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Transduction of Schistosoma japonicum schistosomules with vesicular stomatitis virus glycoprotein pseudotyped murine leukemia retrovirus and expression of reporter human telomerase reverse transcriptase in the transgenic schistosomes.

ABSTRACT: Although draft genome sequences of two of the major human schistosomes, Schistosoma japonicum and Schistosoma mansoni are available, the structures and characteristics of most genes and the influence of exogenous genes on the metabolism of schistosomes remain uncharacterized. Furthermore, which functional genomics approaches will be tractable for schistosomes are not yet apparent. Here, the vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped pantropic retroviral vector pBABE-puro was modified to incorporate the human telomerase reverse transcriptase gene (hTERT) as a reporter, under the control of the retroviral long terminal repeat (LTR). Pseudotyped virions were employed to transduce S. japonicum to investigate the utility of retrovirus-mediated transgenesis of S. japonicum and the activity of human telomerase reverse transcriptase as a reporter transgene in schistosomes. Schistosomules perfused from experimentally infected rabbits were cultured for 6 days after exposure to the virions after which genomic DNAs from virus exposed and control worms were extracted. Analysis of RNA from transduced parasites and immunohistochemistry of thin parasite sections revealed expression of hTERT in the transduced worms. Expression of hTERT was also confirmed by immunoblot analysis. These findings indicated that S. japonicum could be effectively transduced by VSVG-pseudotyped retrovirus carrying the hTERT gene. Given the potential of hTERT to aid in derivation of immortalized cells, these findings suggest that this pantropic retroviral approach can be employed to transduce cells from specific tissues and organs of schistosomes to investigate the influence of transgene hTERT on growth and proliferation of schistosome cells.

SUBMITTER: Yang S

PROVIDER: S-EPMC3836731 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Transduction of Schistosoma japonicum schistosomules with vesicular stomatitis virus glycoprotein pseudotyped murine leukemia retrovirus and expression of reporter human telomerase reverse transcriptase in the transgenic schistosomes.

Yang Shenghui S Brindley Paul J PJ Zeng Qingren Q Li Yuesheng Y Zhou Jun J Liu Yan Y Liu Biyuan B Cai Liting L Zeng Tiebing T Wei Qi Q Lan Lingmei L McManus Donald P DP

Molecular and biochemical parasitology 20100806 2

Although draft genome sequences of two of the major human schistosomes, Schistosoma japonicum and Schistosoma mansoni are available, the structures and characteristics of most genes and the influence of exogenous genes on the metabolism of schistosomes remain uncharacterized. Furthermore, which functional genomics approaches will be tractable for schistosomes are not yet apparent. Here, the vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped pantropic retroviral vector pBABE-puro was modi ...[more]

PMID: 20692298

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Project description:The recent release of draft genome sequences of two of the major human schistosomes has underscored the pressing need to develop functional genomics approaches for these significant pathogens. The sequence information also makes feasible genome-scale investigation of transgene integration into schistosome chromosomes. Retrovirus-mediated transduction offers a means to establish transgenic lines of schistosomes, to elucidate schistosome gene function and expression, and to advance functional genomics approaches for these parasites. We investigated the utility of the Moloney murine leukemia retrovirus (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) for the transduction of Schistosoma mansoni and delivery of reporter transgenes into schistosome chromosomes. Schistosomula were exposed to virions of VSVG-pseudotyped MLV, after which genomic DNA was extracted from the transduced schistosomes. Southern hybridization analysis indicated the presence of proviral MLV retrovirus in the transduced schistosomes. Fragments of the MLV transgene and flanking schistosome sequences recovered using an anchored PCR-based approach demonstrated definitively that somatic transgenesis of schistosome chromosomes had taken place and, moreover, revealed widespread retrovirus integration into schistosome chromosomes. More specifically, MLV transgenes had inserted in the vicinity of genes encoding immunophilin, zinc finger protein Sma-Zic, and others, as well as near the endogenous schistosome retrotransposons, the fugitive and SR1. Proviral integration of the MLV transgene appeared to exhibit primary sequence site specificity, targeting a gGATcc-like motif. Reporter luciferase transgene activity driven by the schistosome actin gene promoter was expressed in the tissues of transduced schistosomula and adult schistosomes. Luciferase activity appeared to be developmentally expressed in schistosomula with increased activity observed after 1 to 2 wk in culture. These findings indicate the utility of VSVG-pseudotyped MLV for transgenesis of S. mansoni, herald a tractable pathway forward toward germline transgenesis and functional genomics of parasitic helminths, and provide the basis for comparative molecular pathogenesis studies of chromosomal lesions arising from retroviral integration into human compared with schistosome chromosomes.

Project description:The recent Ebola virus (EBOV) epidemic in West Africa demonstrates the potential for a significant public health burden caused by filoviral infections. No vaccine or antiviral is currently FDA approved. To expand the vaccine options potentially available, we assessed protection conferred by an EBOV vaccine composed of vesicular stomatitis virus pseudovirions that lack native G glycoprotein (VSV?G) and bear EBOV glycoprotein (GP). These pseudovirions mediate a single round of infection. Both single-dose and prime/boost vaccination regimens protected mice against lethal challenge with mouse-adapted Ebola virus (ma-EBOV) in a dose-dependent manner. The prime/boost regimen provided significantly better protection than a single dose. As N-linked glycans are thought to shield conserved regions of the EBOV GP receptor-binding domain (RBD), thereby blocking epitopes within the RBD, we also tested whether VSV?G bearing EBOV GPs that lack GP1 N-linked glycans provided effective immunity against challenge with ma-EBOV or a more distantly related virus, Sudan virus. Using a prime/boost strategy, high doses of GP/VSV?G partially or fully denuded of N-linked glycans on GP1 protected mice against ma-EBOV challenge, but these mutants were no more effective than wild-type (WT) GP/VSV?G and did not provide cross protection against Sudan virus. As reported for other EBOV vaccine platforms, the protection conferred correlated with the quantity of EBOV GP-specific Ig produced but not with the production of neutralizing antibodies. Our results show that EBOV GP/VSV?G pseudovirions serve as a successful vaccination platform in a rodent model of Ebola virus disease and that GP1 N-glycan loss does not influence immunogenicity or vaccination success.IMPORTANCE The West African Ebola virus epidemic was the largest to date, with more than 28,000 people infected. No FDA-approved vaccines are yet available, but in a trial vaccination strategy in West Africa, recombinant, infectious VSV encoding the Ebola virus glycoprotein effectively prevented virus-associated disease. VSV?G pseudovirion vaccines may prove as efficacious and have better safety, but they have not been tested to date. Thus, we tested the efficacy of VSV?G pseudovirions bearing Ebola virus glycoprotein as a vaccine platform. We found that wild-type Ebola virus glycoprotein, in the context of this platform, provides robust protection of EBOV-challenged mice. Further, we found that removal of the heavy glycan shield surrounding conserved regions of the glycoprotein does not enhance vaccine efficacy.

Project description:Vesicular stomatitis Indiana virus (VSIV), formerly known as vesicular stomatitis virus (VSV) Indiana (VSVIND), is a model virus that is exceptionally sensitive to the inhibitory action of interferons (IFNs). Interferons induce an antiviral state by stimulating the expression of hundreds of interferon-stimulated genes (ISGs). These ISGs can constrain viral replication, limit tissue tropism, reduce pathogenicity, and inhibit viral transmission. Since VSIV is used as a backbone for multiple oncolytic and vaccine strategies, understanding how ISGs restrict VSIV not only helps in understanding VSIV-induced pathogenesis but also helps us evaluate and understand the safety and efficacy of VSIV-based therapies. Thus, there is a need to identify and characterize the ISGs that possess anti-VSIV activity. Using arrayed ISG expression screening, we identified TRIM69 as an ISG that potently inhibits VSIV. This inhibition was highly specific as multiple viruses, including influenza A virus, HIV-1, Rift Valley fever virus, and dengue virus, were unaffected by TRIM69. Indeed, just one amino acid substitution in VSIV can govern sensitivity/resistance to TRIM69. Furthermore, TRIM69 is highly divergent in human populations and exhibits signatures of positive selection that are consistent with this gene playing a key role in antiviral immunity. We propose that TRIM69 is an IFN-induced inhibitor of VSIV and speculate that TRIM69 could be important in limiting VSIV pathogenesis and might influence the specificity and/or efficacy of vesiculovirus-based therapies.IMPORTANCE Vesicular stomatitis Indiana virus (VSIV) is a veterinary pathogen that is also used as a backbone for many oncolytic and vaccine strategies. In natural and therapeutic settings, viral infections like VSIV are sensed by the host, and as a result the host cells make proteins that can protect them from viruses. In the case of VSIV, these antiviral proteins constrain viral replication and protect most healthy tissues from virus infection. In order to understand how VSIV causes disease and how healthy tissues are protected from VSIV-based therapies, it is crucial that we identify the proteins that inhibit VSIV. Here, we show that TRIM69 is an antiviral defense that can potently and specifically block VSIV infection.

Dataset Information

Transduction of Schistosoma japonicum schistosomules with vesicular stomatitis virus glycoprotein pseudotyped murine leukemia retrovirus and expression of reporter human telomerase reverse transcriptase in the transgenic schistosomes.

Publications

Transduction of Schistosoma japonicum schistosomules with vesicular stomatitis virus glycoprotein pseudotyped murine leukemia retrovirus and expression of reporter human telomerase reverse transcriptase in the transgenic schistosomes.

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