Mutations in the IGF-II pathway that confer resistance to lytic reovirus infection - PubMed (original) (raw)

Mutations in the IGF-II pathway that confer resistance to lytic reovirus infection

Jinsong Sheng et al. BMC Cell Biol. 2004.

Abstract

Background: Viruses are obligate intracellular parasites and rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies.

Results: A gene entrapment approach was used to identify candidate cellular genes that affect reovirus infection or virus induced cell lysis. Four of the 111 genes disrupted in clones selected for resistance to infection by reovirus type 1 involved the insulin growth factor-2 (IGF-II) pathway, including: the mannose-6-phosphate/IGF2 receptor (Igf2r), a protease associated with insulin growth factor binding protein 5 (Prss11), and the CTCF transcriptional regulator (Ctcf). The disruption of Ctcf, which encodes a repressor of Igf2, was associated with enhanced Igf2 gene expression. Plasmids expressing either the IGF-II pro-hormone or IGF-II without the carboxy terminal extension (E)-peptide sequence independently conferred high levels of cellular resistance to reovirus infection. Forced IGF-II expression results in a block in virus disassembly. In addition, Ctcf disruption and forced Igf2 expression both enabled cells to proliferate in soft agar, a phenotype associated with malignant growth in vivo.

Conclusion: These results indicate that IGF-II, and by inference other components of the IGF-II signalling pathway, can confer resistance to lytic reovirus infection. This report represents the first use of gene entrapment to identify host factors affecting virus infection. Concomitant transformation observed in some virus resistant cells illustrates a potential mechanism of carcinogenesis associated with chronic virus infection.

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Figures

Figure 1

Figure 1

Persistently infected RIE-1 cells fail to survive in serum-free media. RIE-1 parental cells and cells persistently infected with reovirus type 1 were plated in complete medium (FBS+) or in media in which the serum was omitted (FBS-). Surviving cells were stained with gentian violet after 7 days. Darkly staining wells represent cell survival.

Figure 2

Figure 2

Disruption of the Ctcf gene in 6B72 cells. DNA sequences flanking the U3NeoSV1 provirus in 6B72 cells were cloned and sequenced. The flanking sequences were identical to sequences in the rat genome, placing the provirus in the first intron of the Ctcf gene (A). Filled and open boxes indicate coding and non-coding exons, respectively. Flanking sequences 5' of the U3NeoSV1 provirus (B) include the first exon (shaded) of the gene.

Figure 3

Figure 3

CTCF and Igf2 expression in RIE-1 and 6B72 cells. Levels of CTCF protein were assessed by Western blot analysis (A), normalized to a β-actin control. Levels of Igf2 transcripts were assessed by Northern blot analysis (B) normalized to GAPDH control. Protein content, as assayed by western blot analysis and standardized to β-actin was decreased in the 6B72 cell clone to 30% of control. Reverse transcriptase PCR analysis of Igf2 transcripts (C). The RT-PCR products (arrows) were separated on a 2% agarose gel, revealing an additional transcript in the 6B72 cells. The DNA sequence of the larger RT-PCR product (D) revealed an alternatively spliced transcript (Igf2 sv) generated by splicing of exon 2 to a cryptic 3' splice site located 14 nucleotides upstream of exon 3.

Figure 4

Figure 4

Alternatively spliced product contains a single nucleotide polymorphism in the igf2 coding sequence. Intron sequences incorporated into the alternatively spliced transcript (highlighted in black) alter the translational reading frame of the pro-homone downstream of the coding sequence of the processed IGF-II protein (italics and bold). The Igf2 sv PCR product also contained a G to A base substitution (underlined) that replaces alanine with threonine at codon 62 (boxed) of the mature hormone.

Figure 5

Figure 5

IGF-II modulates reovirus resistance in RIE-1 and 6B72 cells. RIE-1 and 6B72 cells expressing either the IGF-II pro-hormone (proIGF2) or the alternatively spliced transcript (IGF2SV, Figure 3D) were challenged with serial dilutions of reovirus type 1 (upper panel), and the surviving cells were stained with gentian violet 4 days post-infection. The multiplicity of infection (MOI) for each row is indicated. pro-IGF-II converted RIE-I cells to a reovirus resistant phenotype (RIE-1/proIGF2) but had little if any effect on already-resistant 6B72 cells (6B72/proIGF2). Plasmids expressing the alternatively spliced Igf2 transcript had no effect on RIE-1 cells (RIE-1/IGF2SV) but abrogated virus resistance in 6B72 cells (6B72/IGF2SV). The experiment was repeated 3 times. Expression of I_gf2_ transgenes (lower panel) was monitored by Northern blot hybridization, and the expression of Igf2 in RIE-1 cells is shown for comparison. Expression of Igf2 in 6B72 is not shown.

Figure 6

Figure 6

IGF-II sequences lacking the E-peptide can convert RIE-1 cells to a reovirus-resistant phenotype. Two independent clones of RIE-1 cells transfected with plasmids expressing native (IGF-II) and mutant (IGF-IIA62T) proteins without the carboxyl terminal extension-peptide (E-peptide) were challenged with serial dilutions of reovirus type 1 as described in Figure 4. Native IGF-II protected RIE-1 cells from reovirus infection (IGF-II(a) and IGF-II(b)), while the IGF-IIA62T mutant (IGF-IIA62T(a) and IGF-IIA62T(b) did not. Non-infected RIE-1 cells (C), and infected 6B72, and RIE-1 cells were included as controls. Expression of Igf2 transgenes (lower panel) was monitored by Northern blot hybridization, and the expression of Igf2 in RIE-1 cells is shown for comparison. The native IGF-II (small arrow) is slightly larger than the cDNA constructs (larger arrow), whereas the double-sided arrow marks the constitutively expressed GAPDH, as shown. Expression of Igf2 in 6B72 is not shown.

Figure 7

Figure 7

Decreased lytic infection of L-cell clones over expressing the IGF-II gene. Constitutively expressed GAPDH was used to assess loading of RNA in lanes. Survival of lytic infection was determined by infecting 105 L-cells or L-cells expressing the pro-IGF2 transgene with varying concentrations of reovirus type 1 (A) or type 3 (B). The multiplicity of infectious virus particles per cell (MOI) is indicated for each virus serotype. Surviving cells were visualized at 4 (A) or 5 days (B) following infection with gentian violet. Transgene expression was determined by northern blot (C). Experiments were repeated three times and a representative experiment is shown.

Figure 8

Figure 8

6B72 cells have delayed disassembly of reovirus type 1. Fluorescein-labelled reovirus particles were absorbed to RIE-1 (A) or 6B72 cells (B). Persistent fluorescence at 2 hours was found in 6B72 cells, but not in RIE-1 cells. Non-replicating reovirus type 1, at 3 × 104 particles per cell, was adsorbed to RIE-1 (C) or 6B72 (D) cells at 4°C, washed and incubated at 37°C for 2 and 4 hours. Cells were lysed and the state of virus particles determined by western blot. The outer capsid proteins μ1 and σ3 are present in the 6B72 cell preparations at 2 and 4 hours, but not in the RIE-1 cells.

Figure 9

Figure 9

Anchorage-independent growth phenotypes of RIE-1 cell clones. 105 cells were suspended in media containing 1% agarose and plated in 6 well culture dishes. RIE-1 cells acquire the ability to grow in soft agar after being transfected with a vector expressing pro-IGF2 but not the IGF2SV splice-variant (a). The vector inserted in the Ctcf gene (6B72) confers the ability to grow in soft agar, but the phenotype is suppressed by expression of IGF2SV (b). Clones selected for reovirus resistance with gene trap vectors inserted into the Prss11, Igf2r and Anxa2 genes failed to grow in soft agar (c). RIE-1 cells expressing native IGF-II protein without the E-peptide grew in soft agar but the colonies were smaller (d) than produced by pro-IGF-2 (a), while the corresponding IGF-IIA62T protein (E-peptide) did not transform RIE-1 cells to anchorage independence (d). Colonies were photographed (20×) after 7 days except (d) where the cells were photographed after 10 days.

Figure 10

Figure 10

IGF-II increases colony formation of L-cells in soft agar. Forced expression of the rat pro-Igf2 gene (pro-Igf2c) in L-cells increases the number of soft-agar colonies by 4 to 5 fold as compared to the L-cell parent, as shown in this representative photomicrograph at 7 days (10×).

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