Diversity in responses to oncolytic Lassa-vesicular stomatitis virus in patient-derived glioblastoma cells.

The difficulty of glioblastoma treatment makes it a good candidate for novel therapies, such as oncolytic viruses. Vesicular stomatitis virus expressing Lassa virus glycoprotein (Lassa-VSV) showed significant promise in animal models using established glioblastoma cell lines. These experiments were to determine the susceptibility of low-passage, patient-derived cell lines to Lassa-VSV oncolysis. Four patient-derived glioblastoma cell lines were infected with Lassa-VSV that expresses green fluorescent protein (GFP) and analyzed by fluorescence microscopy, flow cytometry, and cell viability assays. Cells were also analyzed as tumorspheres containing primarily glioma stem-like cells. Three low-passage, patient-derived cells were further analyzed with RNA sequencing (RNA-seq). Individual cell lines varied somewhat in their levels of viral gene expression and time course of Lassa-VSV-induced cell death, but each was susceptible to Lassa-VSV. Brain Tumor Center of Excellence (BTCOE) 4765 cells had the highest level of expression of interferon-stimulated genes but were most susceptible to Lassa-VSV-induced cell death, indicating that more susceptible cells do not necessarily have lower interferon pathway activation. Cells cultured as tumorspheres and infected with Lassa-VSV also showed variable susceptibility to Lassa-VSV, but BTCOE 4765 cells were least susceptible. Thus, patient-derived brain tumor cells show variable responses to Lassa-VSV infection, but each of the lines was susceptible to VSV oncolysis.

Mucin-Like Domain of Ebola Virus Glycoprotein Enhances Selective Oncolytic Actions against Brain Tumors.

Given that the Ebola virus (EBOV) infects a wide array of organs and cells yet displays a relative lack of neurotropism, we asked whether a chimeric vesicular stomatitis virus (VSV) expressing the EBOV glycoprotein (GP) might selectively target brain tumors. The mucin-like domain (MLD) of the EBOV GP may enhance virus immune system evasion. Here, we compared chimeric VSVs in which EBOV GP replaces the VSV glycoprotein, thereby reducing the neurotoxicity associated with wild-type VSV. A chimeric VSV expressing the full-length EBOV GP (VSV-EBOV) containing the MLD was substantially more effective and safer than a parallel construct with an EBOV GP lacking the MLD (VSV-EBOVΔMLD). One-step growth, reverse transcription-quantitative PCR, and Western blotting assessments showed that VSV-EBOVΔMLD produced substantially more progeny faster than VSV-EBOV. Using immunodeficient SCID mice, we focused on targeting human brain tumors with these VSV-EBOVs. Similar to the findings of our previous study in which we used an attenuated VSV-EBOV with no MLD that expressed green fluorescent protein (GFP) (VSV-EBOVΔMLD-GFP), VSV-EBOVΔMLD without GFP targeted glioma but yielded only a modest extension of survival. In contrast, VSV-EBOV containing the MLD showed substantially better targeting and elimination of brain tumors after intravenous delivery and increased the survival of brain tumor-bearing mice. Despite the apparent destruction of most tumor cells by VSV-EBOVΔMLD, the virus remained active within the SCID mouse brain and showed widespread infection of normal brain cells. In contrast, VSV-EBOV eliminated the tumors and showed relatively little infection of normal brain cells. Parallel experiments with direct intracranial virus infection generated similar results. Neither VSV-EBOV nor VSV-EBOVΔMLD showed substantive infection of the brains of normal immunocompetent mice.IMPORTANCE The Ebola virus glycoprotein contains a mucin-like domain which may play a role in immune evasion. Chimeric vesicular stomatitis viruses with the EBOV glycoprotein substituted for the VSV glycoprotein show greater safety and efficacy in targeting brain tumors in immunodeficient mice when the MLD was expressed within the EBOV glycoprotein than when EBOV lacked the mucin-like domain.

Defining the caudal hypothalamic arcuate nucleus with a focus on anorexic excitatory neurons.

KEY POINTS: Excitatory glutamate neurons are sparse in the rostral hypothalamic arcuate nucleus (ARC), the subregion that has received the most attention in the past. In striking contrast, excitatory neurons are far more common (by a factor of 10) in the caudal ARC, an area which has received relatively little attention. These glutamate cells may play a negative role in energy balance and food intake. They can show an increase in phosphorylated Stat-3 in the presence of leptin, are electrically excited by the anorectic neuromodulator cholecystokinin, and inhibited by orexigenic neuromodulators neuropeptide Y, met-enkephalin, dynorphin and the catecholamine dopamine. The neurons project local axonal connections that excite other ARC neurons including proopiomelanocortin neurons that can play an important role in obesity. These data are consistent with models suggesting that the ARC glutamatergic neurons may play both a rapid and a slower role in acting as anorectic neurons in CNS control of food intake and energy homeostasis. ABSTRACT: Here we interrogate a unique class of excitatory neurons in the hypothalamic arcuate nucleus (ARC) that utilizes glutamate as a fast neurotransmitter using mice expressing GFP under control of the vesicular glutamate transporter 2 (vGluT2) promoter. These neurons show a unique distribution, synaptic characterization, cellular physiology and response to neuropeptides involved in energy homeostasis. Although apparently not previously appreciated, the caudal ARC showed a far greater density of vGluT2 cells than the rostral ARC, as seen in transgenic vGluT2-GFP mice and mRNA analysis. After food deprivation, leptin induced an increase in phosphorylated Stat-3 in vGluT2-positive neurons, indicating a response to hormonal cues of energy state. Based on whole-cell recording electrophysiology in brain slices, vGluT2 neurons were spontaneously active with a spike frequency around 2 Hz. vGluT2 cells were responsive to a number of neuropeptides related to energy homeostasis; they were excited by the anorectic peptide cholecystokinin, but inhibited by orexigenic neuropeptide Y, dynorphin and met-enkephalin, consistent with an anorexic role in energy homeostasis. Dopamine, associated with the hedonic aspect of enhancing food intake, inhibited vGluT2 neurons. Optogenetic excitation of vGluT2 cells evoked EPSCs in neighbouring neurons, indicating local synaptic excitation of other ARC neurons. Microdrop excitation of ARC glutamate cells in brain slices rapidly increased excitatory synaptic activity in anorexigenic proopiomelanocortin neurons. Together these data support the perspective that vGluT2 cells may be more prevalent in the ARC than previously appreciated, and play predominantly an anorectic role in energy metabolism.

Zika Virus Targeting in the Developing Brain.

Zika virus (ZIKV), a positive-sense RNA flavivirus, has attracted considerable attention recently for its potential to cause serious neurological problems, including microcephaly, cortical thinning, and blindness during early development. Recent findings suggest that ZIKV infection of the brain can occur not only during very early stages of development, but also in later fetal/early neonatal stages of maturation. Surprisingly, after peripheral inoculation of immunocompetent mice on the day of birth, the first cells targeted throughout the brain were isolated astrocytes. At later stages, more neurons showed ZIKV immunoreactivity, in part potentially due to ZIKV release from infected astrocytes. In all developing mice studied, we detected infection of retinal neurons; in many mice, this was also associated with infection of the lateral geniculate, suprachiasmatic nuclei, and superior colliculus, suggesting a commonality for the virus to infect cells of the visual system. Interestingly, in mature mice lacking a Type 1 interferon response (IFNR-/-), after inoculation of the eye, the initial majority of infected cells in the visual system were glial cells along the optic tract. ZIKV microinjection into the somatosensory cortex on one side of the normal mouse brain resulted in mirror infection restricted to the contralateral somatosensory cortex without any infection of midline brain regions, indicating the virus can move by axonal transport to synaptically coupled brain loci. These data support the view that ZIKV shows considerable complexity in targeting the CNS and may target different cells at different stages of brain development.SIGNIFICANCE STATEMENT Zika virus (ZIKV) can cause substantial damage to the developing human brain. Here we examine a developmental mouse model of ZIKV infection in the newborn mouse in which the brain is developmentally similar to a second-trimester human fetus. After peripheral inoculation, the virus entered the CNS in all mice tested and initially targeted astrocytes throughout the brain. Infections of the retina were detected in all mice, and infection of CNS visual system nuclei in the brain was common. We find that ZIKV can be transported axonally, thereby enhancing virus spread within the brain. These data suggest that ZIKV infects multiple cell types within the brain and that astrocyte infection may play a more important role in initial infection than previously appreciated.

Chikungunya, Influenza, Nipah, and Semliki Forest Chimeric Viruses with Vesicular Stomatitis Virus: Actions in the Brain.

Recombinant vesicular stomatitis virus (VSV)-based chimeric viruses that include genes from other viruses show promise as vaccines and oncolytic viruses. However, the critical safety concern is the neurotropic nature conveyed by the VSV glycoprotein. VSVs that include the VSV glycoprotein (G) gene, even in most recombinant attenuated strains, can still show substantial adverse or lethal actions in the brain. Here, we test 4 chimeric viruses in the brain, including those in which glycoprotein genes from Nipah, chikungunya (CHIKV), and influenza H5N1 viruses were substituted for the VSV glycoprotein gene. We also test a virus-like vesicle (VLV) in which the VSV glycoprotein gene is expressed from a replicon encoding the nonstructural proteins of Semliki Forest virus. VSVΔG-CHIKV, VSVΔG-H5N1, and VLV were all safe in the adult mouse brain, as were VSVΔG viruses expressing either the Nipah F or G glycoprotein. In contrast, a complementing pair of VSVΔG viruses expressing Nipah G and F glycoproteins were lethal within the brain within a surprisingly short time frame of 2 days. Intranasal inoculation in postnatal day 14 mice with VSVΔG-CHIKV or VLV evoked no adverse response, whereas VSVΔG-H5N1 by this route was lethal in most mice. A key immune mechanism underlying the safety of VSVΔG-CHIKV, VSVΔG-H5N1, and VLV in the adult brain was the type I interferon response; all three viruses were lethal in the brains of adult mice lacking the interferon receptor, suggesting that the viruses can infect and replicate and spread in brain cells if not blocked by interferon-stimulated genes within the brain.IMPORTANCE Vesicular stomatitis virus (VSV) shows considerable promise both as a vaccine vector and as an oncolytic virus. The greatest limitation of VSV is that it is highly neurotropic and can be lethal within the brain. The neurotropism can be mostly attributed to the VSV G glycoprotein. Here, we test 4 chimeric viruses of VSV with glycoprotein genes from Nipah, chikungunya, and influenza viruses and nonstructural genes from Semliki Forest virus. Two of the four, VSVΔG-CHIKV and VLV, show substantially attenuated neurotropism and were safe in the healthy adult mouse brain. VSVΔG-H5N1 was safe in the adult brain but lethal in the younger brain. VSVΔG Nipah F+G was even more neurotropic than wild-type VSV, evoking a rapid lethal response in the adult brain. These results suggest that while chimeric VSVs show promise, each must be tested with both intranasal and intracranial administration to ensure the absence of lethal neurotropism.

Mood stabilizers inhibit cytomegalovirus infection.

Cytomegalovirus (CMV) infection can generate debilitating disease in immunocompromised individuals and neonates. It is also the most common infectious cause of congenital birth defects in infected fetuses. Available anti-CMV drugs are partially effective but are limited by some toxicity, potential viral resistance, and are not recommended for fetal exposure. Valproate, valpromide, and valnoctamide have been used for many years to treat epilepsy and mood disorders. We report for the first time that, in contrast to the virus-enhancing actions of valproate, structurally related valpromide and valnoctamide evoke a substantial and specific inhibition of mouse and human CMV in vitro. In vivo, both drugs safely attenuate mouse CMV, improving survival, body weight, and developmental maturation of infected newborns. The compounds appear to act by a novel mechanism that interferes with CMV attachment to the cell. Our work provides a novel potential direction for CMV therapeutics through repositioning of agents already approved for use in psychiatric disorders.

Lassa-vesicular stomatitis chimeric virus safely destroys brain tumors.

UNLABELLED: High-grade tumors in the brain are among the deadliest of cancers. Here, we took a promising oncolytic virus, vesicular stomatitis virus (VSV), and tested the hypothesis that the neurotoxicity associated with the virus could be eliminated without blocking its oncolytic potential in the brain by replacing the neurotropic VSV glycoprotein with the glycoprotein from one of five different viruses, including Ebola virus, Marburg virus, lymphocytic choriomeningitis virus (LCMV), rabies virus, and Lassa virus. Based on in vitro infections of normal and tumor cells, we selected two viruses to test in vivo. Wild-type VSV was lethal when injected directly into the brain. In contrast, a novel chimeric virus (VSV-LASV-GPC) containing genes from both the Lassa virus glycoprotein precursor (GPC) and VSV showed no adverse actions within or outside the brain and targeted and completely destroyed brain cancer, including high-grade glioblastoma and melanoma, even in metastatic cancer models. When mice had two brain tumors, intratumoral VSV-LASV-GPC injection in one tumor (glioma or melanoma) led to complete tumor destruction; importantly, the virus moved contralaterally within the brain to selectively infect the second noninjected tumor. A chimeric virus combining VSV genes with the gene coding for the Ebola virus glycoprotein was safe in the brain and also selectively targeted brain tumors but was substantially less effective in destroying brain tumors and prolonging survival of tumor-bearing mice. A tropism for multiple cancer types combined with an exquisite tumor specificity opens a new door to widespread application of VSV-LASV-GPC as a safe and efficacious oncolytic chimeric virus within the brain. IMPORTANCE: Many viruses have been tested for their ability to target and kill cancer cells. Vesicular stomatitis virus (VSV) has shown substantial promise, but a key problem is that if it enters the brain, it can generate adverse neurologic consequences, including death. We tested a series of chimeric viruses containing genes coding for VSV, together with a gene coding for the glycoprotein from other viruses, including Ebola virus, Lassa virus, LCMV, rabies virus, and Marburg virus, which was substituted for the VSV glycoprotein gene. Ebola and Lassa chimeric viruses were safe in the brain and targeted brain tumors. Lassa-VSV was particularly effective, showed no adverse side effects even when injected directly into the brain, and targeted and destroyed two different types of deadly brain cancer, including glioblastoma and melanoma.

Vesicular stomatitis virus variants selectively infect and kill human melanomas but not normal melanocytes.

Metastatic malignant melanoma remains one of the most therapeutically challenging forms of cancer. Here we test replication-competent vesicular stomatitis viruses (VSV) on 19 primary human melanoma samples and compare these infections with those of normal human melanocyte control cells. Even at a low viral concentration, we found a strong susceptibility to viral oncolysis in over 70% of melanomas. In contrast, melanocytes displayed strong resistance to virus infection and showed complete protection by interferon. Several recombinant VSVs were compared, and all infected and killed most melanomas with differences in the time course with increasing rates of melanoma infection, as follows: VSV-CT9-M51 < VSV-M51 < VSV-G/GFP < VSV-rp30. VSV-rp30 sequencing revealed 2 nonsynonymous mutations at codon positions P126 and L223, both of which appear to be required for the enhanced phenotype. VSV-rp30 showed effective targeting and infection of multiple subcutaneous and intracranial melanoma xenografts in SCID mice after tail vein virus application. Sequence analysis of mutations in the melanomas used revealed that BRAF but not NRAS gene mutation status was predictive for enhanced susceptibility to infection. In mouse melanoma models with specific induced gene mutations including mutations of the Braf, Pten, and Cdkn2a genes, viral infection correlated with the extent of malignant transformation. Similar to human melanocytes, mouse melanocytes resisted VSV-rp30 infection. This study confirms the general susceptibility of the majority of human melanoma types for VSV-mediated oncolysis.

Highly attenuated recombinant vesicular stomatitis virus VSV-12'GFP displays immunogenic and oncolytic activity.

Vesicular stomatitis virus (VSV) has shown considerable promise both as an immunization vector and as an oncolytic virus. In both applications, an important concern is the safety profile of the virus. To generate a highly attenuated virus, we added two reporter genes to the 3' end of the VSV genome, thereby shifting the NPMGL genes from positions 1 to 5 to positions 3 to 7. The resulting virus (VSV-12'GFP) was highly attenuated, generating smaller plaques than four other attenuated VSVs. In one-step growth curves, VSV-12'GFP displayed the slowest growth kinetics. The mechanism of attenuation appears to be due to reduced expression of VSV genes downstream of the reporter genes, as suggested by a 10.4-fold reduction in L-protein RNA transcript. Although attenuated, VSV-12'GFP was highly effective at generating an immune response, indicated by a high-titer antibody response against the green fluorescent protein (GFP) expressed by the virus. Although VSV-12'GFP was more attenuated than other VSVs on both normal and cancer cells, it nonetheless showed a greater level of infection of human cancer cells (glioma and melanoma) than of normal cells, and this effect was magnified in glioma by interferon application, indicating selective oncolysis. Intravenous VSV-12'GFP selectively infected human gliomas implanted into SCID mice subcutaneously or intracranially. All postnatal day 16 mice given intranasal VSV-12'GFP survived, whereas only 10% of those given VSV-G/GFP survived, indicating reduced neurotoxicity. Intratumoral injection of tumors with VSV-12'GFP dramatically suppressed tumor growth and enhanced survival. Together these data suggest this recombinant virus merits further study for its oncolytic and vaccine potential.

Viral mutagenesis as a means for generating novel proteins.

We demonstrate that a mutation-prone virus engineered to express a foreign gene is an expedient means for generating novel mutant nonviral proteins in mammalian cells. Using vesicular stomatitis virus to express a gene coding for a fluorescent DsRed protein, a number of green mutant variants including a new variant not previously described were rapidly isolated from infected cells, sequenced, and cloned. Similar methods may be useful in the development of physiologically sensitive fluorescent reporter proteins and directed evolution or mutagenesis of proteins in general.

Mitochondrial DNA depletion analysis by pseudogene ratioing.

The mitochondrial DNA (mtDNA) depletion status of rho(0) cell lines is typically assessed by hybridization or polymerase chain reaction (PCR) experiments, in which the failure to hybridize mtDNA or amplify mtDNA using mtDNA-directed primers suggests thorough mitochondrial genome removal. Here, we report the use of an mtDNA pseudogene ratioing technique for the additional confirmation of rho0 status. Total genomic DNA from a U251 human glioma cell line treated with ethidium bromide was amplified using primers designed to anneal either mtDNA or a previously described nuclear DNA-embedded mtDNA pseudogene (mtDNApsi). The resultant PCR product was used to generate plasmid clones. Sixty-two plasmid clones were genotyped, and all arose from mtDNApsi template. These data allowed us to determine with 95% confidence that the resultant mtDNA-depleted cell line contains less than one copy of mtDNA per 10 cells. Unlike previous hybridization or PCR-based analyses of mtDNA depletion, this mtDNApsi ratioing technique does not rely on interpretation of a negative result, and may prove useful as an adjunct for the determination of rho0 status or mtDNA copy number.