ROBERT M. KRUG,'* MICHAEL SHAW,2 BARBARA BRONI,' GEOFFREY SHAPIRO,' AND OTTO HALLER2t. Graduate Program in Molecular Biology, Memorial ...
Vol. 56, No. 1
JOURNAL OF VIROLOGY, OCt. 1985, p. 201-206
0022-538X/85/100201-06$02.00/0 Copyright C) 1985, American Society for Microbiology
Inhibition of Influenza Viral mRNA Synthesis in Cells Expressing the Interferon-Induced Mx Gene Product ROBERT M.
KRUG,'*
MICHAEL SHAW,2 BARBARA BRONI,' GEOFFREY SHAPIRO,' AND OTTO HALLER2t
Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center,' and Rockefeller University,2 New York, New York 10021 Received 4 April 1985/Accepted 11 June 1985
Interferons a and induce an efficient antiviral state against influenza virus in mouse cells that possess the Mx gene, but not in mouse cells that lack this gene. In Mx-containing cells treated with interferon the amount of viral mRNA synthesized as a result of primary transcription is drastically reduced. Only two viral mRNAs could be detected by Northern analysis and by translating the poly(A)+ RNA from infected cells in wheat germ extracts: a reduced amount of the mRNA for nonstructural protein 1 and an even lower amount of the mRNA for the matrix protein. The other viral mRNAs were not made in detectable amounts. In addition, the rate of viral mRNA synthesis catalyzed by the inoculum transcriptase, measured by in vitro RNA synthesis catalyzed by permeabilized cells, was severely inhibited. In contrast, interferon treatment of cells lacking the Mx gene had little or no effect on either the steady-state level or the rate of synthesis of viral mRNAs made by the inoculum transcriptase. These results indicate that the interferon-induced Mx gene product, a 75,000molecular-weight protein that accumulates in the nucleus, inhibits influenza viral mRNA synthesis which occurs in the nucleus. No Mx-specific effect acting directly on viral protein synthesis in the cytoplasm was observed.
Cells exposed to interferon (IFN) develop an antiviral state in which the replication of most viruses is inhibited to various degrees. In mouse cells the antiviral state induced by IFN-a and IFN-P against influenza virus is controlled by the host gene Mx (6). Only cells that possess this gene develop an efficient antiviral state against influenza virus after exposure to IFN-o and IFN-P, whereas the antiviral state against other viruses, e.g., the rhabdovirus vesicular stomatitis virus, is independent of the Mx gene (7). The Mx gene product is a 75,000-molecular-weight protein that accumulates in the nuclei of IFN-treated cells (5, 9). The mechanism by which the Mx gene product specifically inhibits influenza virus replication has not been established. It was reported that in Mx-containing macrophages IFN causes the inhibition of the translation of apparently functional viral mRNAs in the cytoplasm, but does not affect the viral transcriptional events that occur in the nucleus (17). The nucleus is the site of synthesis of viral mRNA and of the full-length transcripts that serve as the templates for virion RNA (vRNA) replication (3, 8, 12). Viral mRNA synthesis requires initiation by capped RNA primers generated by the cleavage of newly synthesized host cell RNA polymerase II
MATERIALS AND METHODS Viruses and cells. Influenza A virus strain WSN was grown in Maden-Darby bovine kidney cells to a titer of 2 x 109 PFU/ml as assayed on Maden-Darby bovine kidney cells (11). Embryo cells were prepared from 16-day-old embryos of inbred mouse strains A2G (homozygous for the resistance allele Mx+) and A/J (homozygous for allele Mx-) as described previously (2). Cells were grown to three to five passages in Dulbecco modified minimal essential medium supplemented with 10% fetal calf serum. IFN. Mouse IFN type I induced by Newcastle disease virus in C-243 cells (a mixture of IFN-a and IFN-P, partially purified to 107 U/mg of protein) was purchased from Enzo Biochem., Inc., New York, N.Y. The antiviral activity of this preparation was calibrated with a mouse IFN-ao3/ standard from the National Institutes of Health and A/J mouse embryo cells and vesicular stomatitis virus as described previously (21). Analysis of virus-specific protein synthesis in infected cells. Confluent monolayers of mouse embryo fibroblasts were treated for 18 h at 37°C with 1,000 U of IFN-a/I per ml in Dulbecco modified essential medium containing 5% fetal calf serum. In some experiments the IFN was removed and the cells, after being washed five times, were incubated for 3 days (72 h) at 37°C in medium lacking IFN. As controls, cells were subjected to the same manipulations in medium lacking IFN. Cells were infected with 100 PFU of influenza virus per cell. After virus adsorption at 37°C for 60 min, the inoculum was replaced with Dulbecco modified essential medium containing 2% calf serum. At the indicated times, the medium was replaced with Dulbecco modified essential medium deficient in unlabeled methionine supplemented with 100 ,uCi of [35S]methionine per ml (1,200 Ci/mmol). After incubation for 60 min at 37°C, the labeled medium was removed and the cells were harvested by being scraped into cell lysis buffer (19). The samples were sonicated and analyzed by electro-
transcripts (8, 12). In this paper we show that IFN causes a dramatic inhibition of viral mRNA synthesis in mouse embryo cells containing the Mx gene, but not in mouse embryo cells lacking this gene. Primary transcription, the viral mRNA synthesis catalyzed by the inoculum viral RNA transcriptase, is drastically reduced, indicating that the Mx protein, a nuclear protein, inhibits the process of viral mRNA synthesis in the nucleus. No Mx-specific effect acting directly on viral protein synthesis in the cytoplasm was observed. Corresponding author. t Present address: Institute for Immunology and Virology, University of Zurich, 8028 Zurich, Switzerland. *
201
202
J. VIROL.
KRUG ET AL.
phoresis on 17.5% polyacrylamide gels containing 4 M urea (14). Analysis of viral mRNA levels in infected cells. Confluent monolayers of mouse embryo fibroblasts were infected as described above, but were not labeled with [35S]methionine. Where indicated, the cells were treated with 100 ,ug cycloheximide (CM) per ml or 100 ,uM anisomycin (provided by Pfizer Pharmaceuticals) starting 30 min before infection and maintained throughout infection. For Northern analysis, total cellular RNA was extracted with phenol-chloroform, selected on oligo(dT)-cellulose, enzymatically deadenylated, electrophoresed on a 1% agarose-formaldehyde gel, and transferred to a nitrocellulose filter (15, 22). The filter was hybridized with probes specific for five viral mRNAs, those coding for the PB1, PB2, PA, HA, and NS1 proteins. These probes were generated by using vRNA-sense M13 clones as described by Mason et al. (16). With this procedure, a 32P-labeled single-stranded DNA copy of M13 DNA lacking an insert is synthesized by using calf thymus DNA primers and the Klenow fragment of DNA polymerase. The mixture is heat denatured and then reannealed in the presence of a twofold excess of M13 DNA containing a vRNA-sense viral insert. The M13 regions of the resulting hybrid DNA molecules are double stranded and provide the 32p label, whereas the viral sequences remain single stranded and thus are available to hybridize to complementary sequences on the nitrocellulose filter. For the isolation of mRNA for in vitro translation, cells were harvested into a buffer containing 100 mM NaCI, 10 mM Tris hydrochloride (pH 8.8), 2 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, and 1% P-mercaptoethanol (4). Nuclei and cell debris were pelleted out at 2,000 x g for 15 min, and the supernatant was adjusted to 400 mM NaCl and 0.1% sodium dodecyl sulfate before chromatography on oligo(dT)-cellulose. The poly(A)+ RNA was isolated, ethanol precipitated, and dissolved in water before translation in non-preincubated wheat germ extracts (14). Labeled proteins were analyzed by electrophoresis on 17.5% acrylamide gels containing 4 M urea. Measurement of the rate of viral mRNA synthesis. Confluent monolayers were infected as described above. At the indicated time, the cells were removed from the monolayer by trypsinization. Calf serum was added to neutralize the trypsin, and the cells were collected by centrifugation and washed twice with phosphate-buffered saline. The cell pellet was taken up in an equal volume of a buffer (35 mM N-2-hydroxyethylpiperazine-N'-2ethylsulfonic acid [HEPES] [pH 7.4], 80 mM potassium acetate, 5 mM MgCl2, 0.5mM CaCl2, 150 mM sucrose) containing 1 mg of lysolecithin per ml (18). After a 5-min incubation at 0°C to permeabilize the cells, 40 ,ul of the cell suspension was incubated for 30 min at 30°C in a 100-pA reaction mixture containing a final concentration of 30 mM HEPES (pH 7.4), 100 mM potassium acetate, 2.5 mM MgCl2, 0.2 mM CaCl2, 2 mM dithiothreitol, 0.4 mM spermidine hydrochloride, 0.2 mM S-adenosylmethionine, 8 mM creatine phosphate, 0.1 mg of creatine kinase per ml, 20 amino acids at 100,uM each, 0.8 mM ATP, 0.4 mM each of GTP and CTP, and [(x-32P]UTP (1,uM, 400 mCi/ mol). RNA was extracted by making the reactions 1% in sodium dodecyl sulfate, 2 mM in vanadyl ribonucleoside complexes, and 50 ,ug/ml in Pronase and incubating the mixtures at 37°C for 30 min (3). After phenol-chloroform extraction and ethanol precipitation, the samples were digested with RNase-free DNase and extracted with phenol-chloroform. The RNA after ethanol precipitation was annealed with an excess (3 ,ug) of unlabeled vRNA in 55% (vol/vol) formamide, and
after RNase T2 digestion the virus-specific, double-stranded RNAs were analyzed on 3.5% nondenaturing acrylamide gels (20).
RESULTS Effect of IFN on viral protein synthesis. As shown previously (21), treatment of embryo cells from BALB A2G-Mx mice (homozygous for Mx, termed Mx+) with 1,000 U of IFN-oa/p per ml establishes an antiviral state in which only low levels of influenza viral proteins are synthesized. At 6 h after infection with 100 PFU of WSN influenza virus per cell, the only viral protein detected in IFN-treated cells was the NS1 protein, which was synthesized at a low level, whereas in the absence of IFN all the proteins were synthesized at high levels (Fig. 1). At later times of infection (11 h postinfection) in IFN-treated cells, a larger amount of the NS1 protein and a small amount of the Ml protein were synthesized, but the other viral proteins were not detectable. In contrast, in BALB/c embryo cells (lacking the Mx gene, termed Mx-), treatment with 1,000 U of IFN-ao/ per ml only delayed the time course of viral protein synthesis. All the viral proteins were synthesized by 6 h postinfection but in reduced amounts, and the level of synthesis of all the viral proteins at 11 h postinfection was equivalent to that seen at earlier times in the absence of IFN, Effect of IFN on the levels of viral mRNAs. The inhibition of viral protein synthesis seen in IFN-treated Mx+ cells could reflect a block in either the synthesis or the translation of viral mRNAs. To distinguish between these two possibili-
Mx+ 5
6
C
IFN 6 11
Mx-
Ml
NS]_ FIG. 1. Inhibition of viral protein synthesis by IFN-a/f3 in vivo. Mx+ and Mx- cells were treated with IFN-a/13 for 18 h at 37°C and infected with influenza virus as described in Materials and Methods. At 6 and 11 h postinfection, the cells were labeled with [35S]methionine for 60 mnin, and the labeled proteins were analyzed by gel electrophoresis (lanes IFN, 6, and 11). Other Mx+ and Mxcells were pretreated for 18 h with medium lacking IFN and were infected with influenza virus for 5 or 6 h, at which times [35S]methionine labeling was carried out (lanes 5 and 6). Lane C shows the proteins synthesized in cells which were neither treated with IFN nor infected with influenza virus. Cell-equivalent amounts of protein were analyzed in each lane. The viral proteins are indicated by standard abbreviations: polymerase proteins (P), hemagglutinin (HA), nucleocapsid protein (NP), matrix protein (Ml), and nonstructural protein 1 (NS1).
Mx GENE AND FLU mRNA SYNTHESIS
VOL. 56, 1985
ties, we measured the steady-state levels of viral mRNAs by two procedures, Northern analysis and in vitro translation. For the Northern analysis, the blots of poly(A)+ RNA from infected cells were hybridized to single-stranded M13 probes specific for five viral mRNAs, those coding for the NS1, HA, and three P proteins. In Mx+ cells, IFN treatment resulted in the absence of detectable amounts of the HA and P mRNAs and in a large reduction in the amount of the NS1 mRNA at 5 h postinfection (Fig. 2, compare lanes 2 and 4). The same effect was seen when virus infection was carried out in the presence of the protein synthesis inhibitor anisomycin (lanes 1 and 3), conditions under which viral mRNA synthesis is confined to that catalyzed by the inoculum viral transcriptase (primary transcription). This indicates that viral mRNA synthesis itself is inhibited in IFN-treated cells. In contrast, in Mx- cells the levels of viral mRNA in untreated and IFN-treated cells were similar, in both the presence and absence of anisomycin (lanes 5 through 8). Similar results were obtained by analyzing viral mRNA levels by in vitro translation. The four lanes on the right in Fig. 3 show the translation of the viral mRNAs synthesized by the inoculum transcriptase, i.e., during infections in the presence of CM. In Mx+ cells treated with IFN, only the NS1 mRNA and a small amount of the Ml mRNA were detected, whereas in Mx- cells the same amount of all the
203
Mx-- -MxCM
-
+
IFN
-
-
+ +
+ +
+ -
HIA,-
HA- -
NP
NAO-
-
491
A mI
NSi-
14
.I ;w
NS.)-
4& A
'low
Ifto
FIG. 3. Reduction in the levels of functional viral mRNAs in
IFN-treated Mx+ cells. Mx+ and Mx- cells were treated with
Mx+ l
2
3
Mx-
4
5
6
7
8
...
p
*
F;
IFN-o/dB for 18 h at 37°C and were infected with influenza virus in the presence of 100 p.g of CM per ml. As a control, Mx+ cells treated with neither IFN nor CM were infected with influenza virus. At 6 h after infection, poly(A)+ RNA was isolated and translated in wheat germ extracts with [35S]methionine as labeled precursor. The labeled proteins were analyzed by gel electrophoresis. The positions of viral proteins are indicated. HAo and NAo are the nonglycosylated hemagglutinin and neuraminidase, respectively; NS2 is nonstructural protein 2.
HA
N'
NSI
FIG. 2. Reduction in the levels of influenza viral mRNAs in IFN-treated Mx+ cells. Mx+ and Mx- cells were treated with IFN-a/, for 18 h at 37°C and infected with influenza virus in the presence (lanes 1 and 5) or absence (lanes 2 and 6) of 100 ,uM anisomycin. Other Mx+ and Mx- cells were pretreated with medium lacking IFN and were infected with influenza virus in the presence (lanes 3 and 7) or absence (lanes 4 and 8) of 100 p.M anisomycin. At 5 h after infection, total poly(A)+ RNA was isolated, enzymatically deadenylated, and resolved by electrophoresis on a 1% agaroseformaldehyde gel. The poly(A)+ RNA from 2 x 107 cells was applied to each lane. The RNA was transferred to nitrocellulose filters, which were hybridized to 32P-labeled probes specific for the three P (PB1, PB2 and PA), HA, and NS mRNAs. The blot for the Mx+ cell RNAs was exposed to X-ray film about five times longer than the Mx- blot to detect the NS1 mRNA in IFN-treated Mx+ cells. Shorter exposure of the blot from Mx+ cells showed that the pattern of viral mRNAs produced in the absence of anisomycin and IFN (lane 4) is the same as that produced in Mx- cells in the absence of anisomycin and IFN (lane 8). Equal amounts of poly(A)+ RNA were present in the four lanes of the Mx+ blot (lanes 1 through 4), as shown by equivalent levels of hybridization of a nick-translated cDNA clone of P-actin to the RNA in each lane (data not shown).
viral mRNAs was detected in untreated and IFN-treated cells. Translatable mRNAs for viral proteins other than the NS1 and Ml proteins were not detected in the RNA preparation from IFN-treated Mx+ cells even when larger amounts of this RNA (three times more) was added to the in vitro system (data not shown). Again, these results indicate that viral mRNA synthesis is inhibited in IFN-treated Mx+, but not Mx- cells. The extra background bands in the in vitro products are presumably premature termination products and host proteins. Host cell mRNAs, which are not translated in influenza virus-infected cells, have been shown to be functional in in vitro translation (10). It should also be pointed out that P mRNAs are readily detected by translation only when the RNA is obtained from cells in which amplified transcription is prevented by CM (in both Mx+ and Mx- cells), most probably because P mRNAs are not amplified whereas the other viral mRNAs are (13). Effect of IFN on the rate of viral mRNA synthesis. The low levels of viral mRNAs in IFN-treated Mx+ cells could reflect a reduced rate of synthesis or an increased rate of breakdown. To address this issue, we measured the rate of viral mRNA synthesis both in the presence and absence of anisomycin (primary and amplified transcription, respectively). Because it is extremely difficult to label influenza viral mRNAs synthesized during primary transcription by using short pulses of [3H]uridine (8), we measured the rate of viral mRNA synthesis by permeabilizing infected cells with lysolecithin and carrying out viral mRNA synthesis for short
204
J. VIROL.
KRUG ET AL.
Mx+ 1 2 3 4
Mx1
3 4
2
P. HA-
NP-HA
NA-
-NP -NA
M1-
Om
l
-ml -NS1
FIG. 4. Inhibition of the rate of viral mRNA synthesis in IFNtreated Mx+ cells. Mx+ and Mx- cells were treated with IFN-a/P for 18 h at 37°C and were infected with influenza virus in the presence (lanes 1 and 5) or absence (lanes 2 and 6) of 100 p.M anisomycin. Other Mx+ and Mx- cells were pretreated with medium lacking IFN and were infected with influenza virus in the presence (lanes 3 and 7) or absence (lanes 4 and 8) of 100 ,uM anisomycin. At 4.5 h after infection, the cells were collected by trypsinization. After permeabilization with lysolecithin, viral mRNA synthesis was carried out in vitro with [a-32P]UTP as labeled precursor, as described in Materials and Methods. The labeled RNA was extracted and annealed to an excess of unlabeled vRNA. After RNase T2 digestion, the labeled viral double strands were resolved by gel electrophoresis.
periods in vitro with 32P-labeled nucleoside triphosphate precursors (3, 18). The labeled RNA products were hybridized to vRNA, and the resulting double strands after RNase T2 digestion were resolved by gel electrophoresis. Mx+ cells in the absence of both IFN and anisomycin synthesized all eight viral mRNAs (Fig. 4, left panel, lane 4). In addition, the templates for vRNA replication were synthesized; the M and NS template RNAs were clearly resolved from their corresponding mRNAs (Ml and NS1 mRNAs) as slower-migrating species. In the presence of anisomycin, all eight viral mRNAs were synthesized but in greatly reduced amounts, and as expected, template RNAs made (left panel, lane 3). With IFN treatment, the level of viral mRNA synthesis was drastically reduced, both in the presence and absence of anisomycin (left panel, lanes 1 and 2). In the absence of anisomycin, some NS1 mRNA, less Ml mRNA, and a trace amount of the middle-sized viral mRNAs (NA, NP, and HA) were made, and no template RNA was synthesized. In the presence of anisomycin, only a very low amount of the NS1 mRNA was made. In contrast, in Mx- cells IFN treatment had little or no effect on viral mRNA synthesis in the absence of anisomycin (right panel, lanes 2 and 4). In the presence of anisomycin IFN had at were not
most a small effect on the rate of viral mRNA synthesis in Mx- cells: either about a twofold reduction (right panel, lanes 1 and 3) or no detectable reduction (data not shown). These results therefore indicate that IFN drastically inhibits the rate of viral mRNA synthesis in Mx+ cells, but causes at most a small reduction in this rate in Mx- cells.
Persistence of the IFN-induced effect on viral mRNA synthesis in Mx+ cells. The antiviral state induced in Mx+ cells against influenza virus by IFN decays much more slowly than does the antiviral state induced in Mx- cells (1, 2). Thus, 3 days after removal of IFN the antiviral state in Mx+ cells was only partially reduced, whereas in Mx- cells it had largely, if not totally, disappeared. This can be seen by the pattern of viral protein synthesis at 4.5 h after infection (Fig. 5A). In Mx- cells, the delay in the time course of viral protein synthesis observed immediately after IFN treatment had largely vanished (cf. Fig. 1). In contrast, in Mx+ cells many of the viral proteins were still not synthesized. The most significant difference between this pattern of viral protein synthesis and that seen immediately after IFN treatment was that the synthesis of NP protein could be detected after the 3-day decay period (cf. Fig. 1). Thus, the effect of the IFN-induced cells on viral protein synthesis persists in Mx+ 3 days after IFN removal, but has decayed to some extent. In addition, the IFN-induced effect on viral mRNA synthesis in Mx+ cells largely persisted. Northern analysis of the poly(A)+ RNA from cells at 5 h postinfection in both the absence and presence of anisomycin showed a large reduction in the levels of viral mRNAs (Fig. SB, compare lanes 1 and 2 with lanes 3 and 4). In addition to the NS1 mRNA, a small amount of HA mRNA was detectable in the Mx+ cells exposed to IFN, which was not the case immediately after IFN treatment (compare with Fig. 2). In contrast, in Mxcells the level of viral mRNA in IFN-treated cells was the same as that in untreated cells (Fig. 5B, lanes 5 through 8). In vitro translation of the viral mRNAs synthesized in CM-treated cells exposed to IFN confirmed that Mx+, but not Mx-, cells contained reduced levels of the viral mRNAs synthesized by the inoculum transcriptase (Fig. 5C), and analysis of the viral mRNAs synthesized by permeabilized cells indicated that the rate of viral mRNA synthesis in Mx+ cells exposed to IFN continues to be greatly reduced by 3 days after removal of the IFN (Fig. 5D). DISCUSSION The present results indicate that in Mx+ cells treated with IFN-a/c the process of influenza viral mRNA synthesis in the nucleus is inhibited. In Mx+ cells treated with IFN the amount of viral mRNA synthesized by the inoculum transcriptase was greatly reduced. Only two viral mRNAs were detected: a reduced amount of the NS1 mRNA and an even lower amount of the Ml mRNA. In addition, the rate of viral mRNA synthesis, measured by in vitro RNA synthesis catalyzed by permeabilized cells, was severely inhibited. In contrast, treatment of Mx- cells with IFN had little or no effect on either the steady-state level or the rate of synthesis of viral mRNAs made by the inoculum transcriptase. The NS1 and Ml viral mRNAs that continued to be synthesized in IFN-treated Mx+ cells were translated in vivo. In fact, the amount of this in vivo translation was similar to the amount of translation observed when the poly(A)+ RNA from these cells was assayed in wheat germ extracts (compare Fig. 1 and 3). Consequently, we find no evidence for an Mx-specific effect causing a selective block of viral mRNA translation, in conflict with a previous report (17). Viral mRNA translation, however, was affected by the other IFN-induced gene products, which apparently cause a delay in viral translation. Thus, in Mx- cells the various mRNAs were produced at essentially normal levels by 5 to 6 h postinfection but were not translated at normal levels until 11 h postinfection. A similar delay in translation would also
Mx GENE AND FLU mRNA SYNTHESIS
VOL. 56, 1985
Mx
C
IJ
205
.._
HA- *'24p:: D MXi
(A)
Mx+ 1 2
mx-
3
(B)
I
4
Mx+ 2
3
1 2 34
Mx
_74
-17
6
7
I
p
:1
..?,
HLA NPP11 _
0
0
i
VP
kI
OR
Ml NSI-
K.4A 90
I
,Ip HA
.z.
NP NA
NS FIG. 5. Persistence of the IFN-induced inhibition of viral mRNA synthesis in Mx+ cells. Mx+ and Mx- cells were treated with IFN-Wp/, for 18 h at 37°C, the IFN was then removed, and the cells were incubated for 3 days at 37°C in the absence of IFN. Other Mx+ and Mx- cells were treated with medium lacking IFN. Cells were then infected with influenza virus and analyzed as described below. (A) Protein synthesis in vivo. At 4.5 h after infection, IFN-treated (lanes 2 and 4) and untreated (lanes 1 and 3) cells were labeled with [35S]methionine for 60 min, and the labeled proteins were analyzed by gel electrophoresis. (B) Intracellular levels of viral mRNAs. IFN-treated (lanes 1, 2, 5, and 6) and untreated (lanes 3, 4, 7, and 8) cells were infected in the presence (lanes 1, 3, 5, and 7) or absence (lanes 2, 4, 6, and 8) or 100 puM anisomycin. The poly(A)+ RNA isolated at 5 h after infection was analyzed by Northern analysis as described in the legend to Fig. 2. (C) Intracellular levels of functional viral mRNAs. Mx+ and Mx- cells treated with IFN were infected in the presence of 100 ,ug of CM per ml. At 6 h after infection, poly(A)+ RNA was isolated and translated in wheat germ extracts. (D) Rate of viral mRNA synthesis. IFN-treated (lanes 1 and 2) and untreated (lanes 3 and 4) Mx+ cells were infected with influenza virus in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of 100 ,uM anisomycin. At 4.5 h after infection, the cells were collected by trypsinization. After permeabilizing the cells with lysolecithin, viral mRNA synthesis was carried out in vitro, and the RNA products were analyzed as described in the legend to Fig. 4.
be expected to operate in Mx+ cells, but such an effect was largely obscured by the dramatic reduction in the synthesis of the viral mRNAs. When IFN was removed and the cells were allowed to recover for 3 days, the delay in viral mRNA translation mediated by the other IFN-induced gene products in Mx- cells largely disappeared, whereas the inhibition of viral mRNA synthesis occurring in Mx+ cells persisted to a large extent. Because Mx-specific inhibition of influenza virus replication strictly correlates with IFN inducibility of the Mx gene product, a nuclear 75,000-molecular-weight protein (5, 9), the inhibition of influenza viral mRNA synthesis can be ascribed to the action of this Mx protein. It will be of great interest to determine the mechanism of inhibition. One possibility is that the initiation of viral mRNA synthesis by cellular capped RNA primers is blocked, either directly or by interfering with the availability of the primer fragments. The present results, however, argue against this possibility. If initiation were specifically blocked, we would expect to find small amounts of each of the viral mRNAs. Instead, we have found that the synthesis of the smallest viral mRNAs, the NS1 and Ml mRNAs, is preferentially spared. This suggests an inhibition of elongation rather than of initiation.
It should also be emphasized that our results do not conclusively rule out the possibility of a rapidly acting nuclease, as such a nuclease would also be expected to spare smaller mRNAs preferentially. However, the absence of fragments of the larger viral mRNAs in the Northern analyses (Fig. 2 and SB) is inconsistent with the action of a nuclease. Clearly, to determine the mechanism of inhibition of viral mRNA synthesis by the Mx protein, it will be necessary to establish a cell-free in vitro system in which the inhibitory effect occurs.
ACKNOWLEDGMENTS This investigation was supported by Public Health Service grants Al 11772, Al 05600, Al 20201, and CA 08747 from the National Institutes of Health. G.S., an M.D.-Ph.D. student in the Cornell University Graduate School of Medical Sciences, is funded by The Louis and Rachel Rudin Foundation. O.H. was a recipient of a fellowship by the Swiss Academy of Medical Sciences. We thank Purnell Choppin and Andreas Scheid for their encouragement and support. LITERATURE CITED
1. Arnheiter, H., and 0. Haller. 1983. Mx gene control of interferon action: different kinetics of the antiviral state against
206
2. 3. 4. 5. 6.
7. 8. 9.
10. 11.
12.
KRUG ET AL.
influenza virus and vesicular stomatitis virus. J. Virol. 47:626-630. Arnheiter, H., and P. Staeheli. 1983. Expression of interferon dependent resistance to influenza virus in mouse embryo cells. Arch. Virol. 76:127-137. Beaton, A. R., and R. M. Krug. 1984. Synthesis of the templates for influenza virion RNA replication in vitro. Proc. Natl. Acad. Sci. USA 81:4682-4686. Collins, P. L., L. E. Hightower, and L. A. Ball. 1978. Transcription and translation of Newcastle disease virus mRNA's in vitro. J. Virol. 28:324-336. Dreiding, P., P. Staeheli, and P. Haller. 1985. Interferon-induced protein Mx accumulates in nuclei of mouse cells expressing resistance to influenza viruses. Virology 141:192-196. Haller, 0. 1981. Inborn resistance of mice to orthomyxoviruses. Curr. Top. Microbiol. Immunol. 92:25-52. Haller, O., H. Arnheiter, J. Lindenmann, and I. Gresser. 1980. Host gene influences sensitivity to interferon action selectively for influenza virus. Nature (London) 283:660-662. Herz, C., E. Stavnezer, R. M. Krug, and T. Gurney, Jr. 1981. Influenza virus, an RNA virus, synthesizes its messenger RNA in the nucleus of infected cells. Cell 26:391-400. Horisberger, M. A., P. Staeheli, and 0. Haller. 1983. Interferon induces a unique protein in mouse cells bearing a gene for resistance to influenza virus. Proc. Natl. Acad. Sci. USA 80:1910-1914. Katze, M. G., and R. M. Krug. 1984. Metabolism and expression of RNA polymerase II transcripts in influenza virusinfected cells. Mol. Cell. Biol. 4:2198-2206. Krug, R. M. 1971. Influenza viral RNPs newly synthesized during the latent period of viral growth in MDCK cells. Virology 44:125-136. Krug, R. M. 1981. Priming of influenza viral RNA transcription by capped heterologous RNAs. Curr. Top. Microbiol. Immunol.
J. VIROL.
93:125-150. 13. Lamb, R. A., and P. W. Choppin. 1977. Synthesis of influenza virus proteins in infected cells: translation of viral proteins, including three P polypeptides, from RNA produced by primary transcription. Virology 74:504-519. 14. Lamb, R. A., P. R. Etkind, and P. W. Choppin. 1978. Evidence for a ninth influenza viral polypeptide. Virology 91:60-78. 15. Lehrach, H., D. Diamond, J. M. Wozney, and H. Boedtker. 1977. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16:4743-4751. 16. Mason, W. S., C. Aldrich, J. Summers, and J. M. Taylor. 1982. Asymmetric replication of duck hepatitis B virus DNA in liver cells: free minus-strand DNA. Proc. Natl. Acad. Sci. USA 79:3997-4001. 17. Meyer, T., and M. A. Horisberger. 1984. Combined action of mouse a and ,B interferons in influenza virus-infected macrophages containing the resistance gene Mx. J. Virol. 49:709716. 18. Miller, M. R., J. J. Castellot, Jr., and A. B. Pardee. 1978. A permeable animal cell preparation for studying macromolecular synthesis. DNA synthesis and the role of deoxyribonucleotides in S phase initiation. Biochemistry 17:1073-1080. 19. Peluso, R. W., R. A. Lamb, and P. W. Choppin. 1977. Polypeptide synthesis in simian virus 5-infected cells. J. Virol. 23:177-187. 20. Plotch, S. J., and R. M. Krug. 1978. Segments of influenza virus complementary RNA synthesized in vitro. J. Virol. 25:579-586. 21. Staeheli, P., M. A. Horisberger, and 0. Haller. 1984. Mxdependent resistance to influenza viruses is induced by mouse interferons a and P but not y. Virology 132:456-461. 22. Thomas, P. S. 1980. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77:5201-5205.