Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0038362 (stomatitis)
 8,852 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Inactivation of the reverse transcriptase (RT) and integrase (IN) enzymes can abolish the replication of the human immunodeficiency virus (HIV) and, thus, its infectivity. Here, inactivated HIV particles convenient for designing virus-like particle (VLP) based vaccines have been produced. Inactivated HIV-provirus was created by introducing a frame shift mutation. HIV provirus DNA was cut in the pol region by Age I restriction enzyme, followed by filling of sticky ends using the Klenow fragment before ligation. The resulting plasmid was named as pRINNL4-3. HEK-293T cells were used as producer, after being transfected with the modified plasmid. Viral particle production and biological activity were assayed by virus capsid protein (p24) quantification and syncytium formation in MT2 cells, respectively. The immunogenicity of the RINNL4-3 virions was investigated in a mouse model. The mutation was expected to inactivate the virus RT and IN enzymes. The results showed that the VLPs were assembled, as measured by the p24 load of the culture supernatant, and contained functional envelope proteins (Env) as monitored by the syncytium formation. However, these VLPs had no ability to infect target MT2 cells, as well as their VSVG (vesicular stomatitis virus-glycoprotein) pseudotyped counterparts infected HEK-293T cells. A high level of antibody response was observed in immunized mice. Since RINNL4-3 virions are replication incompetent, they are convenient for production and use in biomedical studies. Also, RINNL4-3 is a candidate for a vaccine development due to it contains envelope and structural virus proteins which are crucial for triggering neutralizing antibodies and the cellular immune response.
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PMID:Introducing a frameshift mutation to the POL sequence of HIV-1 provirus and evaluation of the immunogenic characteristics of the mutated virions (RINNL4-3). 2288 41

Human immunodeficiency virus (HIV) is the primary etiologic agent responsible for the AIDS pandemic. In this work, we used a chimeric recombinant protein strategy to test the possibility of irreversibly destroying the HIV-1 virion using an agent that simultaneously binds the Env protein and viral membrane. We constructed a fusion of the lectin cyanovirin-N (CVN) and the gp41 membrane-proximal external region (MPER) peptide with a variable-length (Gly4Ser)x linker (where x is 4 or 8) between the C terminus of the former and N terminus of the latter. The His-tagged recombinant proteins, expressed in BL21(DE3)pLysS cells and purified by immobilized metal affinity chromatography followed by gel filtration, were found to display a nanomolar efficacy in blocking BaL-pseudotyped HIV-1 infection of HOS.T4.R5 cells. This antiviral activity was HIV-1 specific, since it did not inhibit cell infection by vesicular stomatitis virus (VSV) or amphotropic-murine leukemia virus. Importantly, the chimeric proteins were found to release intraviral p24 protein from both BaL-pseudotyped HIV-1 and fully infectious BaL HIV-1 in a dose-dependent manner in the absence of host cells. The addition of either MPER or CVN was found to outcompete this virolytic effect, indicating that both components of the chimera are required for virolysis. The finding that engaging the Env protein spike and membrane using a chimeric ligand can destabilize the virus and lead to inactivation opens up a means to investigate virus particle metastability and to evaluate this approach for inactivation at the earliest stages of exposure to virus and before host cell encounter.
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PMID:Chimeric Cyanovirin-MPER recombinantly engineered proteins cause cell-free virolysis of HIV-1. 2385 80

Three compounds (1, 2, and 3) previously reported to inhibit HIV-1 replication and/or in vitro activity of reverse transcriptase were studied, but only fullerene derivatives 1 and 2 showed strong antiviral activity on the replication of HIV-1 in human CD4(+) T cells. However, these compounds did not inhibit infection by single-round infection vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped viruses, indicating no effect on the early steps of the viral life cycle. In contrast, analysis of single-round infection VSV-G-pseudotyped HIV-1 produced in the presence of compound 1 or 2 showed a complete lack of infectivity in human CD4(+) T cells, suggesting that the late stages of the HIV-1 life cycle were affected. Quantification of virion-associated viral RNA and p24 indicates that RNA packaging and viral production were unremarkable in these viruses. However, Gag and Gag-Pol processing was affected, as evidenced by immunoblot analysis with an anti-p24 antibody and the measurement of virion-associated reverse transcriptase activity, ratifying the effect of the fullerene derivatives on virion maturation of the HIV-1 life cycle. Surprisingly, fullerenes 1 and 2 did not inhibit HIV-1 protease in an in vitro assay at the doses that potently blocked viral infectivity, suggesting a protease-independent mechanism of action. Highlighting the potential therapeutic relevance of fullerene derivatives, these compounds block infection by HIV-1 resistant to protease and maturation inhibitors.
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PMID:Fullerene Derivatives Strongly Inhibit HIV-1 Replication by Affecting Virus Maturation without Impairing Protease Activity. 2743 Dec 32

Research into the properties of Japanese encephalitis virus (JEV) has been facilitated by use of pseudotyped viruses. The signal peptide is a key determinant for membrane targeting and membrane insertion, which could affect packaging of pseudotyped viruses. In this study, we generated three lentiviral vectors pseudotyped with JEV envelope proteins that co-express either a strong signal peptide from vesicular stomatitis virus (VSV)-G (VSVMEpv) or a weak signal peptide of JEV (SPMEpv), or a virus without a signal peptide in front of the JEV prM/E (MEpv). Western blot demonstrated that JEV E protein and HIV p24 were present in the same particles of the three pseudotyped JEV-E based lentiviral vectors. Electron microscopy revealed that the three pseudotyped JEV-E based lentiviral vectors were 120-180nm in diameter. Real-time quantitative reverse transcriptase polymerase chain reaction showed that the titer of VSVMEpv was 17-fold higher than that of MEpv, while the titer of SPMEpv was six-fold higher than that of MEpv. Inclusion of a signal peptide enhanced packaging efficiency of pseudotyped JEV-E based lentiviral vectors. With a strong signal peptide helping they generate a higher number of viral particles. Green fluorescent protein and luciferase expression showed that the transduction titer or relative fluorescence units of VSVMEpv, SPMEpv and MEpv were not significantly different. We suggest that the signal peptide does not influence the infectivity of pseudotyped JEV-E based lentiviral vectors. In addition, our findings indicated that pseudotyped JEVs show preferential tropism for BHK-21 cells, supporting the mimic function displayed by parental JEV. Therefore, our study provided a cost-effective method to generate pseudotyped JEV-E based lentiviral vectors, which may represent a valid model to investigate some of the infectious properties of JEV.
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PMID:Introducing a cleavable signal peptide enhances the packaging efficiency of lentiviral vectors pseudotyped with Japanese encephalitis virus envelope proteins. 2799 24

The titer of a lentivirus vector is often expressed in transducing units per milliliter. This is a functional titer that reflects the lentivirus' ability to transduce a particular cell line under specific conditions. Transduction of other cell lines is likely to be different and will require optimization. 293T cells are used for production of lentivirus stocks, and they can be easily transduced with vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentivirus vectors. Consequently, this cell line is commonly used to determine the functional titer of lentivirus vector stocks produced with this envelope. For lentivirus vectors encoding fluorescent proteins under the control of promoters functional in these cells, titration can be performed using the limiting dilution method or a flow cytometry-based method. For lentivirus vectors lacking a fluorescent marker, or for those carrying promoters that may not be functional in 293T cells, titer can be determined either by real-time polymerase chain reaction (PCR) quantification of viral genomes in genomic DNA from transduced cells or a p24 ELISA-based assay.
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PMID:Titration of Lentivirus Vectors. 2961 Mar 63


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