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The recombinant vaccinia virus gene product, B18R, neutralizes interferon alpha and alleviates histopathological complications in an HIV encephalitis mouse model.

ABSTRACT: Interferon-alpha (IFN-?) has been identified as a neurotoxin that plays a prominent role in human immunodeficiency virus (HIV)-associated neurocognitive disorders and HIV encephalitis (HIVE) pathology. IFN-? is associated with cognitive dysfunction in other inflammatory diseases where IFN-? is upregulated. Trials of monoclonal anti-IFN-? antibodies have been generally disappointing possibly due to high specificity to limited IFN-? subtypes and low affinity. We investigated a novel IFN-? inhibitor, B18R, in an HIVE/severe combined immunodeficiency (SCID) mouse model. Immunostaining for B18R in systemically treated HIVE/SCID mice suggested the ability of B18R to cross the blood-brain barrier (BBB). Real-time PCR indicated that B18R treatment resulted in a decrease in gene expression associated with IFN-? signaling in the brain. Mice treated with B18R were found to have decreased mouse mononuclear phagocytes and significant retention of neuronal arborization compared to untreated HIVE/SCID mice. Increased mononuclear phagocytes and decreased neuronal arborization are key features of HIVE. These results suggest that B18R crosses the BBB, blocks IFN-? signaling, and it prevents key features of HIVE pathology. These data suggest that the high affinity and broad IFN-? subtype specificity of B18R make it a viable alternative to monoclonal antibodies for the inhibition of IFN-? in the immune-suppressed environment.

SUBMITTER: Fritz-French C

PROVIDER: S-EPMC4080846 | biostudies-other | 2014 Jul

REPOSITORIES: biostudies-other

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The recombinant vaccinia virus gene product, B18R, neutralizes interferon alpha and alleviates histopathological complications in an HIV encephalitis mouse model.

Fritz-French Cari C Shawahna Ramzi R Ward Jennifer E JE Maroun Leonard E LE Tyor William R WR

Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research 20140224 7

Interferon-alpha (IFN-α) has been identified as a neurotoxin that plays a prominent role in human immunodeficiency virus (HIV)-associated neurocognitive disorders and HIV encephalitis (HIVE) pathology. IFN-α is associated with cognitive dysfunction in other inflammatory diseases where IFN-α is upregulated. Trials of monoclonal anti-IFN-α antibodies have been generally disappointing possibly due to high specificity to limited IFN-α subtypes and low affinity. We investigated a novel IFN-α inhibito ...[more]

PMID: 24564363

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Project description:BackgroundIn response to viral infections, interferons induce the transcription of several hundred genes in mammalian cells. Specific antiviral functions, however, have only been attributed to a few of them. 90K/LGALS3BP has been reported to be an interferon-stimulated gene that is upregulated in individuals with cancer or HIV-1 infection.ResultsHere, we show that 90K expression dose-dependently decreased the particle infectivity of HIV-1 progeny. The lower infectivity of released particles correlated with reduced virion incorporation of mature envelope glycoproteins gp120 and gp41. Further, proteolytic processing of the gp160 precursor and surface expression of gp120 in the producer cell were impaired in the presence of 90K expression. In contrast, expression of Gag, Nef and Vpu, and virus release were not grossly affected by 90K expression. 90K-imposed restriction occurred in the absence of direct interaction of 90K with HIV-1 Env or entrapment of Env in the ER. The cell-associated, but not the secreted species of 90K, mediated the antiviral effect. A truncated version of human 90K, solely consisting of the two intermediate domains, displayed a similar antiviral activity as the full-length wildtype 90K, indicating that the N-terminal SRCR-like domain and the C-terminal domain are dispensable for 90K's antiviral activity. The murine homolog of 90K, CypCAP (Cyclophilin C-associated protein), neither modulated particle infectivity of HIV-1 nor lowered the virion incorporation of mature gp120, suggesting a species-specific mode of action. 90K was expressed at basal levels in TZM-bl cells and in primary macrophages, and at low levels in CD4⁺ T-cells and PBMCs. 90K's susceptibility to IFN-mediated stimulation of expression was cell type-specific. siRNA-mediated knockdown of 90K in TZM-bl cells and primary macrophages enhanced the incorporation of Env glycoproteins into progeny virions, boosted the particle infectivity of released HIV-1, and accelerated HIV-1 spread. Conversely, treatment of HIV-1 infected macrophages with IFN-α induced 90K expression and lowered the particle infectivity of HIV-1.ConclusionsThus, 90K constitutes a novel antiviral factor that reduces the particle infectivity of HIV-1, involving interference with the maturation and incorporation of HIV-1 Env molecules into virions.

Project description:B18R protein of Vaccinia virus binds to type I interferons and inhibits activation of interferon-mediated signal transduction. Cells which have unimpaired interferon signaling such as primary cell cultures or some industrially important cell lines are capable of development of an antiviral state. An establishment of the antiviral state limits replication of RNA-viruses and can suppress replication of RNA vectors. The interferon inhibitor B18R effectively prevents the establishment of the antiviral state. For this reason, B18R has become a ubiquitous component of protocols for epigenetic reprogramming which use transfections of RNA replicons or mRNA. Despite wide practical applicability, commercially available B18R is predominantly produced in cell cultures and little information has been published on a production and use of bacterially expressed B18R. Objectives of this study were to produce B18R in an E.coli expression system and to confirm the product's biological activity by using it to maintain RNA-vectors in cell cultures capable of the antiviral state. The described method allows the expression and efficient refolding to obtain 10-100 mg of B18R from a small-scale culture and the production process is economically attractive compared to a use of an eukaryotic expression. To check for a presence of the biological activity of bacterially-expressed B18R the protein was used to support persistence of an autonomously replicating RNA-vector in a cell culture which is capable of the antiviral state. A RNA-containing virus, Venezuelan equine encephalitis virus (VEE) can serve as an efficient vector for heterologous expression in cell cultures, although its replication is sensitive to the effects of type I interferons which limit a range of cell lines for a use with this vector. The VEE replicon was utilized to direct an expression of recombinant human granulocyte colony stimulating factor (G-CSF). The producing replicon could persist in HEK293 cells for sufficiently long time only in presence of B18R, whereas addition of B18R not only allowed persistence of the replicon but also increased production from the replicon. A model product granulocyte colony stimulating factor accumulated to 35.5 ?g/ml during a 7 day experiment. This work describes efficacious expression and refolding of the viral cytokine inhibitor and demonstrates a utility of bacterially-expressed B18R.

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The recombinant vaccinia virus gene product, B18R, neutralizes interferon alpha and alleviates histopathological complications in an HIV encephalitis mouse model.

Publications

The recombinant vaccinia virus gene product, B18R, neutralizes interferon alpha and alleviates histopathological complications in an HIV encephalitis mouse model.

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