hnRNPA2B1 Associated with Recruitment of RNA into Exosomes Plays a Key Role in Herpes Simplex Virus 1 Release from Infected Cells.

hnRNPA2B1, an abundant cellular protein, has been reported to recruit RNAs bearing a specific sequence (EXO motif) into exosomes. We characterized an exosome population averaging 100 ± 50 nm in diameter and containing a defined set of constitutive exosome markers. This population packages microRNAs (miRNAs) and can be directed to block targeted gene expression in a dose-dependent fashion. The objective of this study was to characterize the role of hnRNPA2B1 in the recruitment of miRNA. We report the following four key findings. (i) hnRNPA2B1 is not a component of exosomes produced in HEp-2 or HEK293T cells. Hence, hnRNPA2B1 carries its cargo, at most, to the site of exosome assembly, but it is not itself incorporated into exosomes. (ii) The accumulation of exosomes produced by cells in which the gene encoding hnRNPA2B1 has been knocked out (ΔhnRNPA2B1 cells) was reduced 3-fold. (iii) In uninfected HEp-2 cells, hnRNPA2B1 is localized in the nucleus. In cells infected with herpes simplex virus 1 (HSV-1), hnRNPA2B1 was quantitatively exported to the cytoplasm and at least a fraction of hnRNPA2B1 colocalized with a Golgi marker. (iv) Lastly, in ΔhnRNPA2B1 cells, there was a 2- to 3-fold reduction in virus yield but a significant (>10-fold) reduction in HSV-1 released through the apical surface into the extracellular environment. The absence of hnRNPA2B1 had no significant impact on the basolateral export of HSV-1 from infected to uninfected cells by direct cell-to-cell contact. The results suggest that hnRNPA2B1 plays a key role in the transport of enveloped virus from its site of assembly to the extracellular environment.IMPORTANCE In this report, we show that hnRNPA2B1 is not a component of exosomes produced in HEp-2 or HEK293T cells. In herpes simplex virus 1 (HSV-1)-infected cells, hnRNPA2B1 was quantitatively translocated from the nucleus into the cytoplasm. In infected ΔhnRNPA2B1 cells, Golgi-dependent transport of virus from the apical surface to the extracellular medium was significantly reduced. In essence, this report supports the hypothesis that hnRNPA2B1 plays a key role in the egress of exosomes and HSV-1 from infected cells.

Herpes Simplex Virus 1 MicroRNA miR-H28 Exported to Uninfected Cells in Exosomes Restricts Cell-to-Cell Virus Spread by Inducing Gamma Interferon mRNA.

An earlier report showed that herpes simplex virus 1 (HSV-1) expresses two microRNAs (miRNAs), miR-H28 and miR-H29, late in the infectious cycle. The miRNAs are packed in exosomes and, in recipient cells, restrict the transmission of virus from infected cells to uninfected cells. We now report that (i) miR-H28 induced the synthesis of gamma interferon (IFN-γ) in both infected cells and cells transfected with miR-H28, (ii) IFN-γ accumulated concurrently with viral proteins in infected cells, (iii) IFN-γ was produced in HEp-2 cells derived from cancer tissue and in HEK293T cells derived from normal tissue, and (iv) HSV-1 replication was affected by exposure to IFN-γ before infection but not during or after infection. The results presented in this report support the growing body of evidence indicating that HSV-1 encodes functions designed to reduce the spread of infection from infected cells to uninfected cells, possibly in order to maximize the transmission of virus from infected individuals to uninfected individuals.IMPORTANCE In this report, we show that IFN-γ is produced by HSV-1 viral miR-H28 and viral replication is blocked in cells exposed to IFN-γ before infection but not during or after infection. The inevitable conclusion is that HSV-1 induces IFN-γ to curtail its spread from infected cells to uninfected cells. In essence, this report supports the hypothesis that HSV-1 encodes functions that restrict the transmission of virus from cell to cell.

Exosomes containing miRNAs targeting HER2 synthesis and engineered to adhere to HER2 on tumor cells surface exhibit enhanced antitumor activity.

BACKGROUND: Exosomes are small, cellular membrane-derived vesicles with a diameter of 50-150 nm. Exosomes are considered ideal drug delivery systems with a wide range of applications in various diseases, including cancer. However, nonspecific delivery of therapeutic agents by exosomes in vivo remains challenging. Human epidermal growth factor receptor 2 (HER2) is an epidermal growth factor receptor tyrosine kinase, and its overexpression is usually associated with cell survival and tumor progression in various cancers. In this study, we aim to develop novel exosomes with dual HER2-targeting ability as a nanoparticle delivery vehicle to enhance antitumor efficacy in vivo. RESULTS: Here, we report the generation of two kinds of exosomes carrying miRNAs designed to block HER2 synthesis, which consequently showed a distinct anti-tumor effect. The 293-miR-HER2 exosomes package and deliver miRNAs targeting HER2 to recipient cells to block HER2 synthesis. The anti-tumor effect of these exosomes on cancer cells dependent on HER2 for survival but do not affect cells that lack HER2 or that are engineered to express HER2 but are not dependent on it for survival. In contrast, 293-miR-XS-HER2 exosomes carry an additional peptide, which enables them to adhere to HER2 on the surface of cancer cells. Consequently, these exosomes preferentially enter these cells with surface expression of HER2 and further displayed a tumoricidal effect. The 293-miR-XS-HER2 exosomes are significantly more effective than the 293-miR-HER2 exosomes in shrinking HER2-positive tumors implanted in mice. CONCLUSIONS: Collectively, as novel antitumor drug delivery vehicles, HER2 dual-targeting exosomes exhibit increased target-specific delivery efficiency and can be further utilized to develop new nanoparticle-based targeted therapies.

miRNAs Targeting ICP4 and Delivered to Susceptible Cells in Exosomes Block HSV-1 Replication in a Dose-Dependent Manner.

miRNAs are potent tools that in principle can be used to control the replication of infectious agents. The objectives of the studies reported here were to design miRNAs that can block the replication of herpes simplex virus 1 and which could be delivered to infected cells via exosomes. We report the following: (1) We designed three miRNAs targeting the mRNA encoding ICP4, an essential viral regulatory protein. Of the three miRNAs, one miRNA401 effectively blocked ICP4 accumulation and viral replication on transfection into susceptible cells. (2) To facilitate packaging of the miRNA into exosomes, we incorporated into the sequence of miRNA401 an exosome-packaging motif. miRNA401 was shown to be packaged into exosomes and successfully delivered by exosomes to susceptible cells, where it remained stable for at least 72 hr. Finally, the results show that miRNA401 delivered to cells via exosomes effectively reduced virus yields in a miRNA401 dose-dependent fashion. The protocol described in this report can be applied to study viral gene functions without actually deleting or mutagenizing the gene.

miR-H28 and miR-H29 expressed late in productive infection are exported and restrict HSV-1 replication and spread in recipient cells.

We report on the properties and function of two herpes simplex virus-1 (HSV-1) microRNAs (miRNAs) designated "miR-H28" and "miR-H29." Both miRNAs accumulate late in productive infection at a time when, for the most part, viral DNA and proteins have been made. Ectopic expression of miRNA mimics in human cells before infection reduced the accumulation of viral mRNAs and proteins, reduced plaque sizes, and at vey low multiplicities of infection reduced viral yields. The specificity of the miRNA mimics was tested in two ways. First, ectopic expression of mimics carrying mutations in the seed sequence was ineffective. Second, in similar tests two viral miRNAs made early in productive infection also had no effect. Both miR-H28 and miR-H29 are exported from infected cells in exosomes. A noteworthy finding is that both miR-H28 and miR-H29 were absent from murine ganglia harboring latent virus but accumulated in ganglia in which the virus was induced to reactivate. The significance of these findings rests on the principle that the transmission of HSV from person to person is by physical contact between the infected tissues of the donor and those of uninfected recipient. Diminished size of primary or recurrent lesions could be predicted to enhance person-to-person transmission. Reduction in the amount of reactivating latent virus would reduce the risk of retrograde transport to the CNS but would not interfere with anterograde transport to a site at or near the site of initial infection.

Enhanced therapeutic efficacy for glioblastoma immunotherapy with an oncolytic herpes simplex virus armed with anti-PD-1 antibody and IL-12.

Glioblastoma is the most common and aggressive malignant brain tumor and has limited treatment options. Hence, innovative approaches are urgently needed. Oncolytic virus therapy is emerging as a promising modality for cancer treatment due to its tumor-specific targeting and immune-stimulatory properties. In this study, we developed a new generation of oncolytic herpes simplex virus C5252 by deletion of a 15-kb internal repeat region and both copies of γ34.5 genes. Additionally, C5252 was armed with anti-programmed cell death protein 1 antibody and interleukin-12 to enhance its therapeutic efficacy for glioblastoma immune-virotherapy. In vitro and in vivo experiments demonstrate that C5252 has a remarkable safety profile and potent anti-tumor activity against glioblastoma. Mechanistic studies demonstrated that C5252 specifically induces cell apoptosis by caspase-3/7 activation via downregulating ciliary neurotrophic factor receptor α. Furthermore, the enhanced anti-tumor therapeutic efficacy of C5252 in a subcutaneous glioblastoma model and an orthotopic glioblastoma model was confirmed. Moreover, syngeneic mouse models showed that the murine surrogate of C5252 has superior anti-tumor activity compared to the unarmed backbone virus, with enhanced immune activation. Taken together, our findings support C5252 as a promising therapeutic option for glioblastoma treatment, positioning it as a highly promising candidate for clinical translation.

Histone Deacetylase Inhibitor Panobinostat Benefits the Therapeutic Efficacy of Oncolytic Herpes Simplex Virus Combined with PD-1/PD-L1 Blocking in Glioma and Squamous Cell Carcinoma Models.

BACKGROUND: Combination therapy has been widely explored for oncolytic virus (OV), as it can be met with tumor resistance. The HDAC inhibitor (HDACi) panobinostat is a potent pan-deacetylase inhibitor which blocks multiple cancer-related pathways and reverses epigenetic events in cancer progression. METHODS: In this study, oncolytic activity in vitro and antitumor therapeutic efficacy in vivo when combined with oHSV and panobinostat were investigated. RESULTS: (1) Treatment with panobinostat enhanced oHSV propagation and cytotoxicity in human glioma A172 and squamous cell carcinoma SCC9 cells. (2) Combined treatment with oHSV and panobinostat enhanced virus replication mediated by the transcriptional downregulation of IFN-ß- and IFN-responsive antiviral genes in human glioma A172 and squamous cell carcinoma SCC9 cells. (3) Panobinostat treatment induced upregulation of PD-L1 expression in both glioma and squamous cell carcinoma cells. (4) A significantly enhanced therapeutic efficacy was shown in vivo for the murine glioma CT-2A and squamous cell carcinoma SCC7 models when treated with a combination of oHSV, including PD-1/PD-L1 blockade and HDAC inhibition. CONCLUSIONS: Consequently, these data provide some new clues for the clinical development of combination therapy with OVs, epigenetic modifiers, and checkpoint blockades for glioma and squamous cell carcinoma.

Oncolytic herpes simplex virus delivery of dual CAR targets of CD19 and BCMA as well as immunomodulators to enhance therapeutic efficacy in solid tumors combined with CAR T cell therapy.

Background: The CAR T-cell therapy is a promising approach to treating hematologic malignancies. However, the application in solid tumors still has many tough challenges, including heterogenicity in antigen expressions and immunosuppressive tumor microenvironment (TME). As a new cancer treatment modality, oncolytic virotherapy can be engineered to circumvent these obstacles for CAR T cell therapy in solid tumors. Methods: In this study, an oHSV T7011 is engineered to drive ectopic expression of dual-antigens, extracellular domains of CD19 and BCMA, on the solid tumor cell surface to be targeted by approved CAR T cells. In addition, multiple immunomodulators, CCL5, IL-12, and anti-PD-1 antibody are also included to modulate the TME. The antitumor activities of T7011 in combination with CD19 or BCMA CAR T-cell were evaluated in vitro and in vivo. Results: The expression of CD19 or BMCA on the tumor cell surface could be detected after T7011 infection. The level of CCL5 in TME was also increased. Efficacy studies demonstrated that combination with T7011 and CAR-TCD19 or CAR-TBCMA cells showed significant synergistic anti-tumor responses in several solid tumor models. Conclusion: These studies indicated that the new generation of oHSV T7011 can be a promising combinational therapy with CD19 or BCMA-specific CAR T cells for the treatment of a broad range of solid tumors.

Identification of Rab27a as a host factor for oncolytic herpes virus susceptibility to tumor cells.

Oncolytic viruses are emerging as therapeutic agents in oncology. However, resistance of tumor cells to HSV oncolysis pose significant barriers to antitumor response. Thus, study on the mechanisms of therapeutic resistance to oHSV and finding strategies for overcoming these mechanisms are needed. In this study, Rab27a, a small GTPase involved in exosome biogenesis, was noticed to highly correlate with the susceptibility of tumor cells to oHSV. We found that i) lower abundance of Rab27a in oHSV resistant mouse tumor cells was shown when compared to that of sensitive tumor cells through deep-sequencing; ii) the resistance of human tumor cells to oHSV infection is associated with a downregulation of Rab27a expression and overexpression of Rab27a can promote the replication capacity of oHSV; iii) Interestingly, a stabilizer protein of Rab27a, KIBRA, highly accumulated in oHSV resistant tumor cells, which is in contrast with the expression pattern of Rab27a. Furthermore, knockdown of KIBRA expression reduced oHSV replication in oHSV resistant tumor cells. Consequently, Rab27a was found to be relevant with oHSV replication without cell type specificity, and low abundance of Rab27a contributes to oHSV resistance in both mouse and human tumor cells, which will give new insights in the identification of potential targets or biomarkers for oHSV cancer therapy.

Enhancing Therapeutic Efficacy of Oncolytic Herpes Simplex Virus with MEK Inhibitor Trametinib in Some BRAF or KRAS-Mutated Colorectal or Lung Carcinoma Models.

Oncolytic virus (OV) as a promising therapeutic agent can selectively infect and kill tumor cells with naturally inherited or engineered properties. Considering the limitations of OVs monotherapy, combination therapy has been widely explored. MEK inhibitor (MEKi) Trametinib is an FDA-approved kinase inhibitor indicated for the treatment of tumors with BRAF V600E or V600K mutations. In this study, the oncolytic activity in vitro and anti-tumor therapeutic efficacy in vivo when combined with oHSV and MEKi Trametinib were investigated. We found: (1) Treatment with MEKi Trametinib augmented oHSV oncolytic activity in BRAF V600E-mutated tumor cells. (2) Combination treatment with oHSV and MEKi Trametinib enhanced virus replication mediated by down-regulation of STAT1 and PKR expression or phosphorylation in BRAF V600E-mutated tumor cells as well as BRAF wt/KRAS-mutated tumor cells. (3) A remarkably synergistic therapeutic efficacy was shown in vivo for BRAF wt/KRAS-mutated tumor models, when a combination of oHSV including PD-1 blockade and MEK inhibition. Collectively, these data provide some new insights for clinical development of combination therapy with oncolytic virus, MEK inhibition, and checkpoint blockade for BRAF or KRAS-mutated tumors.

Degradation of Herpes Simplex Virus-1 Viral miRNA H11 by Vaccinia Virus Protein VP55 Attenuates Viral Replication.

Among 29 distinct miRNAs expressed by the herpes simplex virus-1 (HSV-1) during lytic infection, miR-H11, together with miR-H1 to miR-H8 are reported to locate in the RNA-induced silencing complex (RISC). miR-H11 is encoded within viral origins of replication and lies entirely within the origins of replication. However, the roles of this miRNA derived from lytic infection with HSV-1 remain unclear. Using the advantage of vaccinia virus protein VP55 (VP55)-mediated degradation of miRNAs, we constructed a recombinant virus expressing VP55 (R5502) to demonstrate that: (1) accumulation of miR-H11 from R5502 was reduced by 540-fold versus that in cells infected with wild-type HSV-1, but miR-H1 to miR-H8 which also located in the RISC were not reduced significantly from R5502 compare with wild-type HSV-1; (2) downregulation of miR-H11 from R5502 infected cells results in markedly lower viral DNA synthesis compared with wild-type HSV-1; and (3) downregulation of miR-H11 also restricted viral spreading, and resulted in low accumulation of representative viral proteins and viral yields. The findings were confirmed through either using of a miR-H11 inhibitor or pre-transfection of a plasmid expressing VP55. These data suggest that miR-H11 plays a currently unidentified role in maintaining sufficient viral DNA synthesis during the course of viral infection.

Effect of Loss-of-function of the Herpes Simplex Virus-1 microRNA H6-5p on Virus Replication.

To date, 29 distinct microRNAs (miRNAs) have been reported to be expressed during herpes simplex virus infections. Sequence analysis of mature herpes simplex virus-1 (HSV-1) miRNAs revealed five sets of miRNAs that are complementary to each other: miR-H6-5p/H1-3p, miR-H6-3p/H1-5p, H2-5p/H14-3p, miR-H2-3p/H14-5p, and miR-H7/H27. However, the roles of individual miRNAs and consequences of this complementarity remain unclear. Here, we focus on two of these complementary miRNAs, miR-H6-5p and miR-H1-3p, using loss-of-function experiments in vitro and in a mouse model of infection using an miRNA sponge approach, including tandem multiplex artificial miRNA-binding sequences that do not match perfectly to the target miRNA inserted downstream of a green fluorescent protein reporter gene. Infection with recombinant virus expressing the miR-H6-5p sponge reduced viral protein levels and virus yield. Decreased accumulation of viral proteins was also observed at early stages of infection in the presence of both an miR-H6-5p inhibitor and plasmid-expressed miR-H1-3p. Moreover, establishment of latency and reactivation did not differ between the recombinant virus expressing the miR-H6-5p sponge and wild-type HSV-1. Taken together, these data suggest that miR-H6-5p has an as-yet-unidentified role in the early stages of viral infection, and its complement miR-H1-3p suppresses this role in later stages of infection. This report extends understanding of the roles of miRNAs in infection by herpes simplex viruses, supporting a model of infection in which the production of virus and its virulent effects are tightly controlled to maximize persistence in the host and population.