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Comparative Study
. 2014 May;88(9):4932-42.
doi: 10.1128/JVI.03508-13. Epub 2014 Feb 19.

Autonomous parvoviruses neither stimulate nor are inhibited by the type I interferon response in human normal or cancer cells

Justin C Paglino  1 , Wells Andres, Anthony N van den Pol
Affiliations
Comparative Study

Autonomous parvoviruses neither stimulate nor are inhibited by the type I interferon response in human normal or cancer cells

Justin C Paglino et al. J Virol. 2014 May.

Abstract

Members of the genus Parvovirus are small, nonenveloped single-stranded DNA viruses that are nonpathogenic in humans but have potential utility as cancer therapeutics. Because the innate immune response to parvoviruses has received relatively little attention, we compared the response to parvoviruses to that of several other types of viruses in human cells. In normal human glia, fibroblasts, or melanocytes, vesicular stomatitis virus evoked robust beta interferon (IFN-β) responses. Cytomegalovirus, pseudorabies virus, and Sindbis virus all evoked a 2-log-unit or greater upregulation of IFN-β in glia; in contrast, LuIII and MVMp parvoviruses did not evoke a detectable IFN-β or interferon-stimulated gene (ISG; MX1, oligoadenylate synthetase [OAS], IFIT-1) response in the same cell types. The lack of response raised the question of whether parvoviral infection can be attenuated by IFN; interestingly, we found that IFN did not decrease parvovirus (MVMp, LuIII, and H-1) infectivity in normal human glia, fibroblasts, or melanocytes. The same was true in human cancers, including glioma, sarcoma, and melanoma. Similarly, IFN failed to attenuate transduction by the dependovirus vector adeno-associated virus type 2. Progeny production of parvoviruses was also unimpaired by IFN in both glioma and melanoma, whereas vesicular stomatitis virus replication was blocked. Sarcoma cells with upregulated IFN signaling that show high levels of resistance to other viruses showed strong infection by LuIII. Unlike many other oncolytic viruses, we found no evidence that impairment of innate immunity in cancer cells plays a role in the oncoselectivity of parvoviruses in human cells. Parvoviral resistance to the effects of IFN in cancer cells may constitute an advantage in the virotherapy of some tumors.

Importance: Understanding the interactions between oncolytic viruses and the innate immune system will facilitate employing these viruses as therapeutic agents in cancer patients. The cancer-selective nature of some oncolytic viruses is based on the impaired innate immunity of many cancer cells. The parvoviruses H-1, LuIII, and MVM target cancer cells; however, their relationship with the innate immune system is relatively uncharacterized. Surprisingly, we found that these parvoviruses do not evoke an interferon response in normal human fibroblasts, glia, or melanocytes. Furthermore, unlike most other types of virus, we found that parvovirus infectivity is unaffected by interferon treatment of human normal or tumor cells. Finally, parvoviral replication was unimpaired by interferon in four human tumor types, including those with residual interferon functionality. We conclude that deficits in the interferon antiviral response of cancer cells do not contribute to parvoviral oncoselectivity in human cells. The interferon-resistant phenotype of parvoviruses may give them an advantage over interferon-sensitive oncolytic viruses in tumors showing residual interferon functionality.

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Figures

FIG 1
FIG 1
No IFN response to parvoviral infection in three normal human cell types. Normal human glia (A), human fibroblasts (B), or human melanocytes (C) were left uninfected (Uninf.) or were infected with the indicated viruses at 5 PFU/cell. mRNA levels for IFN-β and for the interferon-stimulated gene MX1 were determined for samples taken at 36 hpi, and the ratio of the IFN-β and MX-1 mRNA levels to the level of β-actin expression was determined. n.d., none detected; n.s., not significant by ANOVA. Error bars, SEMs.
FIG 2
FIG 2
Detailed analysis of ISG response to infection of human glia. Cells were left uninfected or were infected with LuIII or VSV at 5 PFU/cell. At 18 hpi (VSV) and 24 hpi (LuIII), cells were harvested for analysis of mRNA levels of IFN-β and of the interferon-stimulated genes MX1, OAS, IFIT1, and PKR. All values were normalized to those for β-actin and are expressed as the fold induction over that for uninfected cells. Error bars, SEMs.
FIG 3
FIG 3
High-MOI LuIII parvovirus infection (10 PFU/cell) evokes no IFN response in human glial cells responsive to 4 unrelated viruses. Normal human glial cells were uninfected or were infected at 10 PFU/cell with LuIII, hCMV, PRV, Sindbis virus, or VSV. At 24 hpi, RNA was harvested and the IFN-β mRNA level was determined and normalized to the β-actin mRNA level. For each virus, the fold induction of IFN-β relative to that for uninfected cells is shown. Error bars, SEMs.
FIG 4
FIG 4
Parvoviral infection of normal cells with and without IFN preexposure. Normal human glia (A) and fibroblasts (B and C) were left untreated or were preexposed to exogenous IFN. Cells were infected with LuIII, MVMp, H-1, or VSV at 5 PFU/cell and fixed at 24 hpi. VSV was visualized by use of a GFP reporter, and parvovirus-infected cells were visualized by NS1 immunostaining. Error bars, SEMs. * and n.s., significant and nonsignificant, respectively, by ANOVA.
FIG 5
FIG 5
Preinfection with LuIII for 24 h does not diminish VSV infectivity. Human fibroblasts or glia were LuIII preinfected (10 PFU/cell) or not for 24 h and then inoculated with VSV. The percentage of GFP-positive (VSV-infected) cells was determined at 10 hpi. Error bars, SEMs.
FIG 6
FIG 6
Infectivity of parvovirus LuIII and parvoviral vector AAV-T are unimpaired in human sarcoma cells that are protected from VSV by either intrinsic IFN (SW-R) or extrinsic IFN (SW-S + IFN). (A) Micrographs show SW-R cells, cells of an IFN-secreting cell line that is highly VSV resistant but susceptible to parvoviruses LuIII and AAV-T. (B) Measured infectivity of VSV, LuIII, and AAV-T in VSV-sensitive sarcoma SW-S cells either with IFN (+IFN) or without IFN pretreatment and in VSV-resistant sarcoma SW-R cells. n.s., not significant; *, significant by ANOVA. Error bars, SEMs; pfu/c, number of PFU per cell; Kg/c, number of kilogenomes per cell.
FIG 7
FIG 7
The infectivity of parvoviruses LuIII and AAV-T is unaffected by IFN in human tumors regardless of IFN status. IFN pretreatment protected human melanoma YUSIV cells (A and B) (5 PFU/cell) but not human glioma U87 cells (C) from VSV. The infectivity of LuIII (20 PFU/cell) and AAV (20,000 genomes/cell) was unaffected by IFN pretreatment in both tumor types (B and C). Error bars, SEMs. * and n.s., significant and nonsignificant, respectively, by ANOVA.
FIG 8
FIG 8
Parvoviral selectivity for transformed glioma over normal human glia is unaffected by IFN pretreatment. Human glia or glioma U87 cells were left untreated (−IFN) or were pretreated with interferon (+IFN; 100 U/ml for 4 h) and then infected with parvovirus H-1, MVM, or LuIII at 0.1 PFU/cell. The percentage of infected cells was determined at 24 hpi. The data are plotted in two formats: to illustrate that in no circumstance did IFN pretreatment affect infectivity (A) and to illustrate that the oncoselectivity of all three parvoviruses was unaltered by IFN pretreatment (B). Error bars, SEMs. * and n.s., significant and nonsignificant, respectively, by ANOVA.
FIG 9
FIG 9
Parvovirus LuIII replication is not blocked by IFN pretreatment in a variety of human tumor-derived cell lines. Human gliomas (A172 and U87) and human melanomas (YUSIK and YURIF) were infected at 1 PFU/cell with LuIII (A) or VSV (B) either without IFN (−IFN) or with IFN (+IFN) pretreatment (100 U/ml for 4 h). At 24 hpi, supernatant from VSV-infected cells was harvested for determination of the titer of the VSV progeny (B). At 72 hpi, cells and supernatant from LuIII-infected cells were harvested for determination of the titer of LuIII progeny (A). Error bars, SEMs. * and n.s., significant and nonsignificant, respectively, by ANOVA.
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