Impaired antiviral response and alpha/beta interferon induction in mice lacking beta interferon

Abstract

We have generated mice lacking the gene for beta interferon and report that they are highly susceptible to vaccinia virus infection. Furthermore, in cultured embryo fibroblasts, viral induction of alpha interferon and of 2-5A synthetase genes is impaired. We also show that beta interferon does not prime its own expression.

FIG. 1

Design and use of IFNβ targeting constructs and genotyping of MEFs from an ES clone targeted with pMuβGFP/neo. Details of plasmid construction and conditions for cell growth and transfection are described elsewhere (9). (a) The targeting construct pMuβGFP/neo (NotI linearized; top), the IFNβ locus (center), and the product of homologous recombination between them (bottom) are represented as follows: IFNβ gene (black box), IFNβ promoter region (ellipse), other DNA from the IFNβ locus (thick black lines), GFP gene (white box; from pRSGFP [Clontech]), neo cassette (stippled box), loxP site (black triangle), and pBSKSII+ DNA (thin line). Key sites for BamHI (B) and resulting fragments detectable by probe a (black bar) are shown. (b) As for panel a except for targeting construct pMuβCD2/neo; white box represents human CD2 gene. (c) Flow cytometric analysis of GFP expression in a G418-resistant 293 clone stably transfected with pMuβGFP/neo, either without induction or 48 h after induction by Sendai virus infection. (d) Flow cytometric analysis of CD2 expression in a pool of 293 G418-resistant 293 clones stably transfected with pMubCD2/neo, either without induction or 48 h after induction by Sendai virus infection. Conditions for electroporation, infection, and flow cytometry in panels c and d have been previously described (9) and are available on request. (e) Screening by Southern analysis for ES cell clones targeted with pMuβGFP/neo (18, 22). Analysis of BamHI-digested DNA from 10 G418-resistant clones probed with probe a. The 7.3-kb band representing the unmodified IFNβ locus migrates differently in odd- and even-numbered lanes because one comb was used for odd lanes and another for even lanes. A targeted clone and its diagnostic band are indicated by vertical and horizontal arrows, respectively. (f) As for panel e but with targeting construct pMuβCD2/neo. (g) Duplex PCR detection of IFNβ and GFP genes in genomic DNA. ND, no template DNA. (h) Southern analysis of BamHI-digested MEF DNA probed with probe a or IFN-β probe (a 460-bp BamHI-KpnI fragment of the IFNβ gene). Size markers (M) are shown.

FIG. 2

RT-PCR assays for induction of IFN-α/β, GFP, and 2-5A synthetase transcripts in IFN-β^+/+, IFN-β^+/−, and IFN-β^−/− MEFs. MEFs were infected with Sendai virus and harvested for RNA preparation either immediately (0 h) or 12 h later, as indicated. RNA (1 μg), prepared by lysis in guanidium isothiocyanate and density gradient centrifugation, was reverse transcribed (15-μl reaction mixtures containing avian myeloblastosis virus reverse transcriptase [Promega Biotech]) and, after the indicated dilutions, RT products (1 μl) were assayed by PCR for the following cDNAs: IFN-β, GFP, 2-5A synthetase (2-5AS), all known IFN-α subtypes (Uα), IFN-α-4 subtype (a4), all known IFN-α subtypes excluding IFN-α-4 (Nα4) and, as a positive control, phosphoglycerate kinase (PGK). Conditions for PCRs are summarized in Table 1.

FIG. 3

Vaccinia virus infection in IFN-β^+/+ and IFN-β^−/− mice. Groups of 7- to 9-week-old IFN-β^+/+ (open circles) or IFN-β^−/− mice (closed circles) were intranasally infected with 10³, 10⁴, or 10⁵ PFU of vaccinia virus strain Western Reserve. Every day, mice were individually weighed and monitored for signs of illness, scored from zero to four (ruffled fur, arched backs, and reduced mobility), or death. The mean percentage weight loss of each group ± the standard error of the mean, relative to the weight immediately preceding the infection, and the mean value of signs of illness ± the standard error of the mean in groups of mice infected with the indicated doses of virus, are shown. The horizontal bars indicate those days in which differences were statistically significant when analyzed by Student's t test, and the P values are shown. The number of mice per group that either died or were sacrificed due to severe infection is shown in the insets.

FIG. 4

Vaccinia virus replication in IFN-β^+/+ and IFN-β^−/− mice (7 to 9 weeks old). Groups of IFN-β^+/+ (open circles) and IFN-β^−/− mice (closed circles) were infected intranasally with 10³ or 10⁴ PFU of vaccinia virus strain Western Reserve per animal as previously described (2, 42). On the indicated days postinfection, animals were sacrificed and infectious virus in Dounce-homogenized lungs, spleen, and brain was determined by plaque titration on BS-C-1 cell monolayers. The geometric mean (=) and titers in independent mice, expressed as PFU per organ, are presented. The dashed line indicates the detection limit of the assay.