Riboswitch control of bacterial RNA stability.

Although riboswitches have long been known to regulate translation initiation and transcription termination, a growing body of evidence indicates that they can also control bacterial RNA lifetimes by acting directly to hasten or impede RNA degradation. Ligand binding to the aptamer domain of a riboswitch can accelerate RNA decay by triggering a conformational change that exposes sites to endonucleolytic cleavage or by catalyzing the self-cleavage of a prefolded ribozyme. Alternatively, the conformational change induced by ligand binding can protect RNA from degradation by blocking access to an RNA terminus or internal region that would otherwise be susceptible to attack by an exonuclease or endonuclease. Such changes in RNA longevity often accompany a parallel effect of the same riboswitch on translation or transcription. Consequently, a single riboswitch aptamer may govern the function of multiple effector elements (expression platforms) that are co-resident within a transcript and act independently of one another.

Novel Oncolytic Herpes Simplex Virus 1 VC2 Promotes Long-Lasting, Systemic Anti-melanoma Tumor Immune Responses and Increased Survival in an Immunocompetent B16F10-Derived Mouse Melanoma Model.

Oncolytic virotherapy (OVT) is now understood to be an immunotherapy that uses viral infection to liberate tumor antigens in an immunogenic context to promote the development of antitumor immune responses. The only currently FDA-approved oncolytic virotherapy, T-Vec, is a modified type 1 herpes simplex virus (HSV-1). While T-Vec is associated with limited response rates, its modest efficacy supports the continued development of novel OVT viruses. Herein, we test the efficacy of a recombinant HSV-1, VC2, as an OVT in a syngeneic B16F10-derived mouse model of melanoma. VC2 possesses mutations that block its ability to enter neurons via axonal termini. This greatly enhances its safety profile by precluding the ability of the virus to establish latent infection. VC2 has been shown to be a safe, effective vaccine against both HSV-1 and HSV-2 infection in mice, guinea pigs, and nonhuman primates. We found that VC2 slows tumor growth rates and that VC2 treatment significantly enhances survival of tumor-engrafted, VC2-treated mice over control treatments. VC2-treated mice that survived initial tumor engraftment were resistant to a second engraftment as well as colonization of lungs by intravenous introduction of tumor cells. We found that VC2 treatment induced substantial increases in intratumoral T cells and a decrease in immunosuppressive regulatory T cells. This immunity was critically dependent on CD8+ T cells and less dependent on CD4+ T cells. Our data provide significant support for the continued development of VC2 as an OVT for the treatment of human and animal cancers.IMPORTANCE Current oncolytic virotherapies possess limited response rates. However, when certain patient selection criteria are used, oncolytic virotherapy response rates have been shown to increase. This, in addition to the increased response rates of oncolytic virotherapy in combination with other immunotherapies, suggests that oncolytic viruses possess significant therapeutic potential for the treatment of cancer. As such, it is important to continue to develop novel oncolytic viruses as well as support basic research into their mechanisms of efficacy. Our data demonstrate significant clinical potential for VC2, a novel type 1 oncolytic herpes simplex virus. Additionally, due to the high rates of survival and the dependence on CD8+ T cells for efficacy, our model will enable study of the immunological correlates of protection for VC2 oncolytic virotherapy and oncolytic virotherapy in general. Understanding the mechanisms of efficacious oncolytic virotherapy will inform the rational design of improved oncolytic virotherapies.

The Vc2 Cyclic di-GMP-Dependent Riboswitch of Vibrio cholerae Regulates Expression of an Upstream Putative Small RNA by Controlling RNA Stability.

Cyclic di-GMP (c-di-GMP) is a bacterial second messenger molecule that is important in the biology of Vibrio cholerae, but the molecular mechanisms by which this molecule regulates downstream phenotypes have not been fully characterized. We have previously shown that the Vc2 c-di-GMP-binding riboswitch, encoded upstream of the gene tfoY, functions as an off switch in response to c-di-GMP. However, the mechanism by which c-di-GMP controls expression of tfoY has not been fully elucidated. During our studies of this mechanism, we determined that c-di-GMP binding to Vc2 also controls the abundance and stability of upstream noncoding RNAs with 3' ends located immediately downstream of the Vc2 riboswitch. Our results suggest these putative small RNAs (sRNAs) are not generated by transcriptional termination but rather by preventing degradation of the upstream untranslated RNA when c-di-GMP is bound to Vc2.IMPORTANCE Riboswitches are typically RNA elements located in the 5' untranslated region of mRNAs. They are highly structured and specifically recognize and respond to a given chemical cue to alter transcription termination or translation initiation. In this work, we report a novel mechanism of riboswitch-mediated gene regulation in Vibrio cholerae whereby a 3' riboswitch, named Vc2, controls the stability of upstream untranslated RNA upon binding to its cognate ligand, the second messenger cyclic di-GMP, leading to the accumulation of previously undescribed putative sRNAs. We further demonstrate that binding of the ligand to the riboswitch prevents RNA degradation. As binding of riboswitches to their ligands often produces compactly structured RNA, we hypothesize this mechanism of gene regulation is widespread.

Cyclic di-GMP Regulates TfoY in Vibrio cholerae To Control Motility by both Transcriptional and Posttranscriptional Mechanisms.

3',5'-Cyclic diguanylic acid (c-di-GMP) is a bacterial second messenger molecule that is a key global regulator in Vibrio cholerae, but the molecular mechanisms by which this molecule regulates downstream phenotypes have not been fully characterized. One such regulatory factor that may respond to c-di-GMP is the Vc2 c-di-GMP-binding riboswitch that is hypothesized to control the expression of the downstream putative transcription factor TfoY. Although much is known about the physical and structural properties of the Vc2 riboswitch aptamer, the nature of its expression and function in V. cholerae has not been investigated. Here, we show that Vc2 functions as an off switch to inhibit TfoY production at intermediate and high concentrations of c-di-GMP. At low c-di-GMP concentrations, TfoY production is induced to stimulate dispersive motility. We also observed increased transcription of tfoY at high intracellular concentrations of c-di-GMP, but this induction is independent of the Vc2 riboswitch and occurs via transcriptional control of promoters upstream of tfoY by the previously identified c-di-GMP dependent transcription factor VpsR. Our results show that TfoY is induced by c-di-GMP at both low and high intracellular concentrations of c-di-GMP via posttranscriptional and transcriptional mechanisms, respectively. This regulation contributes to the formation of three distinct c-di-GMP signaling states in V. choleraeIMPORTANCE The bacterial pathogen Vibrio cholerae must transition between life in aquatic environmental reservoirs and life in the gastrointestinal tract. Biofilm formation and bacterial motility, and their control by the second messenger molecule c-di-GMP, play integral roles in this adaptation. Here, we define the third major mechanism by which c-di-GMP controls bacterial motility. This pathway utilizes a noncoding RNA element known as a riboswitch that, when bound to c-di-GMP, inhibits the expression of the transcription factor TfoY. TfoY production switches V. cholerae motility from a dense to a dispersive state. Our results suggest that the c-di-GMP signaling network of V. cholerae can exist in at least three distinct states to regulate biofilm formation and motility.

The Oncolytic herpes simplex virus type-1 (HSV-1) vaccine strain VC2 causes intratumor infiltration of functionally active T cells and inhibition of tumor metastasis and pro-tumor genes VEGF and PDL1 expression in the 4T1/Balb/c mouse model of stage four breast cancer.

Introduction: Oncolytic viruses (OVs) provide new modalities for cancer therapy either alone or in combination with synergistic immunotherapies and/or chemotherapeutics. Engineered Herpes Simplex Virus Type-1 (HSV-1) has shown strong promise for the treatment of various cancers in experimental animal models as well as in human patients, with some virus strains licensed to treat human melanoma and gliomas. In the present study we evaluated the efficacy of mutant HSV-1 (VC2) in a late stage, highly metastatic 4T1 murine syngeneic. Method: VC2 was constructed VC2 using double red recombination technology. For in-vivo efficacy we utilized a late stage 4T1 syngeneic and immunocompetent BALB/cJ mouse model breast cancer model which exhibits efficient metastasis to the lung and other organs. Results: VC2 replicated efficiently in 4T1 cells and in cell culture, achieving titers similar to those in African monkey kidney (Vero) cells. Intra-tumor treatment with VC2 did not appreciably reduce average primary tumor sizes but a significant reduction of lung metastasis was noted in mice treated intratumorally with VC2, but not with ultraviolet-inactivated VC2. This reduction of metastasis was associated with increased T cell infiltration comprised of CD4+ and CD4+CD8+ double-positive T cells. Characterization of purified tumor infiltrating T cells revealed a significant improvement in their proliferation ability compared to controls. In addition, significant T cell infiltration was observed in the metastatic nodules associated with reduction of pro-tumor PD-L1 and VEGF gene transcription. Conclusion: These results show that VC2 therapy can improve anti-tumor response associated with a better control of tumor metastasis. improve T cell responses and reduce pro-tumor biomarker gene transcription. VC2 holds promise for further development as an oncolytic and immunotherapeutic approach to treat breast and other cancers.

Inactivation of the UL37 Deamidase Enhances Virus Replication and Spread of the HSV-1(VC2) Oncolytic Vaccine Strain and Secretion of GM-CSF.

The HSV-1 (VC2) live-attenuated vaccine strain was engineered with specific deletions in the amino termini of glycoprotein K (gK) and membrane protein UL20, rendering the virus unable to enter neurons and establish latency. VC2 replicates efficiently in epithelial cell culture but produces lower viral titers and smaller viral plaques than its parental HSV-1 (F) wild-type virus. VC2 is an effective live-attenuated vaccine against HSV-1 and HSV-2 infections in mice and guinea pigs and an anti-tumor immunotherapeutic and oncolytic virus against melanoma and breast cancer in mouse models. Previously, we reported that the gK/UL20 complex interacts with the UL37 tegument protein, and this interaction is essential for virion intracellular envelopment and egress. To investigate the potential role of the UL37 deamidase functions, the recombinant virus FC819S and VC2C819S were constructed with a C819S substitution to inactivate the UL37 predicted deamidase active site on an HSV-1(F) and HSV-1(VC2) genetic background, respectively. FC819S replicated to similar levels with HSV-1(F) and produced similar size viral plaques. In contrast, VC2C819S replication was enhanced, and viral plaques increased in size, approaching those of the wild-type HSV-1(F) virus. FC819S infection of cell cultures caused enhanced GM-CSF secretion in comparison to HSV-1(F) across several cell lines, including HEp2 cells and cancer cell lines, DU145 (prostate) and Panc 04.03 (pancreas), and primary mouse peritoneal cells. VC2 infection of these cell lines caused GM-CSF secretion at similar levels to FC819S infection. However, the VC2C819S virus did not exhibit any further enhancement of GM-CSF secretion compared to the VC2 virus. These results suggest that the UL37 deamidation functions in conjunction with the gK/UL20 complex to facilitate virus replication and GM-CSF secretion.

Cross protective efficacy of the Non-Neurotropic live attenuated herpes simplex virus type 1 vaccine VC-2 is enhanced by intradermal vaccination and deletion of glycoprotein G.

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2 respectively) cause life-long latent infections resulting in recurrent orofacial and genital blisters or sores. Ensued disease can be painful and may lead to significant mental anguish of infected individuals. Currently, there are no FDA-approved vaccines for either prophylactic or therapeutic use, and recent clinical trials of subunit vaccines failed to achieve endpoints goals. Development of a safe live-attenuated herpes simplex vaccine may provide the antigenic breadth to ultimately protect individuals from acquiring HSV disease. We have previously shown that prophylactic use of the non-neurotropic live attenuated HSV-1 vaccine, VC-2, provides potent and durable protection from genital HSV-2 disease in the guinea pig model. Here, we investigated the effects of intradermal administration as well as the deletion of the viral glycoprotein G (gG) on the efficacy of prophylactic vaccination. Vaccination with either VC-2, VC-2 gG null, or gD2 MPL/Alum offered robust protection from acute disease regardless of route of vaccination. However, both the VC-2 gG-null and the ID vaccination route were more effective compared to the parent VC2 administered by the IM route. Specifically, the VC-2 gG-null administered ID, reduced HSV-2 vaginal replication on day 2 and day 4 as well as mean recurrent lesion scores more effectively than VC2 administered IM. Most importantly, only VC-2 gG null IM and VC-2 ID significantly reduced the frequency of recurrent shedding, the most likely source for virus transmission. Similarly, while all vaccinated groups demonstrated a significant reduction in the number of animals testing PCR-positive for HSV-2 in their dorsal root ganglia following challenge only VC2 ID vaccinated animals demonstrated a significant reduction in DRG viral load. All vaccinations induced neutralizing antibodies to HSV-2 MS when compared to unvaccinated guinea pigs. Therefore, further investigation of VC-2 gG null delivered ID is warranted.

Utility of a Recombinant HSV-1 Vaccine Vector for Personalized Cancer Vaccines.

Current approaches to cancer immunotherapy include immune checkpoint inhibitors, cancer vaccines, and adoptive cellular therapy. These therapies have produced significant clinical success for specific cancers, but their efficacy has been limited. Oncolytic virotherapy (OVT) has emerged as a promising immunotherapy for a variety of cancers. Furthermore, the unique characteristics of OVs make them a good choice for delivering tumor peptides/antigens to induce enhanced tumor-specific immune responses. The first oncolytic virus (OV) approved for human use is the attenuated herpes simplex virus type 1 (HSV-1), Talimogene laherparepvec (T-VEC) which has been FDA approved for the treatment of melanoma in humans. In this study, we engineered the recombinant oncolytic HSV-1 (oHSV) VC2-OVA expressing a fragment of ovalbumin (OVA) as a fusion protein with VP26 virion capsid protein. We tested the ability of VC2-OVA to act as a vector capable of stimulating strong, specific antitumor immunity in a syngeneic murine melanoma model. Therapeutic vaccination with VC2-OVA led to a significant reduction in colonization of tumor cells in the lungs of mice intravenously challenged B16cOVA cells. In addition, VC2-OVA induced a potent prophylactic antitumor response and extended survival of mice that were intradermally engrafted with B16cOVA tumors compared with mice immunized with control virus.

An Attenuated HSV-1-Derived Malaria Vaccine Expressing Liver-Stage Exported Proteins Induces Sterilizing Protection against Infectious Sporozoite Challenge.

Here, we present the construction of an attenuated herpes simplex virus type-1 (HSV-1)-vectored vaccine, expressing three liver-stage (LS) malaria parasite exported proteins (EXP1, UIS3 and TMP21) as fusion proteins with the VP26 viral capsid protein. Intramuscular and subcutaneous immunizations of mice with a pooled vaccine, composed of the three attenuated virus strains expressing each LS antigen, induced sterile protection against the intravenous challenge of Plasmodium yoelii 17X-NL salivary gland sporozoites. Our data suggest that this malaria vaccine may be effective in preventing malaria parasite infection using practical routes of immunization in humans.

The Effect of Herpes Simplex Virus-Type-1 (HSV-1) Oncolytic Immunotherapy on the Tumor Microenvironment.

The development of cancer causes disruption of anti-tumor immunity required for surveillance and elimination of tumor cells. Immunotherapeutic strategies aim for the restoration or establishment of these anti-tumor immune responses. Cancer immunotherapies include immune checkpoint inhibitors (ICIs), adoptive cellular therapy (ACT), cancer vaccines, and oncolytic virotherapy (OVT). The clinical success of some of these immunotherapeutic modalities, including herpes simplex virus type-1 derived OVT, resulted in Food and Drug Administration (FDA) approval for use in treatment of human cancers. However, a significant proportion of patients do not respond or benefit equally from these immunotherapies. The creation of an immunosuppressive tumor microenvironment (TME) represents an important barrier preventing success of many immunotherapeutic approaches. Mechanisms of immunosuppression in the TME are a major area of current research. In this review, we discuss how oncolytic HSV affects the tumor microenvironment to promote anti-tumor immune responses. Where possible we focus on oncolytic HSV strains for which clinical data is available, and discuss how these viruses alter the vasculature, extracellular matrix and immune responses in the tumor microenvironment.

Live Cell Imaging Using Riboswitch-Spinach tRNA Fusions as Metabolite-Sensing Fluorescent Biosensors.

The development of fluorescent biosensors is motivated by the desire to monitor cellular metabolite levels in real time. Most genetically encodable fluorescent biosensors are based on receptor proteins fused to fluorescent protein domains. More recently, small molecule-binding riboswitches have been adapted for use as fluorescent biosensors through fusion to the in vitro selected Spinach aptamer, which binds a profluorescent, cell-permeable small molecule mimic of the GFP chromophore, DFHBI. Here we describe methods to prepare and analyze riboswitch-Spinach tRNA fusions for ligand-dependent activation of fluorescence in vivo. Example procedures describe the use of the Vc2-Spinach tRNA biosensor to monitor perturbations in cellular levels of cyclic di-GMP using either fluorescence microscopy or flow cytometry. In this updated chapter, we have added procedures on using biosensors in flow cytometry to detect exogenously added compounds. The relative ease of cloning and imaging of these biosensors, as well as their modular nature, should make this method appealing to other researchers interested in utilizing riboswitch-based biosensors for metabolite sensing.

First Impressions-the Potential of Altering Initial Host-Virus Interactions for Rational Design of Herpesvirus Vaccine Vectors.

PURPOSE: The earliest host-virus interactions occur during virus attachment and entry into cells. These initial steps in the virus lifecycle influence the outcome of infection beyond delivery of the viral genome into the cell. Herpesviruses alter host signaling pathways and processes during attachment and entry to facilitate virus infection and modulate innate immune responses. We suggest in this review that understanding these early signaling events may inform the rational design of therapeutic and prevention strategies for herpesvirus infection, as well as the engineering of viral vectors for immunotherapy purposes. RECENT FINDINGS: Recent reports demonstrate that modulation of Herpes Simplex Virus Type-1 (HSV-1) entry results in unexpected enhancement of antiviral immune responses. SUMMARY: A variety of evidence suggests that herpesviruses promote specific cellular signaling responses that facilitate viral replication after binding to cell surfaces, as well as during virus entry. Of particular interest is the ability of the virus to alter innate immune responses through these cellular signaling events. Uncovering the underlying immune evasion strategies may lead to the design of live-attenuated vaccines that can generate robust and protective anti-viral immune responses against herpesviruses. These adjuvant properties may be extended to a variety of heterologous antigens expressed by herpesviral vectors.

Structure and activity of contryphan-Vc2: Importance of the d-amino acid residue.

In natural proteins and peptides, amino acids exist almost invariably as l-isomers. There are, however, several examples of naturally-occurring peptides containing d-amino acids. In this study we investigated the role of a naturally-occurring d-amino acid in a small peptide identified in the transcriptome of a marine cone snail. This peptide belongs to a family of peptides known as contryphans, all of which contain a single d-amino acid residue. The solution structure of this peptide was solved by NMR, but further investigations with molecular dynamics simulations suggest that its solution behaviour may be more dynamic than suggested by the NMR ensemble. Functional tests in mice uncovered a novel bioactivity, a depressive phenotype that contrasts with the hyperactive phenotypes typically induced by contryphans. Trp3 is important for bioactivity, but this role is independent of the chirality at this position. The d-chirality of Trp3 in this peptide was found to be protective against enzymatic degradation. Analysis by NMR and molecular dynamics simulations indicated an interaction of Trp3 with lipid membranes, suggesting the possibility of a membrane-mediated mechanism of action for this peptide.

In vitro analysis of riboswitch-Spinach aptamer fusions as metabolite-sensing fluorescent biosensors.

The development of fluorescent biosensors has been motivated by the interest to monitor and measure the levels of specific metabolites in live cells in real time. Common approaches include fusing a protein-based receptor to fluorescent proteins or synthesizing a small molecule reactive probe. Natural metabolite-sensing riboswitches also have been used in reporter-based systems that take advantage of ligand-dependent regulation of downstream gene expression. More recently, it has been shown that RNA-based fluorescent biosensors can be generated by fusing a riboswitch aptamer to the in vitro selected Spinach aptamer, which binds a cell-permeable and conditionally fluorescent molecule. Here, we describe methods to design, prepare, and analyze riboswitch-Spinach aptamer fusion RNAs for ligand-dependent activation of fluorescence in vitro. Examples of procedures to measure fluorescence activation, ligand binding selectivity and affinity, and binding kinetics are given for a cyclic di-GMP-responsive biosensor. The relative ease of in vitro RNA synthesis and purification should make this method accessible to other researchers interested in developing riboswitch-based fluorescent biosensors.