Sequences at gene segment termini inclusive of untranslated regions and partial open reading frames play a critical role in mammalian orthoreovirus S gene packaging.

Mammalian orthoreovirus (MRV) is a prototypic member of the Spinareoviridae family and has ten double-stranded RNA segments. One copy of each segment must be faithfully packaged into the mature virion, and prior literature suggests that nucleotides (nts) at the terminal ends of each gene likely facilitate their packaging. However, little is known about the precise packaging sequences required or how the packaging process is coordinated. Using a novel approach, we have determined that 200 nts at each terminus, inclusive of untranslated regions (UTR) and parts of the open reading frame (ORF), are sufficient for packaging S gene segments (S1-S4) individually and together into replicating virus. Further, we mapped the minimal sequences required for packaging the S1 gene segment into a replicating virus to 25 5' nts and 50 3' nts. The S1 UTRs, while not sufficient, were necessary for efficient packaging, as mutations of the 5' or 3' UTRs led to a complete loss of virus recovery. Using a second novel assay, we determined that 50 5' nts and 50 3' nts of S1 are sufficient to package a non-viral gene segment into MRV. The 5' and 3' termini of the S1 gene are predicted to form a panhandle structure and specific mutations within the stem of the predicted panhandle region led to a significant decrease in viral recovery. Additionally, mutation of six nts that are conserved across the three major serotypes of MRV that are predicted to form an unpaired loop in the S1 3' UTR, led to a complete loss of viral recovery. Overall, our data provide strong experimental proof that MRV packaging signals lie at the terminal ends of the S gene segments and offer support that the sequence requirements for efficient packaging of the S1 segment include a predicted panhandle structure and specific sequences within an unpaired loop in the 3' UTR.

Trauma-Informed Care in Nursing Curricula: Development of a Simulation-Based Educational Framework to Guide Health Professions.

AIM: This research aimed to uncover elements of a comprehensive, trauma-informed (TI) multidisciplinary health professions simulation framework to improve the delivery of care to traumatized patients. BACKGROUND: Trauma is a pervasive public health problem requiring a TI approach. Simulation is an evidence-based teaching strategy that advances knowledge and clinical reasoning. There is a lack of scientifically based simulation education models addressing the delivery of TI care for the health professions. METHOD: A Delphi study utilizing a panel of experts was conducted to identify the most critical elements of a simulation framework. RESULTS: Phase one identified 10 content areas and 111 subcontent areas. Phase two analysis revealed 99 percent of the 111 subcontent areas achieved expert consensus. CONCLUSION: This Delphi study provides the first scientifically based framework to guide the development of a comprehensive, TI, multidisciplinary simulation framework to recognize trauma survivors and subsequently display concern and respect.

Mammalian Orthoreovirus Factories Modulate Stress Granule Protein Localization by Interaction with G3BP1.

Mammalian orthoreovirus (MRV) infection induces phosphorylation of translation initiation factor eIF2α, which promotes the formation of discrete cytoplasmic inclusions, termed stress granules (SGs). SGs are emerging as a component of the innate immune response to virus infection, and modulation of SG assembly is a common mechanism employed by viruses to counter this antiviral response. We previously showed that MRV infection induces SGs early and then interferes with SG formation as infection proceeds. In this work, we found that SG-associated proteins localized to the periphery of virus-encoded cytoplasmic structures, termed virus factories (VFs), where viral transcription, translation, and replication occur. The localization of SG proteins to VFs was dependent on polysome dissociation and occurred via association of the SG effector protein, Ras-GAP SH3-binding protein 1 (G3BP1), with the MRV nonstructural protein σNS, which localizes to VFs via association with VF nucleating protein, µNS. Deletion analysis of the σNS RNA binding domain and G3BP1 RNA (RRM) and ribosomal (RGG) binding domains showed that σNS association and VF localization phenotypes of G3BP1 do not occur solely through RNA or ribosomal binding but require both the RRM and RGG domains of G3BP1 for maximal viral-factory-like structure (VFL) localization and σNS association. Coexpression of σNS and µNS resulted in disruption of normal SG puncta, and in cells lacking G3BP1, MRV replication was enhanced in a manner correlating with strain-dependent induction of host translation shutoff. These results suggest that σNS association with G3BP1 and relocalization of G3BP1 to the VF periphery play roles in SG disruption to facilitate MRV replication in the host translational shutoff environment.IMPORTANCE SGs and SG effector proteins have emerged as important, yet poorly understood, players in the host's innate immune response to virus infection. MRV infection induces SGs early during infection that are dispersed and/or prevented from forming during late stages of infection despite continued activation of the eIF2α signaling pathway. Cellular and viral components involved in disruption of SGs during late stages of MRV infection remain to be elucidated. This work provides evidence that MRV disruption of SGs may be facilitated by association of the MRV nonstructural protein σNS with the major SG effector protein G3BP1 and subsequent localization of G3BP1 and other SG-associated proteins around the peripheries of virus-encoded factories, interrupting the normal formation of SGs. Our findings also reveal the importance of G3BP1 as an inhibitor of MRV replication during infection for the first time.

Characterization of a Replicating Mammalian Orthoreovirus with Tetracysteine-Tagged µNS for Live-Cell Visualization of Viral Factories.

Within infected host cells, mammalian orthoreovirus (MRV) forms viral factories (VFs), which are sites of viral transcription, translation, assembly, and replication. The MRV nonstructural protein µNS comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells, but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced µNS. We set out to develop a method to overcome this obstacle by utilizing the 6-amino-acid (CCPGCC) tetracysteine (TC) tag and FlAsH-EDT2 reagent. The TC tag was introduced into eight sites throughout µNS, and the capacity of the TC-µNS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-µNS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore, the rTC-µNS viruses were utilized to infect cells and examine VF dynamics using live-cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in-depth studies of VF dynamics and host cell interactions.IMPORTANCE MRV has historically been used as a model to study the double-stranded RNA (dsRNA) Reoviridae family, the members of which infect and cause disease in humans, animals, and plants. During infection, MRV forms VFs that play a critical role in virus infection but remain to be fully characterized. To study VFs, researchers have focused on visualizing the nonstructural protein µNS, which forms the VF matrix. This work provides the first evidence of recovery of replicating reoviruses in which VFs can be labeled in live cells via introduction of a TC tag into the µNS open reading frame. Characterization of each recombinant reovirus sheds light on µNS interactions with viral proteins. Moreover, utilizing the TC-labeling FlAsH-EDT2 biarsenical reagent to visualize VFs, evidence is provided of dynamic VF movement and interactions at least partially dependent on intact microtubules.

Highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of nonstructural protein 2 into virions.

Viral structural proteins form the critical intermediary between viral infection cycles within and between hosts, function to initiate entry, participate in immediate early viral replication steps, and are major targets for the host adaptive immune response. We report the identification of nonstructural protein 2 (nsp2) as a novel structural component of the porcine reproductive and respiratory syndrome virus (PRRSV) particle. A set of custom α-nsp2 antibodies targeting conserved epitopes within four distinct regions of nsp2 (the PLP2 protease domain [OTU], the hypervariable domain [HV], the putative transmembrane domain [TM], and the C-terminal region [C]) were obtained commercially and validated in PRRSV-infected cells. Highly purified cell-free virions of several PRRSV strains were isolated through multiple rounds of differential density gradient centrifugation and analyzed by immunoelectron microscopy (IEM) and Western blot assays using the α-nsp2 antibodies. Purified viral preparations were found to contain pleomorphic, predominantly spherical virions of uniform size (57.9 nm ± 8.1 nm diameter; n = 50), consistent with the expected size of PRRSV particles. Analysis by IEM indicated the presence of nsp2 associated with the viral particle of diverse strains of PRRSV. Western blot analysis confirmed the presence of nsp2 in purified viral samples and revealed that multiple nsp2 isoforms were associated with the virion. Finally, a recombinant PRRSV genome containing a myc-tagged nsp2 was used to generate purified virus, and these particles were also shown to harbor myc-tagged nsp2 isoforms. Together, these data identify nsp2 as a virion-associated structural PRRSV protein and reveal that nsp2 exists in or on viral particles as multiple isoforms.

Influenza A virus PB1-F2 protein expression is regulated in a strain-specific manner by sequences located downstream of the PB1-F2 initiation codon.

Translation of influenza A virus PB1-F2 occurs in a second open reading frame (ORF) of the PB1 gene segment. PB1-F2 has been implicated in regulation of polymerase activity, immunopathology, susceptibility to secondary bacterial infection, and induction of apoptosis. Experimental evidence of PB1-F2 molecular function during infection has been collected primarily from human and avian viral isolates. As the 2009 H1N1 (H1N1pdm09) strain highlighted, some swine-derived influenza viruses have the capacity to infect human hosts and emerge as a pandemic. Understanding the impact that virulence factors from swine isolates have on both human and swine health could aid in early identification of viruses with pandemic potential. Studies examining PB1-F2 from swine isolates have focused primarily on H1N1pdm09, which does not encode PB1-F2 but was engineered to carry a full-length PB1-F2 ORF to assess the impact on viral replication and pathogenicity. However, experimental evidence of PB1-F2 protein expression from swine lineage viruses has not been demonstrated. Here, we reveal that during infection, PB1-F2 expression levels are substantially different in swine and human influenza viruses. We provide evidence that PB1-F2 expression is regulated at the translational level, with very low levels of PB1-F2 expression from swine lineage viruses relative to a human isolate PB1-F2. Translational regulation of PB1-F2 expression was partially mapped to two independent regions within the PB1 mRNA, located downstream of the PB1-F2 start site. Our data suggest that carrying a full-length PB1-F2 ORF may not be predictive of PB1-F2 expression in infected cells for all influenza A viruses.

Creation and characterization of a recombinant mammalian orthoreovirus expressing σ1 fusion proteins encoding human epidermal growth factor receptor 2 peptides.

Mammalian orthoreovirus (MRV) is an oncolytic virus that has been tested in over 30 clinical trials. Increased clinical success has been achieved when MRV is used in combination with other onco-immunotherapies. This has led the field to explore the creation of recombinant MRVs which incorporate immunotherapeutic sequences into the virus genome. This work focuses on creation and characterization of a recombinant MRV, S1/HER2nhd, which encodes a truncated σ1 protein fused in frame with three human epidermal growth factor receptor 2 (HER2) peptides (E75, AE36, and GP2) known to induce HER2 specific CD8+ and CD4+ T cells. We show S1/HER2nhd expresses the σ1 fusion protein containing HER2 peptides in infected cells and on the virion, and infects, replicates in, and reduces survival of HER2+ breast cancer cells. The oncolytic properties of MRV combined with HER2 peptide expression holds potential as a vaccine to prevent recurrences of HER2 expressing cancers.

Mammalian orthoreovirus infection in human epidermal growth factor receptor 2 positive (HER2+) breast cancer cells.

Mammalian orthoreovirus (MRV) is a clinically benign oncolytic virus which has been investigated for use in multiple cancer types, including breast cancer (BC). In human clinical trials, MRV has been shown to be safe, and multiple BC patients have shown partial responses to intratumoral and intravenous virus delivery. Combination therapies inclusive of MRV and current FDA approved BC chemotherapies are being investigated to target metastatic, early BC, and triple negative BC. Though MRV is being tested clinically, we still do not fully understand the highly variable patient responses to MRV therapy. One of the most aggressive BC subtypes is HER2+ BC, in which human epidermal growth factor receptor 2 (HER2) is dysregulated, resulting in increased growth, survival, and metastasis of cancer cells. FDA approved therapies, trastuzumab and pertuzumab, target HER2 to prevent signaling of the phosphoinositide 3-kinase (PI3K) pathway. However, recent findings show that accumulation of hypoxia inducible factor-1 alpha (HIF-1α) in HER2+ BC cells contributes to trastuzumab resistance. In this work, we provide evidence that MRV infects, replicates in, and kills HER2 overexpressing cells. MRV infection is also found to have variable effects on signaling pathways that activate or are activated by HER2 expression. Finally, we show that MRV reduces HIF-1α accumulation in all the cell lines tested, including a HER2+ BC cell line. These studies provide further evidence that MRV holds promise for use in conjunction with trastuzumab to treat HER2+ BC patients.

Sequences at gene segment termini inclusive of untranslated regions and partial open reading frames play a critical role in mammalian orthoreovirus S gene packaging.

Mammalian orthoreovirus (MRV) is a prototypic member of the Spinareoviridae family and has ten double-stranded RNA segments. One copy of each segment must be faithfully packaged into the mature virion, and prior literature suggests that nucleotides (nts) at the terminal ends of each gene likely facilitate their packaging. However, little is known about the precise packaging sequences required or how the packaging process is coordinated. Using a novel approach, we have determined that 200 nts at each terminus, inclusive of untranslated regions (UTR) and parts of the open reading frame (ORF), are sufficient for packaging each S gene segment (S1-S4) individually and together into replicating virus. Further, we mapped the minimal sequences required for packaging the S1 gene segment to 25 5' nts and 50 3' nts. The S1 UTRs alone are not sufficient, but are necessary for packaging, as mutations of the 5' or 3' UTRs led to a complete loss of virus recovery. Using a second novel assay, we determined that 50 5'nts and 50 3' nts of S1 are sufficient to package a non-viral gene segment into MRV. The 5' and 3' termini of the S1 gene are predicted to form a panhandle structure and specific mutations within the predicted stem of the panhandle region led to a significant decrease in viral recovery. Additionally, mutation of six nts that are conserved in the three major serotypes of MRV and are predicted to form an unpaired loop in the S1 3'UTR, led to a complete loss of viral recovery. Overall, our data provide strong experimental proof that MRV packaging signals lie at the terminal ends of the S gene segments and offer support that the sequence requirements for efficient packaging of the S1 segment include a predicted panhandle structure and specific sequences within an unpaired loop in the 3' UTR.

Rational design of oral drugs targeting mucosa delivery with gut organoid platforms.

Effective oral drugs and vaccines require high delivery efficiency across the gastrointestinal epithelia and protection of medically effective payloads (i.e., immunogens) against gastric damage. In this study, hollowed nanocarriers (NCs: silica nanospheres and gold nanocages) with poly-l-lysine (PLL) coating and mammalian orthoreovirus cell attachment protein σ1 functionalization (NC-PLL-σ1) were explored as functional oral drug delivery vehicles (ODDVs). The transport of these ODDVs to mucosal lymphoid tissues could be facilitated by microfold cells (M-cells) mediated transcytosis (via σ1-α2-3-linked sialic acids adherence) across gastrointestinal epithelia. PLL coating provided protection and slow-release of rhodamine 6 G (R6G), a model payload. The transport effectiveness of these ODDVs was tested on intestinal organoid monolayers in vitro. When compared with other experimental groups, the fully functionalized ODDV system (with PLL-σ1) demonstrated two significant advantages: a significantly higher transport efficiency (198% over blank control at 48 h); and protection of payloads which led to both better transport efficiency and extended-release of payloads (61% over uncoated carriers at 48 h). In addition, it was shown that the M cell presence in intestinal organoid monolayers (modulated by Rank L stimulation) was a determining factor on the transport efficiency of the ODDVs: more M-cells (induced by higher Rank L) in the organoid monolayers led to higher transport efficiency for ODDV-delivered model payload (R6G). The fully functionalized ODDVs showed great potential as effective oral delivery vehicles for drugs and vaccines.

Mammalian orthoreovirus escape from host translational shutoff correlates with stress granule disruption and is independent of eIF2alpha phosphorylation and PKR.

In response to mammalian orthoreovirus (MRV) infection, cells initiate a stress response that includes eIF2α phosphorylation and protein synthesis inhibition. We have previously shown that early in infection, MRV activation of eIF2α phosphorylation results in the formation of cellular stress granules (SGs). In this work, we show that as infection proceeds, MRV disrupts SGs despite sustained levels of phosphorylated eIF2α and, further, interferes with the induction of SGs by other stress inducers. MRV interference with SG formation occurs downstream of eIF2α phosphorylation, suggesting the virus uncouples the cellular stress signaling machinery from SG formation. We additionally examined mRNA translation in the presence of SGs induced by eIF2α phosphorylation-dependent and -independent mechanisms. We found that irrespective of eIF2α phosphorylation status, the presence of SGs in cells correlated with inhibition of viral and cellular translation. In contrast, MRV disruption of SGs correlated with the release of viral mRNAs from translational inhibition, even in the presence of phosphorylated eIF2α. Viral mRNAs were also translated in the presence of phosphorylated eIF2α in PKR(-/-) cells. These results suggest that MRV escape from host cell translational shutoff correlates with virus-induced SG disruption and occurs in the presence of phosphorylated eIF2α in a PKR-independent manner.

An rRT-PCR assay to detect the matrix gene of a broad range of avian paramyxovirus serotype-1 strains.

The current U.S. Department of Agriculture (USDA)-validated real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay designed to detect the matrix gene of avian paramyxovirus serotype-1 (APMV-1) is the primary screening assay used in the United States. It has previously been shown to be unable to consistently detect all members of class I APMV-1. Diagnostic testing relies on rRT-PCR to quickly detect APMV-1 in wild birds, backyard flocks, live bird markets, commercial poultry, and for export testing. Limitations of the current USDA assay have raised concerns about the potential for some strains of APMV-1 to remain undetected by the primary screening assay. Mismatches in the probe were shown to cause a loss in template binding efficiency, resulting in lack of detection by the assay. Here, we describe the development and analytical validation of a new rRT-PCR assay designed to target a highly conserved region of the matrix gene across a wide range of APMV-1 strains. Limit of detection testing revealed a 3 log10 decrease in sensitivity for one low-virulence strain when compared to the USDA validated assay. Conversely, the assay showed increased sensitivity for a class I isolate and two virulent strains of APMV-1 that were not detected by the USDA-validated assay. The new assay also demonstrated a high degree of specificity by the lack of detection of 43 non-APMV-1 viruses.

Localization of mammalian orthoreovirus proteins to cytoplasmic factory-like structures via nonoverlapping regions of microNS.

Virally induced structures called viral factories form throughout the cytoplasm of cells infected with mammalian orthoreoviruses (MRV). When expressed alone in cells, MRV nonstructural protein microNS forms factory-like structures very similar in appearance to viral factories, suggesting that it is involved in forming the structural matrix of these structures. microNS also associates with MRV core particles; the core proteins mu2, lambda1, lambda2, lambda3, and sigma2; and the RNA-binding nonstructural protein sigmaNS. These multiple associations result in the recruitment or retention of these viral proteins or particles at factory-like structures. In this study, we identified the regions of microNS necessary and sufficient for these associations and additionally examined the localization of viral RNA synthesis in infected cells. We found that short regions within the amino-terminal 220 residues of microNS are necessary for associations with core particles and necessary and sufficient for associations with the proteins mu2, lambda1, lambda2, sigma2, and sigmaNS. We also found that only the lambda3 protein associates with the carboxyl-terminal one-third of microNS and that viral RNA is synthesized within viral factories. These results suggest that microNS may act as a cytoplasmic scaffolding protein involved in localizing and coordinating viral replication or assembly intermediates for the efficient production of progeny core particles during MRV infection.

Reovirus and the Host Integrated Stress Response: On the Frontlines of the Battle to Survive.

Cells are continually exposed to stressful events, which are overcome by the activation of a number of genetic pathways. The integrated stress response (ISR) is a large component of the overall cellular response to stress, which ultimately functions through the phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF2α) to inhibit the energy-taxing process of translation. This response is instrumental in the inhibition of viral infection and contributes to evolution in viruses. Mammalian orthoreovirus (MRV), an oncolytic virus that has shown promise in over 30 phase I-III clinical trials, has been shown to induce multiple arms within the ISR pathway, but it successfully evades, modulates, or subverts each cellular attempt to inhibit viral translation. MRV has not yet received Food and Drug Administration (FDA) approval for general use in the clinic; therefore, researchers continue to study virus interactions with host cells to identify circumstances where MRV effectiveness in tumor killing can be improved. In this review, we will discuss the ISR, MRV modulation of the ISR, and discuss ways in which MRV interaction with the ISR may increase the effectiveness of cancer therapeutics whose modes of action are altered by the ISR.

Inhibition of HIF-1α accumulation in prostate cancer cells is initiated during early stages of mammalian orthoreovirus infection.

Mammalian orthoreovirus (MRV) is a safe and effective cancer killing virus that has completed Phase I-III clinical trials against numerous cancer types. While many patients experience benefit from MRV therapy, pre-defined set points necessary for FDA approval have not been reached. Therefore, additional research into MRV biology and the effect of viral therapy on different tumor genetic subtypes and microenvironments is necessary to identify tumors most amenable to MRV virotherapy. In this work we analyzed the stage of viral infection necessary to inhibit HIF-1α, an aggressive cancer activator induced by hypoxia. We demonstrated that two viral capsid proteins were not necessary and that a step parallel with virus core movement across the endosomal membrane was required for this inhibition. Altogether, this work clarifies the mechanisms of MRV-induced HIF-1α inhibition and provides biological relevance for using MRV to inhibit the devastating effects of tumor hypoxia.

AatA is a novel autotransporter and virulence factor of avian pathogenic Escherichia coli.

Autotransporters (AT) are widespread in Gram-negative bacteria, and many of them are involved in virulence. An open reading frame (APECO1_O1CoBM96) encoding a novel AT was located in the pathogenicity island of avian pathogenic Escherichia coli (APEC) O1's virulence plasmid, pAPEC-O1-ColBM. This 3.5-kb APEC autotransporter gene (aatA) is predicted to encode a 123.7-kDa protein with a 25-amino-acid signal peptide, an 857-amino-acid passenger domain, and a 284-amino-acid beta domain. The three-dimensional structure of AatA was also predicted by the threading method using the I-TASSER online server and then was refined using four-body contact potentials. Molecular analysis of AatA revealed that it is translocated to the cell surface, where it elicits antibody production in infected chickens. Gene prevalence analysis indicated that aatA is strongly associated with E. coli from avian sources but not with E. coli isolated from human hosts. Also, AatA was shown to enhance adhesion of APEC to chicken embryo fibroblast cells and to contribute to APEC virulence.

Mammalian orthoreovirus particles induce and are recruited into stress granules at early times postinfection.

Infection with many mammalian orthoreovirus (MRV) strains results in shutoff of host, but not viral, protein synthesis via protein kinase R (PKR) activation and phosphorylation of translation initiation factor eIF2alpha. Following inhibition of protein synthesis, cellular mRNAs localize to discrete structures in the cytoplasm called stress granules (SGs), where they are held in a translationally inactive state. We examined MRV-infected cells to characterize SG formation in response to MRV infection. We found that SGs formed at early times following infection (2 to 6 h postinfection) in a manner dependent on phosphorylation of eIF2alpha. MRV induced SG formation in all four eIF2alpha kinase knockout cell lines, suggesting that at least two kinases are involved in induction of SGs. Inhibitors of MRV disassembly prevented MRV-induced SG formation, indicating that viral uncoating is a required step for SG formation. Neither inactivation of MRV virions by UV light nor treatment of MRV-infected cells with the translational inhibitor puromycin prevented SG formation, suggesting that viral transcription and translation are not required for SG formation. Viral cores were found to colocalize with SGs; however, cores from UV-inactivated virions did not associate with SGs, suggesting that viral core particles are recruited into SGs in a process that requires the synthesis of viral mRNA. These results demonstrate that MRV particles induce SGs in a step following viral disassembly but preceding viral mRNA transcription and that core particles are themselves recruited to SGs, suggesting that the cellular stress response may play a role in the MRV replication cycle.

Transport of artificial virus-like nanocarriers through intestinal monolayers via microfold cells.

Compared with subcutaneous or intramuscular routes for vaccination, vaccine delivery via the gastrointestinal mucosa has tremendous potential as it is easy to administer and pain-free. Robust immune responses can be triggered successfully once the vaccine carrying an antigen reaches the mucosal associated lymphoid sites (e.g., Peyer's patches). However, the absence of an efficient delivery method has always been an issue for successful oral vaccine development. In our study, inspired by mammalian orthoreovirus (MRV) transport into the gut mucosal lymphoid tissue via Microfold cells (M cells), artificial virus-like nanocarriers (AVNs), consisting of gold nanocages functionalized with the σ1 protein from mammalian reovirus (MRV), were tested as an effective oral vaccine delivery vehicle targeting M cells. AVNs were shown to have a significantly higher transport compared to other experimental groups across mouse organoid monolayers containing M cells. These findings suggest that AVNs have the potential to be an M cell-specific oral vaccine/drug delivery vehicle.

The 3' Untranslated Region of a Plant Viral RNA Directs Efficient Cap-Independent Translation in Plant and Mammalian Systems.

Many plant viral RNA genomes lack a 5' cap, and instead are translated via a cap-independent translation element (CITE) in the 3' untranslated region (UTR). The panicum mosaic virus-like CITE (PTE), found in many plant viral RNAs, binds and requires the cap-binding translation initiation factor eIF4E to facilitate translation. eIF4E is structurally conserved between plants and animals, so we tested cap-independent translation efficiency of PTEs of nine plant viruses in plant and mammalian systems. The PTE from thin paspalum asymptomatic virus (TPAV) facilitated efficient cap-independent translation in wheat germ extract, rabbit reticulocyte lysate, HeLa cell lysate, and in oat and mammalian (BHK) cells. Human eIF4E bound the TPAV PTE but not a PTE that did not stimulate cap-independent translation in mammalian extracts or cells. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) footprinting revealed that both human and wheat eIF4E protected the conserved guanosine (G)-rich domain in the TPAV PTE pseudoknot. The central G plays a key role, as it was found to be required for translation and protection from SHAPE modification by eIF4E. These results provide insight on how plant viruses gain access to the host's translational machinery, an essential step in infection, and raise the possibility that similar PTE-like mechanisms may exist in mRNAs of mammals or their viruses.