Nucleoside analogues are a class of well-established antiviral agents that act by being directly incorporated into the viral genome during the replication process, resulting in chain termination or the induction of lethal mutations. While many nucleoside analogues have exhibited broad-spectrum activity against a wide range of viruses, their effectiveness against SARS-CoV-2 is limited. The lack of activity is hypothesized to be attributed to the proofreading function of viral nsp14 exonuclease. In this study, the role of the nsp14 proofreading in modulating nucleoside antiviral activity was investigated using genetic and pharmacological approaches. Introduction of exonuclease attenuation or disabling mutations to nsp14 led to either severe replication defect or increased sensitivity of SARS-CoV-2 and SARS-CoV replicons to specific nucleoside analogues. In contrast, repurposing of HCV NS5A inhibitors to suppress nsp14 exonuclease activity is insufficient to enhance the potency of nucleoside analogues. These findings provided further support for nsp14 as a target for SARS-CoV-2 antiviral development and highlighted the complex interplay between nsp14 proofreading and RNA replication. ### Competing Interest Statement All authors are employees of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA and may own stock and/or options in Merck & Co., Inc., Rahway, NJ, USA. X.H. and D.W. are inventors on the patent application ''Coronavirus replicons for antiviral screening and testing''.

Respiratory Syncytial Virus (RSV) causes severe respiratory infections and concomitant disease resulting in significant morbidity and mortality in infants, elderly, and immunocompromised adults. Vaccines, monoclonal antibodies, and small-molecule antivirals are now either available or in development to prevent and treat RSV infections. Although rodent and non-rodent preclinical animal models have been used to evaluate these emerging agents, there is still a need to improve our understanding of the pharmacokinetic (PK)-pharmacodynamic (PD) relationships within and between animal models to enable better design of human challenge studies and clinical trials. Herein, we report a PKPD evaluation of MRK-1, a novel small molecule non-nucleoside inhibitor of the RSV L polymerase protein, in the semi-permissive cotton rat and African green monkey models of RSV infection. These studies demonstrate a strong relationship between in vitro activity, in vivo drug exposure, and pharmacodynamic efficacy as well as revealing limitations of the cotton rat RSV model. Additionally, we report unexpected horizontal transmission of human RSV between co-housed African green monkeys, as well as a lack of drug specific resistant mutant generation. Taken together these studies further our understanding of these semi-permissive animal models and offer the potential for expansion of their preclinical utility in evaluating novel RSV therapeutic agents.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are related RNA viruses responsible for severe respiratory infections and resulting disease in infants, elderly, and immunocompromised adults 1 – 3 . Therapeutic small molecule inhibitors that bind to the RSV polymerase and inhibit viral replication are being developed, but their binding sites and molecular mechanisms of action remain largely unknown 4 . Here we report a conserved allosteric inhibitory site identified on the L polymerase proteins of RSV and HMPV that can be targeted by a dual-specificity, non-nucleoside inhibitor, termed MRK-1. Cryo-EM structures of the inhibitor in complexes with truncated RSV and full-length HMPV polymerase proteins provide a structural understanding of how MRK-1 is active against both viruses. Functional analyses indicate that MRK-1 inhibits conformational changes necessary for the polymerase to engage in RNA synthesis initiation and to transition into an elongation mode. Competition studies reveal that the MRK-1 binding pocket is distinct from that of a capping inhibitor with an overlapping resistance profile, suggesting that the polymerase conformation bound by MRK-1 may be distinct from that involved in mRNA capping. These findings should facilitate optimization of dual RSV and HMPV replication inhibitors and provide insights into the molecular mechanisms underlying their polymerase activities.

All herpesviruses encode a conserved DNA polymerase that is required for viral genome replication and serves as an important therapeutic target. Currently available herpesvirus therapies include nucleoside and non-nucleoside inhibitors (NNI) that target the DNA-bound state of herpesvirus polymerase and block replication. Here we report the ternary complex crystal structure of Herpes Simplex Virus 1 DNA polymerase bound to DNA and a 4-oxo-dihydroquinoline NNI, PNU-183792 (PNU), at 3.5 Å resolution. PNU bound at the polymerase active site, displacing the template strand and inducing a conformational shift of the fingers domain into an open state. These results demonstrate that PNU inhibits replication by blocking association of dNTP and stalling the enzyme in a catalytically incompetent conformation, ultimately acting as a nucleotide competing inhibitor (NCI). Sequence conservation of the NCI binding pocket further explains broad-spectrum activity while a direct interaction between PNU and residue V823 rationalizes why mutations at this position result in loss of inhibition.

The purpose of this study was to examine the cathartic and therapeutic effects of written essays and therapy interviews about traumatic events over four successive days as compared with a control writing about trivial events. The subjects were 102 male and female college students. A content analysis showed that both treatment groups expressed more positive and negative emotion than the control as well as showed more cognitive, self-esteem, and behavior changes. Positive emotion, cognitive changes, and self-esteem changes increased over days while negative emotion decreased. Pain and upset about the topic decreased over days for both treatment groups. A post-experimental questionnaire showed that both treatment groups felt more positive about their topics and themselves. On the other hand, there were dramatic differences in pre- to post-session mood with written expression consistently increasing negative mood and decreasing positive mood.

ABSTRACT The current standard of care for hepatitis C virus (HCV) patients is cotreatment with human alpha interferon (IFN-α) and ribavirin. The host factor USP18 functions to regulate the interferon signaling pathway by acting as an off-switch. In order to understand whether the inhibition of USP18 represents a valid target for the enhancement of interferon treatment for chronic viral diseases, we have used a wide range of RNA interference (RNAi) reagents to suppress USP18 gene expression in Huh7 cell lines. We demonstrate that a USP18 knockdown results in IFN-α2a signaling (measured by increased IFN-stimulated response element [ISRE] reporter gene activity, 2′,5′-oligoadenylate synthetase [2-5 OAS] expression, and ISG15 induction) that is increased by ∼100-fold, whereas the antiviral (AV) potency in both the Huh7 HCV subgenomic replicon assay and the Huh7.5 HCV infectious virus assay increased by ∼3-fold. While the degree of the USP18 knockdown of USP18 elicited by the different RNAi reagents correlated with the enhancement of IFN-α2a signaling, it did not correlate with the enhancement of AV activity. The failure of increased IFN-α2a signaling to fully translate into increased AV potency was also observed for encephalomyocarditis virus (EMCV) assays using Huh7.5 cells. These data suggest that the IFN-mediated AV response in Huh7.5 cells has only a limited dependence on USP18 activity.

ABSTRACT We have screened 47 locked nucleic acid (LNA) antisense oligonucleotides (ASOs) targeting conserved (>95% homology) sequences in the hepatitis C virus (HCV) genome using the subgenomic HCV replicon assay and generated both antiviral (50% effective concentration [EC50]) and cytotoxic (50% cytotoxic concentration [CC50]) dose-response curves to allow measurement of the selectivity index (SI). This comprehensive approach has identified an LNA ASO with potent antiviral activity (EC50 = 4 nM) and low cytotoxicity (CC50 >880 nM) targeting the 25- to 40-nucleotide region (nt) of the HCV internal ribosome entry site (IRES) containing the distal and proximal miR-122 binding sites. LNA ASOs targeting previously known accessible regions of the IRES, namely, loop III and the initiation codon in loop IV, had poor SI values. We optimized the LNA ASO sequence by performing a 1-nucleotide walk through the 25- to 40-nt region and show that the boundaries for antiviral efficacy are extremely precise. Furthermore, we have optimized the format for the LNA ASO using different gapmer and mixomer patterns and show that RNase H is required for antiviral activity. We demonstrate that RNase H-refractory ASOs targeting the 25- to 40-nt region have no antiviral effect, revealing important regulatory features of the 25- to 40-nt region and suggesting that RNase H-refractory LNA ASOs can act as potential surrogates for proviral functions of miR-122. We confirm the antisense mechanism of action using mismatched LNA ASOs. Finally, we have performed pharmacokinetic experiments to demonstrate that the LNA ASOs have a very long half-life (>5 days) and attain hepatic maximum concentrations >100 times the concentration required for in vitro antiviral activity.

A new small-molecule inhibitor class that targets virion maturation was identified from a human immunodeficiency virus type 1 (HIV-1) antiviral screen. PF-46396, a representative molecule, exhibits antiviral activity against HIV-1 laboratory strains and clinical isolates in T-cell lines and peripheral blood mononuclear cells (PBMCs). PF-46396 specifically inhibits the processing of capsid (CA)/spacer peptide 1 (SP1) (p25), resulting in the accumulation of CA/SP1 (p25) precursor proteins and blocked maturation of the viral core particle. Viral variants resistant to PF-46396 contain a single amino acid substitution in HIV-1 CA sequences (CAI201V), distal to the CA/SP1 cleavage site in the primary structure, which we demonstrate is sufficient to confer significant resistance to PF-46396 and 3-O-(3',3'-dimethylsuccinyl) betulinic acid (DSB), a previously described maturation inhibitor. Conversely, a single amino substitution in SP1 (SP1A1V), which was previously associated with DSB in vitro resistance, was sufficient to confer resistance to DSB and PF-46396. Further, the CAI201V substitution restored CA/SP1 processing in HIV-1-infected cells treated with PF-46396 or DSB. Our results demonstrate that PF-46396 acts through a mechanism that is similar to DSB to inhibit the maturation of HIV-1 virions. To our knowledge, PF-46396 represents the first small-molecule HIV-1 maturation inhibitor that is distinct in chemical class from betulinic acid-derived maturation inhibitors (e.g., DSB), demonstrating that molecules of diverse chemical classes can inhibit this mechanism.

The use of writing, alone or in conjunction with traditional psychotherapy, has increased substantially in recent years. The most widespread use of writing has been for single-shot ad hoc purposes or to log behavior. The purpose of this review is to summarize a decade of research demonstrating the efficacy of writing about past traumatic experiences on mental and physical health outcomes. It is widely acknowledged in our culture that putting upsetting experiences into words can be healthy. Research from several domains indicates that talking with friends, confiding to a therapist, praying, and even writing about one's thoughts and feelings can be physically and mentally beneficial. This review highlights advances in written disclosure that determine some therapeutic outcomes. In addition, we attempt to explore the mechanisms that predict improved psychological and physical health. Finally, limitations of previous studies are highlighted, and suggestions for future research and application are made.

This study reports data on the efficacy of Strategic Structural Systems Engagement (SSSE), which is designed to bring hard-to-reach families into treatment. The study also explores variables that may contribute to differential effectiveness. Participants were 193 Hispanic families, who were randomly assigned to either experimental or control conditions. Several important findings emerged. First, the overall results replicated earlier findings showing the superiority of SSSE : 81% of SSSE families, compared to 60% of control families, were successfully engaged, χ 2 (1, N = 193) = 7.5, p <.001. Second, SSSE interventions were more successful with non-Cuban Hispanics (97% successfully engaged) than with Cuban Hispanics (64% successfully engaged), χ 2 (1, N = 51) = 7.53, p =.006. Third, an analysis of intervention failures suggests a mechanism by which culture and ethnicity influence clinical processes (resistance to engagement) and may result in differential effectiveness.

The purpose of this study was to compare vocal and written expression of feeling about interpersonal traumatic and trivial events in 20-min sessions over a 4-day period. Similar emotional processing was produced by vocal and written expression of feeling about traumatic events. The painfulness of the topic decreased steadily over the 4 days. At the end, both groups felt better about their topics and themselves and also reported positive cognitive changes. A content analysis of the sessions suggested greater overt expression of emotion and related changes in the vocal condition. Finally, there was an upsurge in negative emotion after each session of either vocal or written expression. These results suggest that previous findings that psychotherapy ameliorated this negative mood upsurge could not be attributed to the vocal character of psychotherapy.

College students with unresolved traumatic experiences were given a brief course of cognitive therapy or asked to talk into a tape recorder. Both procedures were equally effective in reducing negative mood and negative thoughts, although cognitive therapy was somewhat more effective on one outcome measure. The arousal of negative affect was inversely related to positive outcome. The reduction of negative affect and, particularly, negative thoughts was positively related to outcome. In spite of similarities in outcome, the two procedures seemed to operate by somewhat different processes.

The validity and utility of the Psychological Screening Inventory (PSI) was evaluated with a group of 351 male and 185 female adolescents in a juvenile court related agency. In addition, 174 noncourt controls were studied. Court records and psychological evaluations were used to obtain behavioral measures and ratings on the Life History Checklist. The PSI discriminated major emotional problems, offenses, peer relations, and cooperativeness and it was related to several measures of the Life History Checklist. However, the mean differences and correlations were small and of little practical use. Classification accuracy was poor. In a multitrait-multimethod analysis, the PSI showed poor discriminant validity from unrelated measures and method variance. Good discriminant validity from demographic variables was found. It was concluded that there are serious limitations in using the PSI in a juvenile population.

In an effort to understand emotional change, brief psychotherapy was compared with written expression about stressful life events as well as with a control condition of writing about trivial events using college students. The written expression condition was quite effective in temporarily arousing negative affect but not in changing feelings about the traumatic events. Some self-generated cognitive changes did occur. In contrast, psychotherapy aroused less negative affect but showed much more cognitive reappraisal and a dramatic shift to positive affect, as well as in a basic change in attitude about the stressful event. The study supports a model of therapeutic change stressing emotional expression followed by cognitive reappraisal rather than a model of simple affective discharge.