments. On a per-sample basis, vOTU recovery was 32 times higher from viromes compared with total metagenomes. Conclusions Results suggest strong viral “species” boundaries between terrestrial and aquatic ecosystems and to some extent between peat and other soils, with differences less pronounced at higher taxonomic levels. The significant enrichment of aquatic-like vOTUs in more waterlogged peat suggests that viruses may also exhibit niche partitioning on more local scales. These patterns are presumably driven in part by host ecology, consistent with the predicted narrow host ranges. Although more samples and increased sequencing depth improved vOTU recovery from total metagenomes, the substantially higher per-sample vOTU recovery after viral particle enrichment highlights the utility of soil viromics.

59 BASIC BIOLOGICAL SCIENCES↗

Defining Lipidomic Responses to Coronavirus Infection

Highly pathogenic human coronavirus infection can cause a severe atypical, rapid onset pneumonia with a mortality rate of up to 10% for severe acute respiratory syndrome coronavirus 1 (SARS-CoV 1), 35% for Middle East respiratory syndrome coronavirus (MERS-CoV), or 1% for severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2 causative agent of COVID 19). While medical countermeasures successfully controlled the worldwide SARS-CoV epidemic, the MERS-CoV epidemic is still ongoing and continues to be a concern for travelers in the Middle East and the multi-year SARS-CoV 2 pandemic underscore the importance of defining biomarkers that are diagnostic and/or predictive of severe disease outcomes for respiratory viruses. Systems biology approaches provide global snapshots of infection induced changes in host cells/tissues and provide extremely rich datasets for understanding host pathogen interactions. Metabolites, especially lipids, are critical for viral replication but less is understood about infection induced changes to lipids due to limits in lipid species detection and identification. To characterize how individual lipid species and proteins with lipid associated functions contribute to highly pathogenic human coronavirus replication and disease severity, existing datasets were probed to determine cell type specific lipid responses to MERS-CoV infection and verification studies were performed to determine if modification of the host lipid signature (by inhibiting enzymatic functions that produce specific lipid species) can perturb CoV replication in human lung cells. MERS-CoV infects human lung epithelial, endothelial and fibroblast cells. All three cell types were infected with MERS-CoV and samples collected to analyze lipids, proteins, metabolites, and transcripts from 12 to 48 hours post infection. Time matched mock-infected cells were collected in parallel for each cell type. Following sample and statistical analysis, functional enrichment and bioinformatic analysis was performed to identify differentially expressed lipids and proteins. Two lipid species were found to be significantly upregulated following MERS-CoV infection, ceramides, and triacylglycerol both of which are indicative of cells undergoing apoptotic cell death. In contrast, sphingomyelins (lipid molecules that can serve as a precursor for one pathway for ceramide synthesis) had significantly decreased expression in MERS-CoV infected cells. An inhibitor of acid sphingomyelinase (that converts sphingomyelin to ceramides and phosphorylcholine) reduced MERS-CoV replication suggesting that production of ceramides is key for successful viral replication and transmission. Acyl-CoA-synthetase 3 (ACSL3), the only protein whose function is lipid associated and had increased differential expression in our dataset, regulates the synthesis of triacylglycerol (increased expression). Inhibitors that directly block ACSL3 (Triacsin C) but not steps later in the triacylglycerol synthesis pathway (Etomoxir) inhibit MERS-CoV replication suggesting that triacylglycerol production and/or ACSL3 activity is also key for viral replication. ACSL3 expression is also upregulated in lung cancer cells and is predicted to promote continued cell viability which would also enhance viral replication. The differentially expressed lipid and lipid-associated protein species identified in our studies suggest that MERS-CoV infection is activating cellular death pathways to limit the number of cells that become infected but also stimulating the production of lipid-associated enzymes that can prolong host cell viability and the amount of time progeny virions can be produced and released. As the inhibitors that worked against MERS-CoV infection were also efficacious against SARS-CoV 2 infection, countermeasures that target these host pathways may provide novel ways to block highly pathogenic human coronavirus infection and/or prevent severe disease outcomes.

59 BASIC BIOLOGICAL SCIENCES↗

Ribavirin targets sugar transporter 6 to suppress acquisition and transmission of rice stripe tenuivirus by its vector Laodelphax striatellus

Abstract BACKGROUND Rice stripe tenuivirus (RSV) is one of the most destructive pathogens of rice and other cereal crops. The virus is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus ) in a circulative‐propagative manner. Thus, blocking transmission by the insect vector would provide an effective strategy to prevent epidemic outbreaks of the disease. RESULTS In this study, we explored the effect of ribavirin on acquisition and transmission of the virus by specifically inhibiting the expression of sugar transporter 6 (LsSt‐6), which was recently reported as a key vector component for RSV transmission. Ribavirin at the highest concentration tested (250 μmol L ‐1 ) significantly reduced RSV acquisition and transmission efficiency by SBPHs through inhibiting LsSt‐6 messenger RNA (mRNA) level. Survival of the model insect Spodoptera frugiperda cell line (Sf9) was 95.0 ± 2.2 and 85.6 ± 2.1% after exposure to 250 μmol L ‐1 ribavirin or 8‐azaguanine, respectively. Further study confirmed that 250 μmol L ‐1 ribavirin also significantly reduced LsSt‐6 mRNA and protein levels in Sf9 cells. However, 8‐azaguanine did not significantly inhibit viral infectivity and LsSt‐6 mRNA levels in SBPH or the Sf9 cell line. CONCLUSION This result provides evidence that ribavirin has the potential to disrupt LsSt‐6 expression but not others like viral RNAs to prevent acquiring RSV, which leads to less viral accumulation in SBPH tissues and thereby lower transmission efficiency. © 2020 Society of Chemical Industry

Hajano, Jamal‐U‐Ddin↗

Structural basis of RNA conformational switching in the transcriptional regulator 7SK RNP

7SK non-coding RNA (7SK) negatively regulates RNA polymerase II (RNA Pol II) elongation by inhibiting positive transcription elongation factor b (P-TEFb), and its ribonucleoprotein complex (RNP) is hijacked by HIV-1 for viral transcription and replication. Methylphosphate capping enzyme (MePCE) and La-related protein 7 (Larp7) constitutively associate with 7SK to form a core RNP, while P-TEFb and other proteins dynamically assemble to form different complexes. Here, we present the cryo-EM structures of 7SK core RNP formed with two 7SK conformations, circular and linear, and uncover a common RNA-dependent MePCE-Larp7 complex. Further, together with NMR, biochemical, and cellular data, these structures reveal the mechanism of MePCE catalytic inactivation in the core RNP, unexpected interactions between Larp7 and RNA that facilitate a role as an RNP chaperone, and that MePCE-7SK-Larp7 core RNP serves as a scaffold for switching between different 7SK conformations essential for RNP assembly and regulation of P-TEFb sequestration and release.

59 BASIC BIOLOGICAL SCIENCES↗

Ticking off Lyme disease: OspA mRNA vaccine halts infection in mouse model

Lyme disease, a condition caused by Borrelia burgdorferi sensu lato and transmitted to humans via ticks, affects approximately 676,000 individuals annually in the United States and Western Europe.1 Currently, there is no approved Lyme vaccine available for human use. A promising study by Tahir et al., published in Molecular Therapy Nucleic Acids, investigated the efficacy of mRNA and subunit vaccines targeting Lyme disease.2 This research demonstrated complete protection from infection in a tick-fed mouse model using an outer surface protein A (OspA) mRNA vaccine. Although mRNA vaccines have shown success against viral pathogens, their clinical application against bacterial diseases has been limited.3 Thus, this study represents an important step toward developing an effective mRNA vaccine against Lyme disease.

He, Wei↗

Structural basis of RNA cap modification by SARS-CoV-2

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused millions of infections worldwide. In SARS coronaviruses, the non-structural protein 16 (nsp16), in conjunction with nsp10, methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of SARS-CoV-2 nsp16 and nsp10 in the presence of cognate RNA substrate analogue and methyl donor, S-adenosyl methionine (SAM). The nsp16/nsp10 heterodimer is captured in the act of 2'-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We observe large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This induced fit model provides mechanistic insights into the 2'-O methylation of the viral mRNA cap. We also discover a distant (25 Å) ligand-binding site unique to SARS-CoV-2, which can alternatively be targeted, in addition to RNA cap and SAM pockets, for antiviral development.

59 BASIC BIOLOGICAL SCIENCES↗

Structural basis of Gabija anti-phage defence and viral immune evasion

Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation. Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA–GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion.

59 BASIC BIOLOGICAL SCIENCES↗

Integrative mapping reveals molecular features underlying the mechanism of nucleocytoplasmic transport

Nuclear pore complexes (NPCs) enable rapid, selective, and robust nucleocytoplasmic transport. To explain how transport emerges from the system components and their interactions, we used experimental data and theoretical information to construct an integrative Brownian dynamics model of transport through an NPC, coupled to a kinetic model of transport in the cell. The model recapitulates key aspects of transport for a wide range of molecular cargoes, including preribosomes and viral capsids. Our model quantifies how flexible phenylalanine-glycine (FG) repeat proteins create an entropic barrier to passive diffusion and how this barrier is selectively lowered in facilitated diffusion by the many transient interactions of nuclear transport receptors with the FG repeats. Selective transport is enhanced by “fuzzy” multivalent interactions, redundant FG repeat mass, coupling to the energy-dependent RanGTP concentration gradient, and exponential dependence of transport kinetics on the transport barrier. Our model will facilitate rational modulation of the NPC and its artificial mimics.

Raveh, Barak [School of Computer Science and Engin↗

Human Host Cellular Response to HCoV-229E Infection Proteomics (ACS-JM-DP2)

The purpose of this experiment was to evaluate the human host cellular response to wild-type Human coronavirus strain 229E (HCoV-229E) infection. Sample data was obtained for mock and infected immortalized human lung epithelial cells (A549) (MOI 5) nuclear extracts, immortalized human lung fibroblasts cells (MRC5) (MOI5) nuclear extracts, and primary human airway epithelial (HAE) (MOI 3) cells from lung tissue and processed for proteome analysis. Processed datasets are openly accessible from the download button and contain secondary processed proteomic results files and supporting metadata materials. Experimental proteomics samples were prepared using Limited Proteolysis (LiP) methods for Label-free quantification (LFQ) and global proteomic evaluation. Sample data was acquired using a Q-Exactive HF-X mass spectrometer and was processed and compiled using MaxQuant software (v.1.6.17.0). Processed proteomic data downloads include a sample naming key, processed MaxQuant results/parameters, and protein annotated relative abundance files. See corresponding primary data accessions below and Viral Experiment LiP Analysis source code supporting data transparency and reuse. Experimental transcriptomics samples were collected in parallel and processed for RNA sequencing (RNA-Seq) as summarized under ACS-DP1 (https://data.pnnl.gov/group/nodes/dataset/34069).

59 BASIC BIOLOGICAL SCIENCES↗

Enhanced Binding of SARS-CoV-2 Spike Protein to Receptor by Distal Polybasic Cleavage Sites

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein plays a crucial role in binding the human cell receptor ACE2 that is required for viral entry. Many studies have been conducted to target the structures of RBD–ACE2 binding and to design RBD-targeting vaccines and drugs. Nevertheless, mutations distal from the SARS-CoV-2 RBD also impact its transmissibility and antibody can target non-RBD regions, suggesting the incomplete role of the RBD region in the spike protein–ACE2 binding. Here, in order to elucidate distant binding mechanisms, we analyze complexes of ACE2 with the wild-type spike protein and with key mutants via large-scale all-atom explicit solvent molecular dynamics simulations. We find that though distributed approximately 10 nm away from the RBD, the SARS-CoV-2 polybasic cleavage sites enhance, via electrostatic interactions and hydration, the RBD–ACE2 binding affinity. A negatively charged tetrapeptide (GluGluLeuGlu) is then designed to neutralize the positively charged arginine on the polybasic cleavage sites. We find that the tetrapeptide GluGluLeuGlu binds to one of the three polybasic cleavage sites of the SARS-CoV-2 spike protein lessening by 34% the RBD–ACE2 binding strength. This significant binding energy reduction demonstrates the feasibility to neutralize RBD–ACE2 binding by targeting this specific polybasic cleavage site. Our work enhances understanding of the binding mechanism of SARS-CoV-2 to ACE2, which may aid the design of therapeutics for COVID-19 infection.

60 APPLIED LIFE SCIENCES↗

Structure of saguaro cactus virus 3′ translational enhancer mimics 5′ cap for eIF4E binding

The genomes of several plant viruses contain RNA structures at their 3' ends called cap-independent translation enhancers (CITEs) that bind the host protein factors such as mRNA 5' cap-binding protein eIF4E for promoting cap-independent genome translation. However, the structural basis of such 5' cap-binding protein recognition by the uncapped RNA remains largely unknown. Here, we have determined the crystal structure of a 3' CITE, panicum mosaic virus-like translation enhancer (PTE) from the saguaro cactus virus (SCV), using a Fab crystallization chaperone. The PTE RNA folds into a three-way junction architecture with a pseudoknot between the purine-rich R domain and pyrimidine-rich Y domain, which organizes the overall structure to protrude out a specific guanine nucleotide, G18, from the R domain that comprises a major interaction site for the eIF4E binding. The superimposable crystal structures of the wild-type, G18A, G18C, and G18U mutants suggest that the PTE scaffold is preorganized with the flipped-out G18 ready to dock into the eIF4E 5' cap-binding pocket. The binding studies with wheat and human eIF4Es using gel electrophoresis and isothermal titration calorimetry, and molecular docking computation for the PTE–eIF4E complex demonstrated that the PTE structure essentially mimics the mRNA 5' cap for eIF4E binding. Such 5' cap mimicry by the uncapped and structured viral RNA highlights how viruses can exploit RNA structures to mimic the host protein-binding partners and bypass the canonical mechanisms for their genome translation, providing opportunities for a better understanding of virus-host interactions and non-canonical translation mechanisms found in many pathogenic RNA viruses.

3' cap-independent translation enhancers↗

CD8 Lymphocyte Depletion Enhances the Latency Reversal Activity of the SMAC Mimetic AZD5582 in ART-Suppressed Simian Immunodeficiency Virus-Infected Rhesus Macaques

ABSTRACT Inducing latency reversal to reveal infected cells to the host immune system represents a potential strategy to cure human immunodeficiency virus (HIV) infection. In separate studies, we have shown previously that CD8 + T cells may contribute to the maintenance of viral latency and have identified a novel SMAC (second mitochondrial activator of caspases) mimetic/IAP (inhibitor of apoptosis protein) inhibitor (AZD5582) capable of reversing HIV/simian immunodeficiency virus (SIV) latency in vivo by activating the noncanonical (nc) NF-κB pathway. Here, we use AZD5582 in combination with antibody (Ab)-mediated depletion of CD8α + cells to further evaluate the role of CD8 + T cells in the maintenance of viral latency. Six rhesus macaques (RMs) were infected with SIV mac239 and treated with antiretroviral therapy (ART) starting at week 8 postinfection. After 84 to 85 weeks of ART, all animals received a single dose of the anti-CD8α depleting Ab MT807R1 (50 mg/kg of body weight, administered subcutaneously [s.c.]), followed by five weekly doses of AZD5582 (0.1 mg/kg, administered intravenously [i.v.]). Following combined treatment with CD8α depletion and AZD5582, 100% of RMs experienced on-ART viremia levels above 60 copies per ml of plasma. In comparator groups of SIV-infected, ART-suppressed RMs treated with AZD5582 only or CD8α depletion only, on-ART viremia was experienced by 56% and 57% of the animals, respectively. Furthermore, the increased frequency of viremic episodes during the treatment period was greater in the group treated with CD8α depletion plus AZD5582 than in the other groups. Mathematical modeling of virus reactivation suggested that in addition to viral dynamics during acute infection, CD8α depletion influenced the response to AZD5582. This work suggests that the latency reversal induced by activation of the ncNF-κB signaling pathway with AZD5582 can be enhanced by CD8α + cell depletion. IMPORTANCE A favored approach to curing HIV infection aims at inducing viral expression using latency-reversing agents (LRAs) to allow the elimination of infected cells. Here, we tested a combination of two recently identified LRAs, the SMAC mimetic/IAP inhibitor AZD5582, which activates the noncanonical NF-κB pathway, and the antibody (Ab) MT807R1, which depletes CD8α + cells, in SIV-infected rhesus macaques (RMs) on ART. Latency reversal, as defined by increased on-ART viremia, was observed in all six SIV-infected, ART-treated RMs that received this combined treatment. Furthermore, comparison of viral reactivation between these animals and groups of SIV-infected, ART-treated RMs treated with AZD5582 only or CD8α + cell depletion only showed more frequent increases in viremic episodes when the two treatments were combined. This study provides additional evidence that CD8 + T cells may contribute to the maintenance of HIV/SIV latency on ART and potentially inhibit latency reversal during HIV cure approaches.

59 BASIC BIOLOGICAL SCIENCES↗