Myxoma virus infection of primary human fibroblasts varies with cellular age and is regulated by host interferon responses

Abstract

Recent studies have indicated a critical role for interferon (IFN)-mediated antiviral responses in the host range of myxoma virus (MV), a pathogenic poxvirus of rabbits. To investigate the contribution of IFN to MV tropism in nonleporine cells, primary human dermal fibroblasts (HDFs) were tested for permissiveness to MV infection. Low-passage HDFs that underwent fewer than 25 population doublings (PD) were fully permissive for MV infection, supporting productive virus replication and cell-to-cell spread. In contrast, early and late viral gene expression was detectable in high-passage HDF (>75 PD), but MV failed to generate infectious progeny and could not form foci in these cells. Vesicular stomatitis virus (VSV) plaque reduction assays confirmed that constitutive IFN production progressively increased as HDFs were passaged, concurrent with an increase in the expression of transcripts for type I IFN and IFN-responsive genes involved in antiviral responses. These findings correlated with the enhanced sensitivity of higher-passage HDF to inducers of type I IFN responses, such as dsRNA. Furthermore, pretreatment of low-passage HDF with type I IFN abrogated MV spread and replication while treatment of mature HDF with neutralizing antibodies to IFN-beta, but not IFN-alpha, restored the capacity to form foci. These findings emphasize the importance of post-entry events in determining the permissiveness of human cells to MV infection and support a critical role for innate type I IFN responses as key determinants of poxvirus host range and species restriction.

Safety and immunogenicity of myxoma virus as a new viral vector for small ruminants

“…We demonstrated that primary ovine fibroblasts could be infected by both the wild-type T1 strain and the vaccine SG33 strain of MYXV, and that the infection was abortive. It has been shown previously that MYXV can productively infect non-host primary fibroblasts in vitro depending on cellular interferon (IFN) production Johnston et al, 2005). In our work, primary fibroblasts were generated from adult ovine skin; even though they were cultured over only three passages, they should produce type I IFN.…”

Myxoma Virus Virotherapy for Glioma in Immunocompetent Animal Models: Optimizing Administration Routes and Synergy with Rapamycin

“…The mechanisms by which rapamycin affects innate immune responses within the gliomas and the surrounding normal host tissues are not completely understood; however, we do know that type I IFN is an important component of the innate response to MYXV. MYXV replication in mammalian cells is naturally sensitive to inhibition by IFN (38), and the host antiviral responses mediated by type I IFN are a major determinant of its restrictive host range outside the natural rabbit host (18). Moreover, RIG-I mediates the coinduction of tumor necrosis factor and type I IFN elicited by MYXV in primary human macrophages, and together, these two induced cytokines shut down virus replication in all primary untransformed human cells tested to date (41).…”

“…The firefly luciferase gene plasmid (pGL3 enhancer vector; Promega) was cotransfected into RG2 cells as reported before (37). All the viruses used in this study were constructed in the laboratory of G. McFadden, and virus was propagated and titrated by focus formation on BGMK cells as described previously (5,16,21,22,38,39). UV-inactivated MYXV [dead virus (DV)] was prepared by irradiating virus with UV light for 2 h. Rapamycin was obtained from LC Laboratories, and RAD001 dry powder and cyclophosphamide were obtained from Sigma.…”

Targeting Human Medulloblastoma: Oncolytic Virotherapy with Myxoma Virus Is Enhanced by Rapamycin

“…Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with antibodies recognizing the viral proteins M-T7 and Serp-1 (7,38), phosphorylated and total Akt (Cell Signaling Technology), and phosphorylated and total 70-kDa ribosomal protein S6 kinase (p70S6K; Cell Signaling Technology). Immunoreactive proteins were detected by enhanced chemiluminescence.…”