Bacteriophage Evcara is a podovirus isolated on Microbacterium paraoxydans NRRL B-24275. Its genome is 16,285 bp in length and contains 22 predicted protein-coding genes. Evcara, has been assigned to cluster GI with Microbacterium foliorum phages PineapplePizza and Curie that share 10 homologues with the well-characterized Bacillus subtilis phage phi29.

Over the last two decades, there have been numerous initiatives to improve undergraduate student outcomes in STEM. One model for scalable reform is the inclusive Research Education Community (iREC). In an iREC, STEM faculty from colleges and universities across the nation are supported to adopt and sustainably implement course-based research – a form of science pedagogy that enhances student learning and persistence in science. In this study, we used pathway modeling to develop a qualitative description that explicates the HHMI Science Education Alliance (SEA) iREC as a model for facilitating the successful adoption and continued advancement of new curricular content and pedagogy. In particular, outcomes that faculty realize through their participation in the SEA iREC were identified, organized by time, and functionally linked. The resulting pathway model was then revised and refined based on several rounds of feedback from over 100 faculty members in the SEA iREC who participated in the study. Our results show that in an iREC, STEM faculty organized as a long-standing community of practice leverage one another, outside expertise, and data to adopt, implement, and iteratively advance their pedagogy. The opportunity to collaborate in this manner and, additionally, to be recognized for pedagogical contributions sustainably engages STEM faculty in the advancement of their pedagogy. Here, we present a detailed pathway model of SEA that, together with underpinning features of an iREC identified in this study, offers a framework to facilitate transformations in undergraduate science education.

Phage Culver, with a siphovirus morphology, was isolated using Gordonia terrae CAG3. Culver is assigned to phage cluster CQ1 based on gene content similarity to actinobacteriophages. Notably, Culver is predicted to encode eight tRNAs, lysin A by two adjacent genes, and, unlike other CQ1 phages, two putative integrase genes.

Course-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessment—(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learning—along with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of course-based research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate students’ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education.

We report the discovery of Gordonia phage Conley, a siphovirus isolated from Cullowhee Creek in the Fall of 2022. The 60,078bp genome contains 90 predicted protein-coding genes all transcribed in the same direction and has been assigned to genetic cluster DJ based on gene content similarity.

The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching.

Phage Vardy is a lytic siphovirus isolated from creek soil in Cullowhee, NC, using Gordonia rubripertincta NRRL B-16540. Vardy's 60,144-bp genome contains 90 predicted genes and five copies of a 50-bp motif that may regulate gene expression. Based on gene content similarity, Vardy is assigned to cluster DJ.

The mycobacteriophages InvictusManeo (K5 subcluster) and Netyap (L2 subcluster) were isolated from soils in Cullowhee Creek, Cullowhee, North Carolina. Both exhibit Siphoviridae morphology and infect Mycobacterium smegmatis mc2155. The InvictusManeo genome is 61,147 bp and contains 96 predicted protein-coding genes, whereas the Netyap genome is 76,366 bp with 131 predicted protein-coding genes.

Regulation of bacteriophage gene expression involves repressor proteins that bind and downregulate early lytic promoters. A large group of mycobacteriophages code for repressors that are unusual in also terminating transcription elongation at numerous binding sites (stoperators) distributed across the phage genome. Here we provide the X-ray crystal structure of a mycobacteriophage immunity repressor bound to DNA, which reveals the binding of a monomer to an asymmetric DNA sequence using two independent DNA binding domains. The structure is supported by small-angle X-ray scattering, DNA binding, molecular dynamics, and in vivo immunity assays. We propose a model for how dual DNA binding domains facilitate regulation of both transcription initiation and elongation, while enabling evolution of other superinfection immune specificities.

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.

Recent structural studies of the bacteriophage T7 DNA replication system have shed light on how multiple proteins assemble to copy two antiparallel DNA strands. In T7, acidic C-terminal tails of both the primase-helicase and single-stranded DNA binding protein bind to two basic patches on the DNA polymerase to aid in replisome assembly, processivity, and coordinated DNA synthesis. Although these electrostatic interactions are essential for DNA replication, the molecular details for how these tails bind the polymerase are unknown. We have determined an X-ray crystal structure of the T7 DNA polymerase bound to both a primer/template DNA and a peptide that mimics the C-terminal tail of the primase-helicase. The structure reveals that the essential C-terminal phenylalanine of the tail binds to a hydrophobic pocket that is surrounded by positive charge on the surface of the polymerase. We show that alterations of polymerase residues that engage the tail lead to defects in viral replication. In the structure, we also observe dTTP bound in the exonuclease active site and stacked against tryptophan 160. Using both primer/extension assays and high-throughput sequencing, we show how mutations in the exonuclease active site lead to defects in mismatch repair and an increase in the level of mutagenesis of the T7 genome. Finally, using small-angle X-ray scattering, we provide the first solution structures of a complex between the single-stranded DNA binding protein and the DNA polymerase and show how a single-stranded DNA binding protein dimer engages both one and two copies of DNA polymerase.

SignificanceAfter eradication of smallpox, zoonotic orthopoxviruses, including cowpox virus (CPXV) and monkeypox virus, continue to be a source for infection in humans. Poxviruses dedicate a significant part of their genome encoding proteins that effectively undermine host immune defenses. Here, we demonstrate that a CPXV-encoded protein, M2, can specifically bind B7.1 (CD80) and B7.2 (CD86), cell surface proteins expressed mainly by professional antigen-presenting cells that serve as ligands of the T cell costimulatory receptor CD28 and inhibitory receptor CTLA4. Functionally, we found that M2 blocks CD28-mediated T cell activation in vitro and in a mouse model of cowpox infection.

Viruses have evolved mechanisms of MHCI inhibition in order to evade recognition by cytotoxic CD8+ T cells (CTLs), which is well-illustrated by our prior studies on cowpox virus (CPXV) that encodes potent MHCI inhibitors. Deletion of CPXV viral MHCI inhibitors markedly attenuated in vivo infection due to effects on CTL effector function, not priming. However, the CTL response to CPXV in C57BL/6 mice is dominated by a single peptide antigen presented by H-2Kb. Here we evaluated the effect of viral MHCI inhibition on immunodominant (IDE) and subdominant epitopes (SDE) as this has not been thoroughly examined. We found that cross-priming, but not cross-dressing, is the main mechanism driving IDE and SDE CTL responses following CPXV infection. Secretion of the immunodominant antigen was not required for immunodominance. Instead, immunodominance was caused by CTL interference, known as immunodomination. Both immunodomination and cross-priming of SDEs were not affected by MHCI inhibition. SDE-specific CTLs were also capable of exerting immunodomination during primary and secondary responses, which was in part dependent on antigen abundance. Furthermore, CTL responses directed solely against SDEs protected against lethal CPXV infection, but only in the absence of the CPXV MHCI inhibitors. Thus, both SDE and IDE responses can contribute to protective immunity against poxviruses, implying that these principles apply to poxvirus-based vaccines.

Nature 505, 691–695 (2014); doi:10.1038/nature12862 In this Letter, we carried out bioinformatic analyses on interferon-stimulated gene screening data sets for multiple viruses, including a data set for West Nile virus (WNV) (Supplementary Table 8 in ref. 1). We recently discovered that the WNV-GFP stock used in our 2011 study 1 was actually Venezuelan equine encephalitis virus (VEEV-GFP).

Although viral MHC class I inhibition is considered a classic immune-evasion strategy, its in vivo role is largely unclear. Mutant cowpox virus lacking its MHC class I inhibitors is markedly attenuated during acute infection because of CD8(+) T-cell-dependent control, but it was not known how CD8(+) T-cell responses are affected. Interestingly, we found no major effect of MHC class I down-regulation on priming of functional cowpox virus-specific CD8(+) T cells. Instead, we demonstrate that, during acute infection in vivo, MHC class I down-regulation prevents primed virus-specific CD8(+) T cells from recognizing infected cells and exerting effector responses to control the infection.

Prior studies have shown that 2′-O methyltransferase activity of flaviviruses, coronaviruses, and poxviruses promotes viral evasion of Ifit1, an interferon-stimulated innate immune effector protein. Viruses lacking 2′-O methyltransferase activity exhibited attenuation in primary macrophages that was rescued in cells lacking Ifit1 gene expression. Here, we examined the role of Ifit1 in restricting pathogenesis in vivo of wild type WNV (WNV-WT) and a mutant in the NS5 gene (WNV-E218A) lacking 2′-O methylation of the 5′ viral RNA cap. While deletion of Ifit1 had marginal effects on WNV-WT pathogenesis, WNV-E218A showed increased replication in peripheral tissues of Ifit1−/− mice after subcutaneous infection, yet this failed to correlate with enhanced infection in the brain or lethality. In comparison, WNV-E218A was virulent after intracranial infection as judged by increased infection in different regions of the central nervous system (CNS) and a greater than 16,000-fold decrease in LD50 values in Ifit1−/− compared to wild type mice. Ex vivo infection experiments revealed cell-type specific differences in the ability of an Ifit1 deficiency to complement the replication defect of WNV-E218A. In particular, WNV-E218A infection was impaired in both wild type and Ifit1−/− brain microvascular endothelial cells, which are believed to participate in blood-brain barrier (BBB) regulation of virus entry into the CNS. A deficiency of Ifit1 also was associated with increased neuronal death in vivo, which was both cell-intrinsic and mediated by immunopathogenic CD8+ T cells. Our results suggest that virulent strains of WNV have largely evaded the antiviral effects of Ifit1, and viral mutants lacking 2′-O methylation are controlled in vivo by Ifit1-dependent and -independent mechanisms in different cell types.

A genetic absence of the common IFN-α/β signaling receptor (IFNAR) in mice is associated with enhanced viral replication and altered adaptive immune responses. However, analysis of IFNAR(-/-) mice is limited for studying the functions of type I IFN at discrete stages of viral infection. To define the temporal functions of type I IFN signaling in the context of infection by West Nile virus (WNV), we treated mice with MAR1-5A3, a neutralizing, non cell-depleting anti-IFNAR antibody. Inhibition of type I IFN signaling at or before day 2 after infection was associated with markedly enhanced viral burden, whereas treatment at day 4 had substantially less effect on WNV dissemination. While antibody treatment prior to infection resulted in massive expansion of virus-specific CD8(+) T cells, blockade of type I IFN signaling starting at day 4 induced dysfunctional CD8(+) T cells with depressed cytokine responses and expression of phenotypic markers suggesting exhaustion. Thus, only the later maturation phase of anti-WNV CD8(+) T cell development requires type I IFN signaling. WNV infection experiments in BATF3(-/-) mice, which lack CD8-α dendritic cells and have impaired priming due to inefficient antigen cross-presentation, revealed a similar effect of blocking IFN signaling on CD8(+) T cell maturation. Collectively, our results suggest that cell non-autonomous type I IFN signaling shapes maturation of antiviral CD8(+) T cell response at a stage distinct from the initial priming event.

Viral fusogenic membrane proteins have been proposed as tools to increase the potency of oncolytic viruses, but there is a need for mechanisms to control the spread of fusogenic viruses in normal versus tumor cells. We have previously shown that a mutant of the paramyxovirus simian virus 5 (SV5) that harbors mutations in the P/V gene from the canine parainfluenza virus (P/V-CPI-) is a potent inducer of type I interferon (IFN) and apoptosis and is restricted for spread through normal but not tumor cells in vitro. Here, we have used the cytopathic P/V-CPI- as a backbone vector to test the hypothesis that a virus expressing a hyperfusogenic glycoprotein will be a more effective oncolytic vector but will retain sensitivity to IFN. A P/V mutant virus expressing an F protein with a glycine-to-alanine substitution in the fusion peptide (P/V-CPI--G3A) was more fusogenic than the parental P/V-CPI- mutant. In two model prostate tumor cell lines which are defective in IFN production (LNCaP and DU145), the hyperfusogenic P/V-CPI--G3A mutant had normal growth properties at low multiplicities of infection and was more effective than the parental P/V-CPI- mutant at cell killing in vitro. However, in PC3 cells which produce and respond to IFN, the hyperfusogenic P/V-CPI--G3A mutant was attenuated for growth and spread. Killing of PC3 cells was equivalent between the parental P/V-CPI- mutant and the hyperfusogenic P/V-CPI--G3A mutant. In a nude mouse model using LNCaP cells, the hyperfusogenic P/V-CPI--G3A mutant was more effective than P/V-CPI- at reducing tumor burden. In the case of DU145 tumors, the two vectors based on P/V-CPI- were equally effective at limiting tumor growth. Together, our results provide proof of principle that a cytopathic SV5 P/V mutant can serve as an oncolytic virus and that the oncolytic effectiveness of P/V mutants can be enhanced by a fusogenic membrane protein without compromising sensitivity to IFN. The potential advantages of SV5-based oncolytic vectors are discussed.

The paramyxovirus simian virus 5 (SV5) establishes highly productive persistent infections of epithelial cells without inducing a global inhibition of translation. Here we show that an SV5 mutant (the P/V-CPI(-) mutant) with substitutions in the P subunit of the viral polymerase and the accessory V protein also establishes highly productive infections like wild-type (WT) SV5 but that cells infected with the P/V-CPI(-) mutant show an overall shutdown of both host and viral translation at late times postinfection. Reduced host and viral protein synthesis with the P/V-CPI(-) virus was not due to lower levels of mRNA or caspase-dependent apoptosis and correlated with phosphorylation of the translation initiation factor eIF-2alpha. WT SV5 was a poor activator of the eIF-2alpha kinase protein kinase R (PKR). By contrast, the P/V-CPI(-) mutant induced PKR phosphorylation, which correlated with the time course of translation inhibition but was independent of interferon signaling. In HeLa cells that expressed the PKR inhibitor influenza A virus NS1 or reovirus sigma3, the rate of host protein synthesis at late times after infection with the P/V-CPI(-) mutant was restored to approximately 50% that of control HeLa cells. By contrast, the rates of P/V-CPI(-) viral protein synthesis in HeLa cells expressing NS1 or sigma3 were dramatically enhanced, between 5- and 20-fold, while levels of viral mRNA were increased only slightly (NS1-expressing cells) or remained constant (sigma3-expressing cells). Similar results were found using HeLa cells where PKR levels were reduced due to knockdown by small interfering RNA. Expression of either the WT P or the WT V protein from the genome of the P/V-CPI(-) mutant resulted in lower levels of PKR activation and rates of host and viral protein synthesis that closely matched those seen with WT SV5. Despite higher rates of translation, cells infected with the V- or P-complemented virus accumulated viral mRNAs to lower levels than that seen with the parental P/V-CPI(-) mutant. We present a model in which the paramyxovirus P/V gene products limit induction of PKR by limiting the synthesis of aberrant viral mRNAs and double-stranded RNA and thus prevent the shutdown of translation by a mechanism that differs from that of other PKR inhibitors such as NS1 and sigma3.

Since the first polygenic risk score (PRS) in 2007, research in this area has progressed significantly. The increasing number of SNPs that have been identified by large scale GWAS analyses has fuelled the development of a myriad of PRSs for a wide variety of diseases and, more recently, to PRSs that potentially identify differential response to specific drugs. PRSs constitute a composite genomic biomarker and potential applications for PRSs in clinical practice encompass risk prediction and disease screening, early diagnosis, prognostication, and drug stratification to improve efficacy or reduce adverse drug reactions. Nevertheless, to our knowledge, no PRSs have yet been adopted into routine clinical practice. Beyond the technical considerations of PRS development, the major challenges that face PRSs include demonstrating clinical utility and circumnavigating the implementation of novel genomic technologies at scale into stretched healthcare systems. In this review, we discuss progress in developing disease susceptibility PRSs across multiple medical specialties, development of pharmacogenomic PRSs, and future directions for the field.