Analysis of gene expression in fetal and adult cells infected with rubella virus

Abstract

Congenital infection with rubella virus (RUB) leads to persistent infection and congenital defects and we showed previously that primary human fetal fibroblasts did not undergo apoptosis when infected with RUB, which could promote fetal virus persistence [Adamo, P., Asís, L., Silveyra, P., Cuffini, C., Pedranti, M., Zapata, M., 2004. Rubella virus does not induce apoptosis in primary human embryo fibroblasts cultures: a possible way of viral persistence in congenital infection. Viral Immunol. 17, 87-100]. To extend this observation, gene chip analysis was performed on a line of primary human fetal fibroblasts (10 weeks gestation) and a line of human adult lung fibroblasts (which underwent apoptosis in response to RUB infection) to compare gene expression in infected and uninfected cells. A total of 632 and 516 genes were upregulated or downregulated in the infected fetal and adult cells respectively in comparison to uninfected cells, however only 52 genes were regulated in both cell types. Although the regulated genes were different, across functional gene categories the patterns of gene regulation were similar. In general, regulation of pro- and anti-apoptotic genes following infection appeared to favor apoptosis in the adult cells and lack of apoptosis in the fetal cells, however there was a greater relative expression of anti-apoptotic genes and reduced expression of pro-apoptotic genes in uninfected fetal cells versus uninfected adult cells and thus the lack of apoptosis in fetal cells following RUB infection was also due to the prevailing background of gene expression that is antagonistic to apoptosis. In support of this hypothesis, it was found that of a battery of five chemicals known to induce apoptosis, two induced apoptosis in the adult cells, but not in fetal cells, and two induced apoptosis more rapidly in the adult cells than in fetal cells (the fifth did not induce apoptosis in either). A robust interferon-stimulated gene response was induced following infection of both fetal and adult cells and many of the genes upregulated in both cell types were those involved in establishment of an antiviral state; this is the first demonstration of an interferon response at this early stage of human embryonic development. In both fetal and adult cells, interferon controlled but did not eliminate virus spread and apoptosis was not induced in infected fetal cells in the absence of interferon. In addition to the interferon response, chemokines were induced in both infected fetal and adult cells. Thus, it is possible that fetal damage following congenital RUB infection, which involves cell proliferation and differentiation, could be due to induction of the innate immune response as well as frank virus infection.

FIG. 1

Growth of RUB in human cell lines. Cultures of Hs888Lu, HEF, Hs143we, and Vero cells were infected with RUB (MOI = 10 PFU/cell) and titer of virus present in the culture medium was determined by plaque assay at 1, 3, 5, and 7 dpi (A) and the percentage of apoptotic cell was determined at 5 dpi (B). The data presented in Panel A are representative of 2 independent repetitions and the data presented in Panel B are the average of 3 independent repetitions.

FIG. 2

Interferon induction in RUB-infected and poly IC treated cells. Cultures of Hs888Lu and HEF were infected with RUB (MOI = 10 PFU/cell) or treated with poly IC (50 μg/ml) and IFN’s in the culture medium were determined at the indicated time points. The data presented are the average of two (poly IC) or three (RUB infection) independent repetitions.

FIG. 3

Growth of RUB in cells pretreated with IFN-containing medium. Growth medium from mock-infected or RUB-infected Hs888Lu and HEF was harvested 3 dpi and virus was UV inactivated at 20,000 μJ/cm² for 20 min on ice. Fresh Hs888Lu cells were then incubated with UV-inactivated medium from mock- or RUB-infected Hs888Lu cells for 24 hours followed by infection with RUB (10 PFU/cell) and likewise, fresh HEF were incubated with UV-inactivated medium from mock-or RUB infected HEF for 24 hours followed by infection with RUB. Titer of virus present in the culture medium was determined by plaque assay at 1, 3, and 5 dpi (A) and the percentage of infected cells was determined at 3 dpi (B). The data presented in Panel A are representative of 2 independent repetitions. The data presented in Panel B are the average of 3 independent repetitions.

FIG. 4

Growth of RUB in cells pretreated with anti-IFN serum. Cultures of Hs888Lu cells and HEF were incubated with anti-Type I IFN antiserum for 24 hours and then infected with RUB (MOI = 10 PFU/cell) in the presence of the anti-interferon serum. At 3 dpi, titer of virus present in the culture medium was quantitated by plaque assay (A) and the percentage of infected cells was determined (B). In both cases, the data presented are the average of 2 independent repetitions. Cells were stained with Giemsa for microscopy at 7 dpi (C) and the percentages of apoptotic cells were determined by TUNEL assay at 5 dpi (D; average of 2 independent repetitions).