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.

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.

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.

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.

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.

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.