Molecular detection and identification of type I interferon mRNAs

12

GATA 11(1): 12-19, 1994

Molecular Detection and Identification of Type I Interferon mRNAs M A R Y C. L A I , S T U A R T J. B O Y E R , and M A N F R E D W. B E I L H A R Z The development of a technique for identifying murine type I interferon messenger RNAs is described that involves the following essential steps: (a) the reverse transcription of total R N A extracts using oligo(dT)le_ls as a primer, (b) the amplification of any type I interferon cDNAs produced by polymerase chain reaction, and (c) the identification of interferon subtypes by hybridization of the polymerase chain reaction products to specific oligonucleotides. The technique was used to characterize the expression of the mouse interferon subtypes etl, ct4, o0, ct6, and f~ in murine L929 cells that had been infected with Newcastle disease virus. The data derived from this study are in excellent agreement with earlier R N A protection experiments performed in the same system to characterize expression of the same genes. The present technique has advantages over those used previously, including superior sensitivity, speed, and far smaller input R N A requirements. The technique is not only applicable to other in vitro systems, but is appropriate for use in vivo.

Introduction The interferons (IFNs) are small, robust, soluble proteins found in all vertebrates. The presently accepted nomenclature is based on sequence analysis of the known IFN genes [1]. Four major classes have been defined: IFNs-a, IFN-to, IFN13, and IFN-~. The IFNs-et consist of multiple closely related species, at least 12 nonallelic genes in humans and 11 in mice, with further pseudogenes present in both cases. The IFNs-to, absent in mice, are represented by six genes in humans; however, all but one appear to be pseudogenes. The IFN-13 gene is present in humans and mice as

From the Departmentof Microbiology,Universityof Western Australia, Nedlands, Western Australia. Address correspondence to Dr. M.W. Beilharz, Department of Microbiology, Queen Elizabeth 1I Medical Centre, Universityof Western Australia,Nedlands, WA 6009, Australia. Received 17 November 1993;revised and accepted 21 January 1994.

a single copy gene. These three classes--IFNs-a, to, and 13---are also referred to as type I IFNs because their sequences show a high degree of relatedness. In humans, all of the genes are clustered on the short arm of chromosome 9 and all of them lack introns--an unusual feature for eukaryotic genes. The known type I IFN gene organization in mice is very similar. IFN-7, the fourth class of IFN, is quite distinct and is also designated as type II IFN. In both mice and humans, the IFN--¢ gene has the more usual intron structure and is not located on the same chromosome as the type I IFNs. The IFN-~/ sequence is only distantly related to other IFN sequences and the protein is physically and biologically distinct from other IFNs. The biologic properties attributed to IFNs fall into three categories: (a) antiviral effects, (b) antiproliferative effects, and (c) immunomodulatory effects. The subtypes of type I IFNs are also known to differ in their potencies [2-8], level of expression [9-11], regulation of gene expression [12], and ability to bind to receptors [13]. The rationale for the existence of the numerous type I IFN subtypes within a species is not fully understood. It has been suggested that each subtype may have a specific role. For example, in sheep and cattle, the trophoblast protein implicated in signaling maternal recognition of pregnancy was recently shown to be a type I IFN [14, 15]. Similarly, it has been shown that there are two IFN-ot subtypes constitutively expressed in organs of normal individuals and this may suggest a physiologic role for them [16]. For studies on the role of IFN subtypes, techniques have been developed to detect the type and level of the IFNs-a expressed in induced and noninduced cells as well as organs. Previously, Northern blot hybridization and S1 mapping have been used to detect and identify IFN mRNAs expressed in virus-induced cells [9-11] as well as in normal organs/blood cells [16]. This approach required large amounts of input RNA, however, and was difficult to apply in many situations. We sought to develop a faster and more widely applicable technique for the detection and characterization of type I IFNs. Reverse transcription (RT) of small RNA samples followed by polymerase chain reaction (PCR) was used for detection. Specific subtype identification was then achieved by differential hybridization to a panel of subtype-specific oligonucleotides. We chose to develop and characterize this system using mouse L929 cells infected with Newcas-

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13 GATA 11(1): 12-19, 1994

Molecular Detection of Interferon Subtype

tie disease virus (NDV) because the type I IFN subtypes expressed had been previously well characterized by S1 nuclease protection methods [10, 11, 17, 18]. The data available enabled us not only to check subtypes expressed, but also to estimate the level of sensitivity of the new detection method and the degree to which quantitation of relative amounts was possible. Materials and Methods

(Promega, Madison, WI, USA) in the presence of 20 U RNasin (Promega) and 6 mM MgCI 2 at 37°C for l0 min. The DNase was then heat inactivated by incubating in boiling water for 20 min. The complete removal of any contaminating DNA was checked by amplifying 1 I~g of total RNA directly. When amplification of total RNA samples confirmed that they were free of contaminating DNA, they were used for cDNA synthesis.

cDNA Synthesis

Induction of Murine Type I Interferons Mouse L929 cells were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS) in 80-mm 2 tissue culture flasks. Confluent monolayers of cells were washed in phosphatebuffered saline (PBS) and then infected with an Australian isolate of NDV to a final NDV concentration of 107.5 EIDso/flask of L929 cells in 1 ml of serum-free DMEM. After incubation at 37°C for 90 min, the cells were washed as above and incubated in DMEM supplemented with 2% FCS. At various times after incubation, the cells were washed in PBS and removed for RNA extraction. Control cells were treated similarly except that the NDV was replaced with serum-free media.

RNA Extraction Total cytoplasmic RNA from control and infected cells was extracted using RNAzol B (BIOTECX Lab, TX, USA). In brief, 2 ml of RNAzol B was added to lyse the cells in the tissue culture flask. The cells were solubilized by passing the lysate through a pipette. One-tenth volume of chloroform was added to the lysate in a 1.5-ml tube. The mixture was vigorously shaken for 15 s, incubated on ice for 5 min, and centrifuged for 15 min at 12,000 g at 4°C. The upper aqueous phase containing the RNA was removed and the RNA was precipitated by adding an equal volume of isopropanol at 4°C for 15 min. The RNA, pelleted by centrifuging at 12,000 g for 15 min, was washed by vortexing in 75% ethanol. The washed RNA pellet, recovered by further centrifuging at 7500 g for 8 rain at 4°C, was dried briefly under vacuum before being resuspended in nuclease-free water. The RNA was quantitated spectrophotometrically at 260/280 nm and analyzed by formaldehyde agarose gel electrophoresis. Any contaminating DNA present was removed by incubating l p.g of total RNA with 2 units (U) of RNase-free DNase RQ1

Total cytoplasmic RNA (I p,g) was utilized to synthesize cDNA by using 100 pmol of either a type I IFN-specific oligonucleotide, random hexamers, or oligo(dT)12_18 in the following reaction mixture of 30 ~l containing 15 U AMV reverse transcriptase (Promega), 1 × RT buffer, 5 mM MgC12, 30 U RNasin, and 1 mM of each dNTP. Following a 60-min incubation at 42°C, the reaction was terminated by heating to 95°C for 5 min. The cDNA was then stored at -70°C.

Oligonucleotide Synthesis A 308-base-pair (bp) fragment corresponding to nucleotides 78-388 of the published sequences of the known murine IFNs-et (MulFNs-e0 genes was amplified using the primers 5'-TCTCTCCTGCCTGAAGGAC-3' (upstream primer, corresponding to the region coding for amino acids [aa] 26-32) and 5'-ACACAGTGATCCTGTGGAA-3' ( d o w n s t r e a m primer, corresponding to the region coding for aa 124-130). MulFN-tx4 contains an internal deletion corresponding to aa 103-107 of other MulFNs-et, this deletion does not effect the annealing of the amplifying oligonucleotides. Similarly, a 390-bp fragment corresponding to nucleotides 19--408 of the published sequence of the murine IFN-13 gene [19] was amplified using the primers 5'CAGCTCCAAGAAAGGACGAA-3' (upstream primer, corresponding to the region coding for aa 7-13) a n d 5 ' - G T A G C T G T T G T A C T T C A T G A G - 3 ' (downstream primer, corresponding to the region coding for aa 130-136). These primers, as well as the oligonucleotides designed for use in identifying the IFN-o~ subtypes (Table 1), were synthesized using a Millipore Cyclone Plus DNA synthesizer and 13 cyanoethyl phosphoramidite chemical synthesis methodology. The oligonucleotides were cleaved and then deprotected by the standard ammonium hydroxide treatment at 55°C and desalted using NAP-10 columns (Pharmacia, Sweden).

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14 GATA 11(1): 12-19, 1994

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Table 1. Identifying Oligonucleotides for Murine Interferons (MulFNs)-etl,et4, et5, and c~6 Type I MulFN-a subtypes

Sequences of oligonucleotides used as specific hybridization probes for identification of type MulFN subtypes

Size in bp

T m in °C

Location of oligonucleotides in the aa sequence

al c~4 ct5 ct6

5'-A TTT CCC CTG ACC CAG GAA GAT G-3' 5'-CC TGT GTG ATG CAG GAA CCT CC-Y 5'-T GAA GTC CAT CAG CAG CTC AAT-3' 5'-CAG GTA GAG ATA CAG GCA CTT CC-3'

23 22 22 23

70 70 70 70

108-116 98-110 87-94 103-110

aa, amino acid; and bp, base pair.

5'-End Labeling and Purification of Oligonucleotides Oligonucleotides designed for use in identification of the MulFN-a subtypes (Table I) were 5'-end labeled with I~-3zp]ATP and T4-polynucleotide kinase (both from Amersham, UK). Radiolabeled oligonucleotides were purified by using BIO-Spin 6 columns (Bio-Rad, USA) and were used at a concentration of 10 6 dpm/ml in the hybridization solution.

DNA Amplification The PCR amplification reaction was performed in 50 ~1 total volume by using a Perkin Elmer-Cetus model 900 temperature cycler. The effect of MgCIz concentration (1, 1.5, 2, and 2.5 mM) on the amplification of control recombinant MulFN-a DNA by using different number of cycles of amplification (I0, 20, and 30) as well as at two different annealing temperatures (52° and 55°C) was evaluated. The sensitivity of the PCR technique was tested by amplifying different concentrations (1, I0, and 100 ng) of control MulFN-a DNA by using different numbers of cycles of amplification (10, 20, and 30). The other components used in the standard reaction were 200 mM of each dNTP, 1 × Taq polymerase buffer, 1 U Taq polymerase, and 50 pmol of each primer. The parameters for amplification were denaturation at 95°C for 5 min, annealing for 2 min, and extension at 70°C for 4 min for the first cycle, followed by denaturation at 95°C for 1 min, annealing for 1 min, and extension at 70°C for 2 min for the subsequent number of cycles tested. The c D N A from NDV-infected cells, used at a final dilution of 1 in 20, was amplified at the optimized conditions.

Analysis of PCR Products PCR amplification products were visualized by agarose gel electrophoresis, ethidium-bromide

staining, and UV illumination. PCR amplification products were then further characterized by fixing to synthetic membranes and hybridization to the panel of subtype-specific oligonucleotides. The hybridization and washing conditions were optimized using PCR-amplified products from control MulFN-ot DNAs. The concentration of the amplified products was estimated by electrophoresis in 2% agarose gel together with known standards. Equivalent amounts of each amplified subtype of MulFN-ct DNA were denatured and dot blotted according to manufacturers specifications onto H y b o n d N ÷ nitrocellulose membrane (Amersham) by using a dot-blot apparatus (Biorad). After air drying at room temperature, the membrane was prehybridized in 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS), and 1 M NaC1 for at least 30 min at 65°C. Hybridization was carried out at 65°C (Tin - 5°C for all the oligonucleotides used) with shaking in 6× SSC, 7% SDS, 100 ~g/ml denatured salmon-sperm DNA, and 10 6 dpm/ml [732p]ATP-labeled oligonucleotide. After overnight hybridization, the blot was washed twice for 10 min each at room temperature in 6× SSC, 1% SDS, followed by two washes at temperature r a n g i n g f r o m T m - 10°C t o T m + 5 ° C i n 6 × S S C ,

1% SDS. The blot was then autoradiographed at -70°C overnight. After overnight exposure, the film was developed. Results

Optimization of Amplification Conditions All sequenced MulFN-et genes have regions of highly conserved sequence at both the 5' and 3' ends of the structural genes. It was therefore possible to synthesize a pair of 19-nucleotide primers that could be used to amplify all known MulFN-a gene sequences (see Materials and Methods for details on these oligonucleotides). Conditions optimal for the use of these MulFN-et oligonucleotides were determined by amplification of linear

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15 GATA 11(1): 12-19, 1994

Molecular Detection of Interferon Subtype

MuIFN-ot DNA fragments obtained from plasmid clones of these genes [10]. Figure 1 shows the expected 308-bp PCR-amplified product from the MuIFN-otl gene at varying template concentrations, numbers of cycles of amplification, and MgCl 2 concentrations. A MgCl 2 concentration of 2.0 mM (lane 16 compared with lanes 14, 15, and 17) was chosen as optimal. Thirty cycles of amplification were chosen as standard since this represented maximum sensitivity with minimum template concentration (lanes 5-13). The annealing temperature was set at 55°C for maximum specificity and sensitivity (data not shown). Note that in lanes 1-3 of Figure 1, the negative control shows no 308-bp band after 10, 20, and 30 cycles of amplification. Similar experiments were performed for the other cloned MuIFN-ot subtypes (a4, a5, and or6) and for the pair of primers specific for MuIFN-13 amplification (see Materials and Methods for oligonucleotide details). Optimal annealing temperature, MgC12 concentration, and cycle number were found to be identical to those developed for the MuIFN-ct primers (data not shown).

ribosomal RNA band (18 S), indicating that the RNAs are intact. The A260/A280 ratios were also routinely better than 2.0.

cDNA Synthesis and Amplification The RNA samples from NDV-infected L929 cells at 0 h, 8 h, and 16 h after (post) infection (p.i.), shown in Figure 2A, were used (following concentration adjustments to 1 ~g) in RT reactions with three different types of primers. Figure 2B shows the electrophoresis of PCR-amplified products after RT of RNA samples from panel A with (a) a MulFN-ot cDNA-specific primer, the downstream or 3' amplification primer (lanes 2-7); (b) oligo(dT)12_18 primer (lanes 8-13), and (c) randomhexamer primer (lanes 14-19). The best results, in terms of intensity of the expected amplified products, were achieved with oligo(dThE_~S and hence this was used in all RT reactions as the primer of choice.

Detection of Individual Subtypes Total RNA Extracts Having established suitable amplification conditions, their application to reverse-transcribed total RNA extracts was to be examined. High-quality total RNA extraction was achieved using the procedures and reagents described in Materials and Methods. Figure 2A shows electrophoresed total RNA preparations (2 ~g) derived from L929 cells at various times after NDV infection. Note that at all time points, the large ribosomal RNA band (28 S) has significantly higher intensity than the small

Since the MulFN-ot amplification primers were capable of detecting all known subtypes of MulFNs~, it was necessary to develop a detection system that was capable of discriminating the components in given PCR products. Oligonucleotides complementary to regions of sequence unique to given subtypes were designed. Table I shows the identifying oligonucleotides for MulFN-otl, et4, et5, and or6 and the regions of sequence from which they were derived. The specificity of the four oligonucleotides for each MulFN-ot subtype that it was designed to

1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 17 720 b.p. 480 b.p. 360 b.p. Figure 1. Optimizations of polymerase chain reaction (PCR) conditions utilizing a linear murine interferon-or DNA fragment. PCR pro-Tr-d'~s were electrophoresed in 2% agarose gels. Lanes 1-13 (excluding lane 4, which contains a phage SPP-I EcoRI-restricted DNA size ladder) show levels of PCR amplification achieved at varying concentrations of target DNA at three different cycle numbers. The number of cycles of amplification used were 10 (lanes 1, 5, 8, and 11), 20 (lanes 2, 6, 9, and 12), and 30 (lanes 3, 7, 10, and 13). The amounts of target DNA in each of these were 0 ng (lanes 1-3), 100 ng (lanes 5-7), 1 ng (lanes 8-10), and 0.1 ng (lanes 11-13). Lanes 14-17 show the effect of the MgCl2 concentration on the amplification level obtained. The number of cycles used was 30 and the concentration of target DNA was set at 100 ng. MgClz concentration varied from 1.0 mM (lane 14) to 2.5 mM (lane 17) in increments of 0.5 raM. The sizes in base pairs (bp) of the three visible bands of SPP-1 EcoRI-restricted DNA size ladder in lane 4 are shown opposite the arrows on the left.

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16 GATA 11(1): 12-19, 1994

M.C. Lai et al.

1234

A 28S

• e

18S----i~

B

1 2 3 4 5 6 7 8 9 10111213141516171819

720 b.p. 480 b.p. 360 b.p.

Figure 2. (A) Electrophoresis of total RNA preparations (2 ~,g) derived from L929 cells at 0 h (lane 1), 8 h (lane 3), and 16 h (lane ~ewcastle disease virus (NDV) infection. Lane 2 contains 1 ~,g of ribosomal RNA from calf liver (Pharmacia, Sweden). The formaldehyde agarose gel used shows both the large ribosomal RNA band (28 S) and the small ribosomal RNA band 08 S), indicating that the RNAs are intact. (B) Electrophoresis of polymerase chain reaction amplification products following reverse transcription of RNA samples from NDV-infected L929 cells at 0 h after (post) infection (p.i.) (lanes 2, 3, 8, 9, 14, and 15), 8 h p.i. (lanes 4, 5, 10, 11, 16, and 17), and 16 h p.i. (lanes 6, 7, 12, 13, 18, and 19) with (a) a murine interferon-ct cDNA-specific primer, the downstream or 3' amplification primer (lanes 2-7); (b) oligo(dT)lz_lS primer (lanes 8-13), and (c) random-hexamer primer (lanes 14-19). Each sample was amplified for 25 cycles and 30 cycles shown left to right from lane 2 onward. The sizes in base pairs (bp) of three visible bands of the phage SPP-I EcoRI-restricted DNA size ladder are shown in lane 1 as indicated by the arrows on the left.

identify was tested under different temperatures of hybridization and washing. [~/-32p]ATP-labeled MulFN-ot4 oligonucleotide (IP-ot4) was very specific for MulFN-et4 subtype as it hybridized only to the MulFN-ot4 subtype at temperatures of ~>60°C. Similarly, [~/-32p]ATP-labeled MulFN-~I oligonucleotide (IP-etl), MulFN-ot5 oligonucleotide (IP-a5), and MulFN-a6 oligonucleotide (IPor6) were specific to MulFN-otl, MulFN-ot5, and MulFN-ot6 DNA, respectively, at 65°C hybridization and 65°C washing temperatures. Figure 3 shows the specificity of these [~-32p]ATP-labeled oligonucleotides when 65°C hybridization and wash temperatures were used against blots of PCR-amplified cloned MulFN-et gene fragments. Note that below the 50-ng level of target DNA, the specificity of each oligonucleotide is absolute. Based on these results, hybridization membranes were prepared by dot blotting 10 ~1 of amplified products from virus-induced cells, hybridized at 65°C and washed at 65°C for all identifying oligonucleotides. Figure 4A shows a typical set of results from such a hybridization experiment. The data are for hybridization to the specific IP-et4 oligonucleotide and show a rapid induction of

MulFN-ot4 mRNA by 4 h p.i. An initial peak of mRNA production occurs at - 8 h, followed by a diminution (lowest at 20 h) and a second peak at 26-28 h. This is followed by an abrupt shutdown of mRNA production at 30 h. Figure 4B shows the control hybridization with IP-o~4 that was done in the same hybridization reaction to ensure absolute specificity for detection of this subtype. (The control hybridization has the same layout as for Figure 3.) Similar hybridizations with controls were performed for each of the other MulFN-ot-identifying oligonucleotides. MulFN-13-specific amplifications were also performed for each of the time points shown in Figure 4. The results of these experiments are summarized in Figure 5. The biphasic production of MulFN mRNAs is apparent for the subtypes or4, or6, and 13. The otl and or5 subtypes show evidence of expression at a low level at the time of the first peak (8-10 h) but a second peak of expression is not apparent. The levels of detection seen for each subtype suggest that 13and or4 are the most highly produced subtypes, with or6 being the next most abundant, while oL1 and or5 are the least expressed. These data on relative levels

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17 GATA 11(1): 12-19, 1994

Molecular Detection of Interferon Subtype

Discussion 50 ng

.5 ng

0-

•

5 ng .5 ng

-(x5

ooo

.......O

5 ng .5 ng

•

50 ng

O

5 ng

•

.

.

.

.

.

.

.

.

.

-(x4

'~

• IP-(xl

D

.S ng

Figure 3. Autoradiograph of dot-blot hybridization of various amounts of amplified murine interferon (MuIFN)-otl, ct4, a5, and ct6 DNA to [',/-32p]ATP-labeled oligonucleotide probes specific for MulFN-et6 (A), MuIFN-ct5 (B), MuIFN-et4 (C), and MuIFN-cxl (D). The number to the left of each panel indicates the concentration of target DNA in nanograms (ng). The symbols to the right of each panel identify the specific oligonucleotide probe used in each hybridization. Symbols on top of all panels indicate the cloned MuIFN-ct gene fragments used as hybridization targets.

of expression of the type I subtypes are in excellent agreement with previous nuclease protection experiments done in the same system [10, 11, 17, 18]. The agreement of these two sets of data suggests that the a- and 13-subtype-amplifying oligonucleotides are operating at comparable efficiencies (see the Discussion). 0

2

4

6

The functional role(s) of individual type I IFN subtypes is poorly understood, particularly in vivo. There is a high degree of sequence homology between the 11 known MulFN-et genes, making differential detection and quantitation a technically difficult task. Indeed, at the level of functional protein, a set of monoclonal antibodies specific to individual subtypes and capable of neutralization in a bioassay has not been developed. At the mRNA level, Northern blot and nuclease protection assays utilizing cDNA probes have been used, but such studies are labor intensive because they require relatively large amounts of RNA (often for the separation o f a polyA fraction) and they are not particularly sensitive for low levels of IFN-et mRNA. With respect to future in vivo studies, these latter limitations are often the most important. The RT-PCR-based system described here overcomes many of these technical problems. The t w o MulFN-a-specific amplification primers show equal degrees of PCR amplification on cloned templates of the subtypes tested as judged by band intensity on agarose gels. In comparative experiments, the two MulFN-13-specific amplification oligonucleotides, tested on a MulFN-13 cDNA clone fragment, showed the same level of template amplification relative to the MulFN-ot amplifications (data not shown). The validity of these observations regarding equivalent PCR amplification of all subtypes examined is substantiated by the comparative levels of subtype expression determined in this study. Based on relative intensities, it was estimated that MulFN-et4 and MulFN-13 were the two most highly expressed subtypes in the L-ceI1-NDV system studied. The levels of MulFN-et4 and 13 were 5- to 10-fold greater than those seen for MulFN-a6, a5, and etl (depending on the time point chosen for comparison, see Fig-

8 10 12 16 20 22 24 26 28 30 36 48

(xl(x4o.Scc6

10 o

50 ng

10-1

5 ng 0.5 rig

A

B Figure 4. (A) Autoradiograph of dot-blot hybridization of amplified cDNA fi-om Newcastle disease virus.infected L929 cells with [~.32p]ATP. I~~-~IP-(x4. The numbers above the panel show time points of 0-48 h after (post) infection (p.i.). The numbers to the left of the panel indicate undiluted (10°) dot blots of 10 ~! from the amplification reaction and 10-fold-diluted samples (10- ~). (B) Autoradiograph of dot-blot hybridizations of amplified postive control murine interferon (MuIFN)-~x DNAs performed at the same time as the above test membrane. Note that only the MuIFN-~x4 column shows hybridization. The layout for this panel is as for Figure 3.

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18 G A T A 11(1): 12-19, 1994

M.C. Lai et al,

I ,I

7.

,lltl

6.

0

5

10

15

20

25

30

48

Time (h) Post Infection

Figure 5. Graph summarizing the relative levels of expression of murine interferon (MulFN)-al (A), a4 (IlL ct5 (x), and et6 (©) in ew/q"e'ff'c~tledisease virus-infected L929 cells. The relative levels of expression were determined by comparison of signal intensities, using laser densitometry, of dot-blotted polymerase chain reaction products with known quantities of control DNA hybridized with the respective oligonucleotidesused for identification of the four IFN-a subtypes. The levels 0-6 refer to the signal intensities when the following amounts of control MulFN-et DNA were used: 0, blank; 1, 0.005 ng; 2, 0.05 ng; 3, 0.5 ng; 4, 5 ng; 5, 50 ng; and 6, >50 ng. The expression of MulFN-13(11)over the same time course is represented by the bars above the graphs for the et subtypes at the corresponding time points. ure 5). These relative levels of expression are in full agreement with previous nuclease protection studies [10, 11]. The fact that results from these previous unamplified R N A extracts match those from the present study indicates that the amplification process is not distorting the relative abundances of subtype detected. The observation that two discrete peaks of type I I F N m R N A e x p r e s s i o n o c c u r in this NDVinfected cells system is novel. Previous studies [10, 11, 17, 18] involving nuclease protection and N o r t h e r n blots did not go b e y o n d the 20-h time point, whereas the present study involved a time course spanning 72 h p.i. The s e c o n d a r y peak around 26-28 h p.i. was observed for only the more highly expressed subtypes (MuIFN-~, or4, and or6) and not for the more weakly expressed or5 and etl subtypes. The timing o f the second peak in I F N m R N A synthesis would appear to correspond to a viral replication cycle; that is, at the multiplicity of infection used in the experiment, at 24 h p.i. a second wave of I F N m R N A synthesis occurs in cells that were not infected initially and are probably " p r i m e d " for superinduction by the inter-

feron produced during the first peak. In relation to this point, it should be noted that a cytopathic effect is seen in these cultures at about 48-72 h p.i. (data not shown). The earliest time point of IFN m R N A detection in the present study is at 2 h p.i. (see Figure 5). This is considerably earlier than the earliest time points of m R N A detection reported in the (unamplified) previous studies where 6 h was the first time point at which m R N A was detected [10]. This increased sensitivity of detection is not doubt due to the PCR amplification used in this study, because the specific activity of radiolabeled probes used for detection in all studies would not have varied much. This final observation supports the notion that the system d e v e l o p e d in the present study is of considerable sensitivity and specificity. The R T - P C R - s p e c i f i c oligonucleotide type I IFN detection system described in this article has now been applied to further in vitro and in vivo studies in this laboratory. The system should enable a detailed study of type I I F N subtype expression under a variety of conditions.

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19 GATA 11(1): 12-19, 1994

Molecular Detection of Interferon Subtype

This work was supported by a grant from the National Health and Medical Research Council of Australia (M.W.B. and M.C.L.).

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8. Swaminathan N, Lai CM, Beilharz MW, Boyer SJ, Klinken SP: Antiviral Res 19:149--159, 1992 9. Hiscott J, Cantell K, Weissman C: Nucleic Acids Res 12: 3727-3746, 1984 10. Kelley KA, Pitha PM: Nucleic Acids Res 13:825-839, 1985 11. Zwarthoff EC, Mooren ATA, Trapman J: Nucleic Acids Res 13:791-804, 1985 12. Goren TG, Fischer DG, Rubinstein M: Biochim Biophys Acta 887:80-85, 1986 13. Hannigan GE, Gewert DR, Fish EN, Read SE, Williams BRG: Biochem Biophys Res Commun 110:537-544, 1983 14. Imakawa K, Anthony RV, Kazemi M, Marotti KR, Polites HG, Roberts RM: Nature 330:377-379, 1987 15. Cross JC, Roberts RM: Proc Natl Acad Sci USA 88:38173821, 1991 16. Tovey MG, Streuli M, Gresser I, Gugenheim J, Blanchard B, Guymarho J, Vignaux F, Gigou M: Proc Natl Acad Sci 84:5038-5042, 1987 17. Bisat F, Raj NBK, Pitha PM: Nucleic Acids Res 16:60676083, 1988 18. Raj NBK, Engelhardt J, Au WC, Levy DE, Pitha PM: J Biol Chem 264:16,658--16,666, 1989 19. Higashi T, Sokawa Y, Watanabe Y, Kawade Y, Ohno S, Takaoka C, Taniguchi T: J Biol Chem 258:9522-9529, 1983

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