Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog β-d-N4-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (β-d-N4-hydroxycytidine-5′-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.

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Mutagenic ribonucleosides can act as broad-based antiviral agents. They are metabolized to the active ribonucleoside triphosphate form and concentrate in the genomes of RNA viruses during viral replication. β-D-N 4-hydroxycytidine (NHC, the initial metabolite of molnupiravir) is more than 100-fold more active than ribavirin or favipiravir against SARS-CoV-2, with antiviral activity correlated to the level of mutagenesis in virion RNA. However, NHC also displays host mutational activity in an animal cell culture assay, consistent with RNA and DNA precursors sharing a common intermediate of a ribonucleoside diphosphate. These results indicate that highly active mutagenic ribonucleosides may hold risk for the host.

Although antiretroviral therapy (ART) is highly effective at suppressing HIV-1 replication, the virus persists as a latent reservoir in resting CD4+ T cells during therapy. This reservoir forms even when ART is initiated early after infection, but the dynamics of its formation are largely unknown. The viral reservoirs of individuals who initiate ART during chronic infection are generally larger and genetically more diverse than those of individuals who initiate therapy during acute infection, consistent with the hypothesis that the reservoir is formed continuously throughout untreated infection. To determine when viruses enter the latent reservoir, we compared sequences of replication-competent viruses from resting peripheral CD4+ T cells from nine HIV-positive women on therapy to viral sequences circulating in blood collected longitudinally before therapy. We found that, on average, 71% of the unique viruses induced from the post-therapy latent reservoir were most genetically similar to viruses replicating just before ART initiation. This proportion is far greater than would be expected if the reservoir formed continuously and was always long lived. We conclude that ART alters the host environment in a way that allows the formation or stabilization of most of the long-lived latent HIV-1 reservoir, which points to new strategies targeted at limiting the formation of the reservoir around the time of therapy initiation.

Validating the sampling depth and reducing sequencing errors are critical for studies of viral populations using next-generation sequencing (NGS). We previously described the use of Primer ID to tag each viral RNA template with a block of degenerate nucleotides in the cDNA primer. We now show that low-abundance Primer IDs (offspring Primer IDs) are generated due to PCR/ sequencing errors. These artifactual Primer IDs can be removed using a cutoff model for the number of reads required to make a template consensus sequence. We have modeled the fraction of sequences lost due to Primer ID resampling. For a typical sequencing run, less than 10% of the raw reads are lost to offspring Primer ID filtering and resampling. The remaining raw reads are used to correct for PCR resampling and sequencing errors. We also demonstrate that Primer ID reveals bias intrinsic to PCR, especially at low template input or utilization. cDNA synthesis and PCR convert ca. 20% of RNA templates into recoverable sequences, and 30-fold sequence coverage recovers most of these template sequences. We have directly measured the residual error rate to be around 1 in 10,000 nucleotides. We use this error rate and the Poisson distribution to define the cutoff to identify preexisting drug resistance mutations at low abundance in an HIV-infected subject. Collectively, these studies show that >90% of the raw sequence reads can be used to validate template sampling depth and to dramatically reduce the error rate in assessing a genetically diverse viral population using NGS. IMPORTANCEAlthough next-generation sequencing (NGS) has revolutionized sequencing strategies, it suffers from serious limitations in defining sequence heterogeneity in a genetically diverse population, such as HIV-1 due to PCR resampling and PCR/sequencing errors. The Primer ID approach reveals the true sampling depth and greatly reduces errors. Knowing the sampling depth allows the construction of a model of how to maximize the recovery of sequences from input templates and to reduce resampling of the Primer ID so that appropriate multiplexing can be included in the experimental design. With the defined sampling depth and measured error rate, we are able to assign cutoffs for the accurate detection of minority variants in viral populations. This approach allows the power of NGS to be realized without having to guess about sampling depth or to ignore the problem of PCR resampling, while also being able to correct most of the errors in the data set. Studies of viral population diversity are increasingly using nextgeneration sequencing (NGS) technologies to extend the depth of population sampling. Key aspects of understanding within-host viral population diversity are knowing the true depth of template/genome sampling and documenting the accuracy of the sequencing method to validate the detection of rare variants. Current approaches using NGS in viral population studies usually require a preceding PCR amplification step. Thus, PCR errors, including nucleotide misincorporation ...

Full-length human immunodeficiency virus type 1 (HIV-1) RNA serves as the genome or as an mRNA, or this RNA undergoes splicing using four donors and 10 acceptors to create over 50 physiologically relevant transcripts in two size classes (1.8 kb and 4 kb). We developed an assay using Primer ID-tagged deep sequencing to quantify HIV-1 splicing. Using the lab strain NL4-3, we found that A5 (/) is the most commonly used acceptor (about 50%) and A3 () the least used (about 3%). Two small exons are made when a splice to acceptor A1 or A2 is followed by activation of donor D2 or D3, and the high-level use of D2 and D3 dramatically reduces the amount of and transcripts. We observed distinct patterns of temperature sensitivity of splicing to acceptors A1 and A2. In addition, disruption of a conserved structure proximal to A1 caused a 10-fold reduction in all transcripts that utilized A1. Analysis of a panel of subtype B transmitted/founder viruses showed that splicing patterns are conserved, but with surprising variability of usage. A subtype C isolate was similar, while a simian immunodeficiency virus (SIV) isolate showed significant differences. We also observed transsplicing from a downstream donor on one transcript to an upstream acceptor on a different transcript, which we detected in 0.3% of 1.8-kb RNA reads. There were several examples of splicing suppression when the intron was retained in the 4-kb size class. These results demonstrate the utility of this assay and identify new examples of HIV-1 splicing regulation. During HIV-1 replication, over 50 conserved spliced RNA variants are generated. The splicing assay described here uses new developments in deep-sequencing technology combined with Primer ID-tagged cDNA primers to efficiently quantify HIV-1 splicing at a depth that allows even low-frequency splice variants to be monitored. We have used this assay to examine several features of HIV-1 splicing and to identify new examples of different mechanisms of regulation of these splicing patterns. This splicing assay can be used to explore in detail how HIV-1 splicing is regulated and, with moderate throughput, could be used to screen for structural elements, small molecules, and host factors that alter these relatively conserved splicing patterns.

Drug resistance continues to be a growing global problem. The efficacy of small molecule inhibitors is threatened by pools of genetic diversity in all systems, including antibacterials, antifungals, cancer therapeutics, and antivirals. Resistant variants often include combinations of active site mutations and distal "secondary" mutations, which are thought to compensate for losses in enzymatic activity. HIV-1 protease is the ideal model system to investigate these combinations and underlying molecular mechanisms of resistance. Darunavir (DRV) binds wild-type (WT) HIV-1 protease with a potency of <5 pM, but we have identified a protease variant that loses potency to DRV 150 000-fold, with 11 mutations in and outside the active site. To elucidate the roles of these mutations in DRV resistance, we used a multidisciplinary approach, combining enzymatic assays, crystallography, and molecular dynamics simulations. Analysis of protease variants with 1, 2, 4, 8, 9, 10, and 11 mutations showed that the primary active site mutations caused ∼50-fold loss in potency (2 mutations), while distal mutations outside the active site further decreased DRV potency from 13 nM (8 mutations) to 0.76 μM (11 mutations). Crystal structures and simulations revealed that distal mutations induce subtle changes that are dynamically propagated through the protease. Our results reveal that changes remote from the active site directly and dramatically impact the potency of the inhibitor. Moreover, we find interdependent effects of mutations in conferring high levels of resistance. These mechanisms of resistance are likely applicable to many other quickly evolving drug targets, and the insights may have implications for the design of more robust inhibitors.

153) 27Coronaviruses (CoVs) traffic frequently between species resulting in novel disease 28 outbreaks, most recently exemplified by the newly emerged SARS-CoV-2. Herein, we 29show that the ribonucleoside analog β-D-N 4 -hydroxycytidine (NHC, EIDD-1931) has 30 broad spectrum antiviral activity against SARS-CoV 2, MERS-CoV, and 31 . : bioRxiv preprint related zoonotic group 2b or 2c Bat-CoVs, as well as increased potency against a 32 coronavirus bearing resistance mutations to another nucleoside analog inhibitor. In 33 mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic 34 administration of EIDD-2801, an orally bioavailable hydroxycytidine-5'-isopropyl ester), improved pulmonary function, and reduced virus 36 titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were 37 associated with increased transition mutation frequency in viral but not host cell RNA, 38 supporting a mechanism of lethal mutagenesis. The potency of NHC/EIDD-2801 against 39 multiple coronaviruses, its therapeutic efficacy, and oral bioavailability in vivo, all 40 highlight its potential utility as an effective antiviral against SARS-CoV-2 and other 41 future zoonotic coronaviruses. 42 43 emergence, broadly active antivirals are clearly needed for rapid response to new CoV 63 outbreaks in humans and domesticated animals. 64Currently, there are no approved therapies specific for any human CoV. β-D-N4-65 hydroxycytidine (NHC, EIDD-1931) is orally bioavailable ribonucleoside analog with 66 broad-spectrum antiviral activity against various unrelated RNA viruses including 67 influenza, Ebola, CoV and Venezuelan equine encephalitis virus (VEEV) 10-13 . For VEEV, 68 the mechanism of action (MOA) for NHC has been shown to be through lethal 69 mutagenesis where deleterious transition mutations accumulate in viral RNA 11,14 . Here, 70we demonstrate that NHC exerts potent, broad-spectrum activity against SARS-CoV, 71 MERS-CoV and their related bat-CoV in primary human airway epithelial cell cultures 72 (HAE), a biologically relevant model of the human conducting airway. In addition, we 73 show that NHC is potently antiviral against the newly emerging SARS-CoV-2 as well as 74 against coronavirus bearing resistance mutations to the potent nucleoside analog 75 inhibitor, remdesivir (RDV). In SARS-or MERS-CoV infected mice, both prophylactic 76 and therapeutic administration EIDD-2801, an oral NHC-prodrug (b-D-N 4 -77 hydroxycytidine-5'-isopropyl ester) improved pulmonary function and reduced virus titer 78 and ameliorated disease severity. In addition, therapeutic EIDD-2801 reduced the 79 pathological features of ALI in SARS-CoV infected mice. Using a high-fidelity deep 80 sequencing approach (Primer ID), we found that increased mutation rates coincide with 81 decreased MERS-CoV yields in vitro and protective efficacy in vivo supporting the MOA 82 of lethal mutagenesis against emerging CoV 13 . The broad activity and therapeutic 83 efficacy of NHC/EIDD-2801 highlight its potential to diminish epidemic diseas...