VIROLOGY
64, 378-387 (1975)
Studies
on the lntranuclear
Influenza
Virus-Specific
ROBERT M. KRUG Memorial
Localization
AND
of
Proteins RUY SOEIRO
Sloan-Kettering Cancer Center, New York, New York and Departments of Medicine Biology, Albert Einstein College of Medicine, Bronx, New York Accepted
December
and Cell
2, 1974
The major influenza virus-specific proteins appearing in the nucleus of infected cells are the virion nucleocapsid protein (NP) and a putative nonstructural protein (NS,) of approximately 25,006 daltons. In the present study, we have determined the intranuclear localization of NP and NS, in two cell lines, canine kidney cells (MDCK cells) and HeLa cells. Nuclear fractionation was monitored by using ribosomal precursor RNA and heterogeneous nuclear RNA as markers for nucleolar and nucleoplasmic components, respectively; and nucleoli were purified by equilibrium centrifugation in Renografin gradients. The purified nucleoli contained essentially no NP protein, indicating that this protein is mostly, if not totally, nucleoplasmic in location. Some of the nuclear NS, remained associated with the purified nucleoli. Of the NS, in the nucleus of infected HeLa cells, 35% remained with the nucleoli, whereas in MDCK cells only 6% remained. Reconstruction experiments further suggest that the NS, found tightly associated with nucleoli did not arise as an artifact of nuclear fractionation.
should be amenable to verification by means of biochemical experiments employA feature distinguishing influenza virus ing fractionation of infected cell nuclei. infection from that of other nononcogenic However, previous biochemical studies RNA viruses is that virus-specific products have not been definitive. In these studies, are found in both the nucleus and the only simple differential centrifugation was cytoplasm (Dimmock, 1967; Fraser, 1967; employed to fractionate nuclei into crude Gregoriades, 1973; Krug, 1971, 1972; Krug nucleoplasmic and nucleolar fractions, and and Etkind, 1973; Lazarowitz et al., 1971; the purity of the fractions was not moniTaylor et al., 1969, 1970). The nucleocapsid tored (Krug and Etkind, 1973; Taylor et al., 1970). Nucleolar preparations obtained protein (NP) of the virus and a putative nonstructural protein (NSI) of approxi- by this crude procedure are known to be mately 25,000 daltons are the major virus- contaminated by membranous structures specific proteins which have been identi- and possibly other nonnucleolar compofied in the nucleus (Gregoriades, 1973; nents (Penman et al., 1966), so that it is Krug and Etkind, 1973; Lazarowitz et al., not clear that all the virus-specific proteins 1971; Taylor et al., 1969, 1970). Their found in crude nucleolar preparations are, in fact, associated with nucleoli. Confunction in the nucleus is not known. The results of analysis of whole cells by versely, as influenza virus infection leads immunofluorescence are consistent with to an eventual disaggregation of nucleoli NP being primarily in the nucleoplasm and (Compans and Dimmock, 1969), the nuwith NSI being primarily in the nucleolus cleoplasmic fraction might have been con(Dimmock, 1969; Fraser, 1967; Morgan et taminated with nucleolar components, due either to virus-induced nucleolar breakal., 1961)_These intranuclear localizations INTRODUCTION
378 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
INTRANUCLEAR
LOCALIZATION
down within the infected cell or to the instability of nucleoli from infected cells during the fractionation procedure. Furthermore, these earlier studies did not address the possibility of adsorption of NS 1 to nucleoli during the process of nuclear disruption. This is important since NS1 is known to adsorb to ribosomes in the cytoplasm (Krug and Etkind, 1973). In the present study, we have determined the intranuclear localization of NP and NS, after a more precise fractionation of infected cell nuclei. We have monitored the fractionation of nuclei by using ribosomal precursor RNA (principally 45 S RNA) as a marker for nucleolar-specific macromolecular components, and the RNA sedimenting greater than 45 S, presumed to be heterogeneous nuclear RNA (HnRNA), as a marker for nucleoplasmic components (Penman, 1969). In addition, we have employed further procedures for purification of nucleoli, specifically detergent treatment and equilibrium centrifugation in Renografin gradients (Soeiro and Basile, 1973). This analysis was carried out with two different cell lines: MDCK cells, the cells employed in our previous studies (Krug and Etkind, 1973), and HeLa cells. MATERIALS
AND
METHODS
Cells and Virus
The procedures for culture of the MDCK (canine kidney) and MDBK (bovine kidney) cell lines, for virus assay, for growth of stock WSN (influenza A) virus in MDBK cells, and for growth and purification of 3H-labeled amino acid-labeled WSN virus have been described (Krug, 1971; Krug and Etkind, 1973). HeLa cells were maintained in suspension culture in Eagle’s spinner medium containing 5% fetal calf serum; for use in experiments, the cells were transferred to Eagle’s medium containing 7% fetal calf serum in 100 x 15-mm petri dishes and were allowed to settle to form a complete monolayer. MDBK-grown WSN virus multiplies in both MDCK and HeLa cells (Choppin, 1969; Krug, 1971; Choppin and Pons, 1970). In MDCK cells, the latent period is 4 hr, and 200-250 plaque-forming units
OF NP AND NS,
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(PFU)/cell, the maximal virus yields, are produced by 7 hr (Krug, 1971). In HeLa cells, the latent period is 5-5.5 hr and the maximal virus yields are 3-6 PFU/cell. Isolation
of Nuclear Fractions
Confluent monolayers of MDCK or HeLa cells were infected with WSN virus at a multiplicity of 30-60 PFU/cell, or were mock infected. The growth medium and the procedure for infection have been described (Krug, 1972). For the labeling of virus-specific proteins in the nucleus, infected cells were labeled with [35S]methionine for 15 min, followed by a chase period of 45 min in the presence of excess unlabeled methionine in order to allow the labeled proteins to migrate into the nucleus (Krug, 1972; Krug and Etkind, 1973). At the start of the chase period, [3H]adenosine was added at a final concentration of 20 &i/ml in order to label the nuclear RNA species which serve to monitor the nuclear fractionation. In the experiments described in this paper, the pulse label was at 4 hr postinfection (p.i.) (MDCK cells) or at 5.5 hr p.i. (HeLa cells), times at which only virus-specific proteins are synthesized. Similar results were obtained at 5 hr p.i. with MDCK cells and at 4 hr p.i. with HeLa cells. As maximum migration of virus-specific proteins to the nucleus occurs after 10 min of the chase period (Krug, 1972; Krug and Etkind, 1973), any chase period longer than 10 min yielded the same results. For the experiments in which only nuclear RNA was labeled, [3H]adenosine at 20 &i/ml was added to infected or mock-infected cells for the time periods indicated in each figure legend. Cells were collected into reticulocyte standard buffer (RSB) (0.01 MKCl; 0.0015 M MgCl,; 0.01 M Tris-HCl, pH 7.4), and detergent-washed nuclei were obtained as described previously (Krug, 1972). The nuclei were disrupted using electrophoretitally purified deoxyribonuclease in a highsalt buffer: 0.5 M NaCl; 0.05 M MgCl,; 0.01 M Tris, pH 7.4. This high-salt buffer replaced the buffer used previously for the demonstration of intranuclear viral RNP’s (Krug, 1971). The nuclear lysate was lay-
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AND SOEIRO
ered onto a 15-30% sucrose gradient in Materials high-salt buffer and was centrifuged for 15 [35S]Methionine, [3H]adenosine, [3H]min at 17,000 rpm in the SW 27.1 rotor at uridine, and 3H-labeled amino acids in a 4”. The top 3.6 ml of the gradient (containreconstituted protein hydrolyzate were ing 90-95% of the HnRNA) comprised the purchased from New England Nuclear nucleoplasm (Penman, 1969; Soeiro and Corporation, Boston, MA. Renografin Basile, 1973). The pellet, which contained (76%) was obtained from E. R. Squibb and the crude nucleoli, was resuspended in 2.2 Sons, Inc., New York, NY. ml of Renografin-buffer (0.1 M NaCl; 0.001 RESULTS M CaCl, ; 0.01 M Tris-HCl, pH 7.4) containing 1% Triton X-100. This suspension Nuclear Fractionation of Virus-Infected Cells was layered onto a Renografin gradient and was subjected to centrifugation for 16 hr at The nuclei of infected cells were frac34,000 rpm in the SW 41 rotor at 4” (Soeiro tionated using high-ionic-strength buffer as and Basile, 1973). In most experiments, a described in Materials and Methods. Figdiscontinuous Renografin gradient with ure 1 shows the distribution of virus-spetwo layers, 60% and 40% (v/v) (5 ml each) cific proteins and of nuclear RNA’s after was employed. The nucleoli sedimented to centriguation of crude nucleoli from inthe interface between these two layers (see fected cells in preformed, discontinuous Results). In one experiment (Fig. 3) a Renografin gradients. The labeled RNA continuous Renografin gradient (30-50%) was concentrated in one major peak at the was employed. After centrifugation, the interface between the 60% and 40% Renogradients were analyzed for radioactivity. grafin layers. Analysis of the RNA in this The continuous Renografin gradients were peak demonstrated only specific nucleolar analyzed for density by refractive index RNA species (Fig. 2). Ribosomal precurmeasurement (Soeiro, unpublished experisor RNA, sedimenting at 45 S and in the ments). 32 S to 28 S region, were the major species found. Sucrose density-gradient analysis Analysis of Proteins by Polyacrylamide Gel does not adequately resolve the other Electrophoresis RNA’s known to exist in nucleoli, the ribosomal precursor RNA’s intermediate beThe proteins in the various nuclear fractions were concentrated by ethanol precipitween 45 S and 32 S RNA (most evident in tation and were analyzed by electrophoreFig. 2A). Only minimal amounts of RNA sis on 7.5% acrylamide gels as described sedimenting greater than 45 S (putative previously (Krug and Etkind, 1973). HnRNA) were detectable. Thus, as was found previously for uninfected HeLa cells Analysis of RNA’s by Sucrose Density (Soeiro and Basile, 1973), the nuclear maGradients terial collecting at the interface between The nuclear fraction was made 2% in 60% and 40% Renografin is comprised of sodium dodecyl sulfate (SDS) and 0.5 M in purified nucleoli. In addition, the Renografin-banded inurea and warmed to 22”. Marker 28 S and 18 S ribosomal RNA was added as an fected cell nucleoli contained virtually all the ribosomal precursor RNA in the nualiquot of an SDS-urea-treated cytoplascleus: 90-95% of the 45 S RNA and 80mic extract obtained from uninfected HeLa cells labeled with 0.5 pCi/ml of 85% of the 32-28 S RNA in the nucleus [14C]uridine for 24 hr. The mixture was were recovered in the purified nucleoli. layered onto a 15-30% sucrose gradient in Similar results were obtained with unin0.1 M NaCl; 0.01 M Tris-HCl, pH 7.4, fected cell nuclei. Thus, by these criteria, 0.5% SDS; and the gradient was cen- the purity of the nucleoli and nucleoplasm trifuged for 16-17 hr at 18,500 rpm in the obtained from infected cell nuclei was comSW 27 rotor at 22”. The gradients were parable to that obtained for these fractions from uninfected cell nuclei (Soeiro and fractionated and were analyzed for radioBasile, 1973; present study). Therefore, activity.
INTRANUCLEAR
LOCALIZATION
(6) HeLa Cells
4
8
OF NP AND NS1
381
_
12 Tube
16
20
24
28
number
FIG. 1. Analysis of crude nucleolar fractions from infected cells by equilibrium centrifugation in preformed, discontinuous Renografin gradients. The procedures for labeling infected cells with [3H]adenosine and [‘“S]methionine, for isolating crude nucleolar fractions, and for centrifugation of these fractions in preformed, discontinuous Renografin gradients were as described in Materials and Methods. Sedimentation is from right to left.
infected cell nucleoli appeared to be stable to the fractionation procedure. Intranuclear Localization Proteins
of Virus-Specific
As shown in Fig. 1, virus-specific proteins were associated with the purified nucleoli. Sixty to seventy per cent of the virus-specific proteins in the crude nucleolar preparations from both cell lines banded with the nucleoli. The remaining 30-40s were separated from the nucleoli band and appeared predominantly in the fractions at the top of the Renografin gradient. In order to further verify that the virus-specific proteins were indeed banding with the nucleoli, the nucleolar preparation was subjected to equilibrium centrifugation in a continuous, rather than a discontinuous, Renografin gradient (Fig. 3).
The nucleoli, whether obtained from infected or control cells, formed a broad band in the Renografin gradient with the peak at a density of 1.24 g/cc. No change in the distribution of virus-specific proteins was effected by the use of continuous Renografin gradients. Again, 60-70s of the virusspecific proteins banded isopycnically with the nucleoli. The identity of the virus-specific proteins associated with the purified nuclear fractions was determined by PAGE, with the proteins of 3H-labeled amino acidlabeled WSN virions included as internal markers. The virus-specific proteins in the nucleoplasm of infected HeLa cells are presented in Fig. 4. The two major virusspecific proteins were the nucleocapsid protein (NP, molecular weight 60,000) and a protein of approximately 25,000 daltons
382
KRUG AND SOEIRO
which migrates ahead of the virion MP protein in SDS-acrylamide gels (Krug and Etkind, 1973; Lazarowitz et al., 1971; Skehel, 1972). On the basis of this observed difference in migration of MP and the nuclear virus-specific protein, we will denote the latter protein NSI (nonstructural protein 1) as was done in our previous publication (Krug and Etkind, 1973). A recent report has suggested, however, that this protein may not be distinct from MP (Gregoriades, 1973). Proteins NP and NS, were present in the nucleoplasm in approx‘Is-
I
70
MP
x JOE 0 D i wl
15.
: NJ 20
FIG. 2. RNA in Renografin-banded nucleoli from infected cells. Infected MDCK cells and HeLa cells were labeled with [JH]adenosine from 4.25-5.0 hr, and from 5.75-6.5 hr, respectively. The nucleoli were obtained after centrifugation in preformed, discontinuous Renografin gradients as described in Fig. 1. To the nucleoli was added SDS (2%) and urea (0.5 kfl, and “C-labeled ribosomal RNA; and the RNA species were analyzed on sucrose density gradients as described in Materials and Methods.
.
k.
30
40
50
60
70
so
90
100 110 120 130
FIG. 4. Polyacrylamide gel electrophoresis (PAGE) of the proteins in the nucleoplasm of infected HeLa cells. The procedures for labeling infected cells with [%]methionine, for obtaining the nucleoplasm from these cells, and for PAGE analysis were as described in Materials and Methods. *H-Labeled WSN virion proteins were coelectrophoresed with the nucleoplasmic proteins, and the arrows represent the positions of the virion proteins NP and MP. 1130
‘..
\.\ h
\
Tube number
FIG. 3. Analysis of the crude nucleolar fraction from infected HeLa cells by equilibrium centrifugation in a preformed, continuous Renografin gradient. The procedures were the same as described in Fig. 1, except that the Renografin gradient employed was a 30-50s continuous gradient.
383
INTRANUCLEARLOCALIZATIONOFNPANDNS1 imately equimolar amounts. In this and subsequent analyses, we shall not concern ourselves with the two minor virus-specific proteins in the nucleus, NS2 (at 125 mm) and P (at 28 mm) (Krug and Etkind, 1973). The latter protein was somewhat obscured by other large proteins, protein aggregates, and proteins associated with the small amount of plasma membranes which might be contaminating these nuclear preparations (Krug and Etkind, 1973). After purification of the nucleoli from infected HeLa cells by banding in a Renografin gradient (peak A, Fig. 5), essentially only a single virus-specific protein, NS,, remained associated with the nucleoli. A small amount of NP was also present, but NP was distributed approximately equally throughout the main fractions of the Renografin gradient (note that the PAGE of fractions A, B, and C in Fig. 5 have different scales), strongly suggesting that this protein was not specifically associated with any material in the gradient. As NP 60
CA-I
24
was found predominantly in the nucleoplasm (see below), the NP in the Renografin gradient is most probably a contaminant from the nucleoplasm which was incompletely removed from the nucleoli during the initial purification steps. Consequently, NP is most probably totally nucleoplasmic in location. In contrast to NP, protein NS, was concentrated in two peaks in the gradient: in the nucleolus (peak A) and in the top component (peak C) of the Renografin gradient. Table 1 summarizes the distribution of NP and NSI among the nuclear fractions of infected HeLa cells. Purification of the nucleoli clearly removed almost all of the NP protein, leaving NSI as the predominant virus-specific protein in the nucleoli. However, only 35% of the NS, in the nucleus is associated with the nucleolus, with 40% being in the nucleoplasm and 17% being in the top component of the Renografin gradient. The fact that such a large percentage of NS, was nonnucleolar A Nucleolus
t
Ljd
7 1
4
I3
12
I-B-I A 16
Tube number
20
24
28
20
40
60
SO
100
I20
Mobhty, mm
FIG. 5. The virus-specificproteinsassociated with the purified nucleoli from infected HeLa cells and associated with other components separated from these nucleoli by equilibrium centrifugation in a Renografin gradient. Infected HeLa cells were labeled with [*%]methionine, and the crude nucleoli from these cells was subjected to equilibrium centrifugation in a preformed, discontinuous Renografin gradient as described in Materials and Methods. The distribution of virus-specific proteins in the Renografin gradient is shown on the left. On the right is shown the PAGE analysis of fractions from the Renografin gradient, pooled as shown. The PAGE analysis, with ‘H-labeled WSN virion proteins as internal markers, was as described in Materials and Methods.
384
KRUG
AND SOEIRO
suggested the possibility that the association of NS, with the nucleoli might result from the adsorption of nucleoplasmic NS, to the nucleoli, analogous to the known adsorption of NS, to cytoplasmic ribosomes (Krug and Etkind, 1973). To test this possibility, we carried out the following reconstruction experiment. Infected cell nucleoplasm containing radiolabeled NS, (and NP) in high-salt buffer was prepared and was used as the high-salt buffer for the disruption and fractionation of either uninfected or infected nuclei radiolabeled in RNA. The nucleoli were then purified by banding in Renografin gradients. Less than 0.2% of the radiolabeled proteins from the infected cell nucleoplasm were found to be associated with the purified nucleoli. This result strongly suggests that the association of NS, with the nucleolus did not simply result from adsorption. It rather strongly suggests that the NS1 associated with the nucleolus is at its true intranuclear location. The results obtained with the nuclei from infected MDCK cells differ from those obtained with infected HeLa cells in one significant aspect: less of the nuclear NS, was associated with the purified nucleoli (Table 2). Only 6% of the NS, remained associated with the Renografinbanded nucleoli, while a comparable amount of NS1 was found in the top TABLE
1
DISTRIBUTION OF NP AND NS, BETWEEN THE NUCLEOPLASM AND THE NUCLEOLUS OF INFECTED H&A c ELLSa
Nuclear fraction
Nucleoplasm Crude nucleoli Purified nucleoli
% of total in nucleus NP
NS,
76 15 5
40 52 35
a Infected HeLa cells were labeled with [Y?~]methionine, and the nuclear fractions were obtained as described in Materials and Methods. The nuclear fractions included the material sedimenting in the high-salt sucrose gradient between the nucleoplasm and the crude nucleoli. The proteins in each of these fractions were analyzed by PAGE as described in Materials and Methods. The results shown represent the average of four separate experiments.
TABLE DISTRIBUTION NUCLEOPLASM
2
OF NP AND N’S, BETWEEN THE AND THE NUCLEOLUS OF INFECTED MDCK CELLS’
Nuclear fraction
Nucleoplasm Crude nucleoli Purified nucleoli
% of total in nucleus NP
NS,
90 2 1
77 11 6
“Infected MDCK cells were labeled with [,,S Jmethionine, and the proteins in all of the nuclear fractions from these cells were analyzed by PAGE as described in Materials and Methods. The results shown represent the average of three separate experiments.
component of the Renografin gradient. Nonetheless, NS, was the predominant virus-specific protein banding with the nucleoli. The molar ratio of NS1 to NP associated with the nucleolus was approximately 12 to 1. Reconstruction experiments suggested that, as with HeLa cells, the NS, associated with the nucleoli did not become bound during the nuclear disruption procedure. Effect of Infection thesis
on Nucleolar
RNA Syn-
Influenza virus infection reportedly inhibits the processing of ribosomal precursor RNA occurring in the nucleolus, with little effect on the synthesis of 45 S RNA (Stephenson and Dimmock, 1974). To determine whether there was any other difference in the nucleoli from infected HeLa and MDCK cells, we compared the effect of influenza virus infection on the nucleolar ribosomal RNA processing in these two cell lines. As shown in Fig. 6, after infection of HeLa cells, the rate of appearance of 32 S in the nucleolus slowly declined 30% during the first 6.7 hr of infection, with little effect on the synthesis of 45 S RNA. In contrast, in MDCK cells the effects of infection on ribosomal RNA processing were more rapid and drastic (Fig. 7). In uninfected cells, the predominant ribosomal RNA species appearing during a 0.7-hr labeling period was 32-28 S RNA and a distinct 45 S RNA species could not be discerned, suggesting
INTRANUCLEAR
4
6
12
16
LOCALIZATION
4
6
12
OF NP AND NS,
16
4
6
12
16
Tube number
FIG. 6. The effect of influenza virus infection on the nucleolar RNA synthesis of HeLa cells. Mock-infected HeLa cells, and infected HeLa cells at 4 and 5.5 hr were labeled with 20 &i/ml of [SH]adenosine for 1.2 hr. Purified (Renografin-banded) nucleoli were prepared from these cells, and the labeled RNA in the nucleoli was analyzed on sucrose density gradients as described in Materials and Methods. Infected 4-4 7hxrs
Tube number
FIG. 7. The effect of influenza virus infection on the nucleolar RNA synthesis of MDCK cells. Mock-infected and infected MDCK cells (at 4 hr) were labeled with 20 rCi/ml of [*H]adenosine for 0.7 hr. The labeled nucleolar RNA was analyzed on sucrose density gradients as described in Fig. 6.
that the rate of processing was rapid relative to the rate of synthesis of 45 S RNA. In contrast, in infected cells, only a minimal amount of 32-28 S RNA appeared during a 0.7-hr labeling period despite the appearance of a distinct 45 S RNA species, indicating an almost complete inhibition of ribosomal RNA processing. Thus, in MDCK cells, in which less NS, was found to be associated with purified nucleoli, the effect of infection on the nucleolar function of ribosomal RNA processing was more pronounced. DISCUSSION
Uninfected HeLa cell nuclei can tionated into two major fractions based on their RNA content, are ered to represent the nucleoplasm
be fracwhich, considand the
nucleolus (Penman, 1969; Soeiro and Basile, 1973). In the present study, we have used the same procedures to fractionate the nuclei of two different cell lines infected by influenza virus. From these cells we obtained two major nuclear fractions with RNA species distribution similar to that obtained from uninfected HeLa cells, indicating that these procedures may be utilized to yield the nucleoplasm and the nucleolus from influenza virus-infected cells. This result was obtained despite the fact that influenza virus infection reportedly leads to an eventual disaggregation of nucleoli (Compans and Dimmock, 1969). However, at the times of infection studied in the present work, little or no nucleolar breakdown was detectable by electronmicroscopic examination of infected cells
386
KRUG AND SOEIRO
(Krug, unpublished experiments). The distribution of virus-specific proteins in these nuclear fractions suggest specific locations of these proteins in the nucleus of infected cells. The removal of essentially all of the nucleocapsid protein (NP) from the nucleolus during the nuclear fractionation strongly suggests that NP is totally nucleoplasmic in location. This conclusion is consistent with most of the immunofluorescence studies (Dimmock, 1969; Fraser, 1967; Morgan et al., 1961). On the other hand, the finding of the NS, protein in nucleoli after banding in Renografin gradients indicates that at least some of the NS, is tightly associated with the nucleolus. Furthermore, the mixing experiments suggest strongly that the NS1 associated with the nucleoli did not result from simple adsorption of NS, from the nucleoplasm. The finding of NS, in the nucleolus is also consistent with immunofluorescence analysis (Dimmock, 1969). Not all of the nuclear NS, was found to be associated with purified nucleoli. Of the NS, in the nucleus of infected HeLa cells, 35% was found to be associated with the nucleolus whereas much less, only 6%, of the nuclear NS1 was found to be associated with the nucleolus of infected MDCK cells. Whether this distribution of NSI between the nucleoplasm and the nucleolus observed after nuclear fractionation reflects the true distribution of NS, within the infected cell nucleus cannot be ascertained with certainty. It must be taken into consideration that the nucleolar purification procedure employed in the present study has been designed to eliminate loosely bound proteins but to retain all ribosomal precursor RNA species (Soeiro and Basile, 1973). Electron micrographs of nucleolar preparations purified from uninfected HeLa cells by banding in Renografin gradients reveal mainly nucleolar cores (Soeiro, unpublished experiments). Therefore, some loosely bound proteins associated with the granular portion of the nucleolus may be released in an attempt to define only those proteins which are tightly bound to the organelle. Although our results indicate that the nucleoli from infected cells retain all the ribosomal precursor RNA, we
have not examined the effect of influenza virus infection on the stability of the association of proteins, both host and viral, with the nucleolus. For example, it is possible that the more extensive inhibition of ribosomal RNA processing observed in MDCK cells after virus infection may be reflected in a less-stable association of proteins (including NSI) to the nucleolus. Consequently, the percentage of the nuclear NS, associated with nucleoli in uiuo may be higher than that found after this nuclear fractionation. However, the most important observation of the present study is that a significant portion of NS 1remains tightly bound to nucleoli even after extensive purification of this organelle. Further experiments are necessary to determine the functional role of NS1 in the nucleolus during viral infection and to decide whether the nucleolar NS1 protein is involved in the known inhibition of ribosomal RNA processing that occurs during influenza virus infection (Stephenson and Dimmock, 1974; present study). ACKNOWLEDGMENTS We thank Barbara Benson for expert technical assistance. This investigation was supported by Grants CA 08748 (RMK) and CA 10933 (RS) from the National Cancer Institute and Grant AI-11772-01 from the National Institute of Allergy and Infectious Disease (RMK). REFERENCES 1. CHOPPIN, P. W. (1969). Replication of influenza virus in a continuous cell line: high yield infective virus from cells inoculated at high multiplicity. Virology 39, 130-134. 2. CHOPPIN, P. W., and PONS, M. W. (1970). The RNAs of infective and incomplete influenza virions grown in MDBK and HeLa cells. Virology 42,603-610. 3. COMPANS, R. W., and DIMMOCK, N. J. (1969). An electron microscopic study of single-cycle infection of chick embryo fibroblasts by influenza virus. Virology 39, 499-515. 4. DIMMOCK, N. J. (1969). New virus-specific antigens in cells infected with influenza virus. Virology 39, 224-234. 5. FRASER, K. B. (1967). Immunofluorescence of abortive and complete infections by influenza virus: extraction from virus and infected cell and acidic chloroform-methanol. Virology 54, 369-383.
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LOCALIZATION
7. KRUG, R. M. (1971). Influenza viral RNP’s newly synthesized during the latent period of viral growth in MDCK cells. Virology 44, 125-136. 8. KRUC, R. M. (1972). Cytoplasmic and nucleoplasmic viral RNP’s in influenza virus-infected MDCK cells. Virology 60, 103-113. 9. KRUG, R. M., and ETKIND, P. E. (1973). Cytoplasmic and nuclear virus-specific proteins in influenza virus-infected MDCK cells. Virology
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Press, New York. 13. PENMAN,S., SMITH,I., HOLTZMAN,E., and GREENBERG,H. (1966). RNA metabolism in the HeLa cell nucleus and nucleolus. Nat. Cancer Inst. Monogr.
23,489~512.
14. SKEHEL, J. J. (1972). Polypeptide synthesis in influenza virus-infected cells. Virology 49, 23-36.
15. SOEIRO,R., and BASILE,C. (1973). Non-ribosomal 56, 334-348. nucleolar proteins in HeLa cells. J. Mol. Biol. 79, 507-519. 10. LAZAROW~~Z, S. G., COMPANS,R. W., and CHOP16. STEPHENSON, J. R., and DIMMOCK,N. J. (1974). PIN, P. W. (1971). Influenza virus structural and non-structural proteins in infected cells Effect of influenza virus on cellular RNA synthesis. In “Negative Strand Viruses” (B. W. J. and their plasma membranes. Virology 46, Mahy and R. D. Barry, eds.), Academic Press, 830-843. 11. MORGAN,C., Hsu, K. C., RIFKIND,R. A., KNOX, A. New York, in press. W., and ROSE,H. M. (1961). The application of 17. TAYLOR,J. M., HAMPSON,A. W., and WHITE,D. 0. ferritin conjugated antibody to electron micro(1969). The polypeptides of influenza virus. I. scopic studies of influenza virus in infected Cytoplasmic synthesis and nuclear accumulacells. II. The interior of the cell. J. Ezp. Med. tion. Virology 39, 419-425. 114. 833-836. 18. TA~OR, J. M., HAMPSON,A. W., LAYTON,J. E., 12. PENMAN,S. (1969). Preparation of purified nuclei and WHITE, D. 0. (1970). The polypeptides of and nucleoli from mammalian cells. In “Funinfluenza virus. IV. An analysis of nuclear damental Techniques in Virology” (K. Habel accumulation. Virology 42, 744-752. and N. P. Salzman, eds.), pp. 35-48. Academic
64, 378-387 (1975)
Studies
on the lntranuclear
Influenza
Virus-Specific
ROBERT M. KRUG Memorial
Localization
AND
of
Proteins RUY SOEIRO
Sloan-Kettering Cancer Center, New York, New York and Departments of Medicine Biology, Albert Einstein College of Medicine, Bronx, New York Accepted
December
and Cell
2, 1974
The major influenza virus-specific proteins appearing in the nucleus of infected cells are the virion nucleocapsid protein (NP) and a putative nonstructural protein (NS,) of approximately 25,006 daltons. In the present study, we have determined the intranuclear localization of NP and NS, in two cell lines, canine kidney cells (MDCK cells) and HeLa cells. Nuclear fractionation was monitored by using ribosomal precursor RNA and heterogeneous nuclear RNA as markers for nucleolar and nucleoplasmic components, respectively; and nucleoli were purified by equilibrium centrifugation in Renografin gradients. The purified nucleoli contained essentially no NP protein, indicating that this protein is mostly, if not totally, nucleoplasmic in location. Some of the nuclear NS, remained associated with the purified nucleoli. Of the NS, in the nucleus of infected HeLa cells, 35% remained with the nucleoli, whereas in MDCK cells only 6% remained. Reconstruction experiments further suggest that the NS, found tightly associated with nucleoli did not arise as an artifact of nuclear fractionation.
should be amenable to verification by means of biochemical experiments employA feature distinguishing influenza virus ing fractionation of infected cell nuclei. infection from that of other nononcogenic However, previous biochemical studies RNA viruses is that virus-specific products have not been definitive. In these studies, are found in both the nucleus and the only simple differential centrifugation was cytoplasm (Dimmock, 1967; Fraser, 1967; employed to fractionate nuclei into crude Gregoriades, 1973; Krug, 1971, 1972; Krug nucleoplasmic and nucleolar fractions, and and Etkind, 1973; Lazarowitz et al., 1971; the purity of the fractions was not moniTaylor et al., 1969, 1970). The nucleocapsid tored (Krug and Etkind, 1973; Taylor et al., 1970). Nucleolar preparations obtained protein (NP) of the virus and a putative nonstructural protein (NSI) of approxi- by this crude procedure are known to be mately 25,000 daltons are the major virus- contaminated by membranous structures specific proteins which have been identi- and possibly other nonnucleolar compofied in the nucleus (Gregoriades, 1973; nents (Penman et al., 1966), so that it is Krug and Etkind, 1973; Lazarowitz et al., not clear that all the virus-specific proteins 1971; Taylor et al., 1969, 1970). Their found in crude nucleolar preparations are, in fact, associated with nucleoli. Confunction in the nucleus is not known. The results of analysis of whole cells by versely, as influenza virus infection leads immunofluorescence are consistent with to an eventual disaggregation of nucleoli NP being primarily in the nucleoplasm and (Compans and Dimmock, 1969), the nuwith NSI being primarily in the nucleolus cleoplasmic fraction might have been con(Dimmock, 1969; Fraser, 1967; Morgan et taminated with nucleolar components, due either to virus-induced nucleolar breakal., 1961)_These intranuclear localizations INTRODUCTION
378 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
INTRANUCLEAR
LOCALIZATION
down within the infected cell or to the instability of nucleoli from infected cells during the fractionation procedure. Furthermore, these earlier studies did not address the possibility of adsorption of NS 1 to nucleoli during the process of nuclear disruption. This is important since NS1 is known to adsorb to ribosomes in the cytoplasm (Krug and Etkind, 1973). In the present study, we have determined the intranuclear localization of NP and NS, after a more precise fractionation of infected cell nuclei. We have monitored the fractionation of nuclei by using ribosomal precursor RNA (principally 45 S RNA) as a marker for nucleolar-specific macromolecular components, and the RNA sedimenting greater than 45 S, presumed to be heterogeneous nuclear RNA (HnRNA), as a marker for nucleoplasmic components (Penman, 1969). In addition, we have employed further procedures for purification of nucleoli, specifically detergent treatment and equilibrium centrifugation in Renografin gradients (Soeiro and Basile, 1973). This analysis was carried out with two different cell lines: MDCK cells, the cells employed in our previous studies (Krug and Etkind, 1973), and HeLa cells. MATERIALS
AND
METHODS
Cells and Virus
The procedures for culture of the MDCK (canine kidney) and MDBK (bovine kidney) cell lines, for virus assay, for growth of stock WSN (influenza A) virus in MDBK cells, and for growth and purification of 3H-labeled amino acid-labeled WSN virus have been described (Krug, 1971; Krug and Etkind, 1973). HeLa cells were maintained in suspension culture in Eagle’s spinner medium containing 5% fetal calf serum; for use in experiments, the cells were transferred to Eagle’s medium containing 7% fetal calf serum in 100 x 15-mm petri dishes and were allowed to settle to form a complete monolayer. MDBK-grown WSN virus multiplies in both MDCK and HeLa cells (Choppin, 1969; Krug, 1971; Choppin and Pons, 1970). In MDCK cells, the latent period is 4 hr, and 200-250 plaque-forming units
OF NP AND NS,
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(PFU)/cell, the maximal virus yields, are produced by 7 hr (Krug, 1971). In HeLa cells, the latent period is 5-5.5 hr and the maximal virus yields are 3-6 PFU/cell. Isolation
of Nuclear Fractions
Confluent monolayers of MDCK or HeLa cells were infected with WSN virus at a multiplicity of 30-60 PFU/cell, or were mock infected. The growth medium and the procedure for infection have been described (Krug, 1972). For the labeling of virus-specific proteins in the nucleus, infected cells were labeled with [35S]methionine for 15 min, followed by a chase period of 45 min in the presence of excess unlabeled methionine in order to allow the labeled proteins to migrate into the nucleus (Krug, 1972; Krug and Etkind, 1973). At the start of the chase period, [3H]adenosine was added at a final concentration of 20 &i/ml in order to label the nuclear RNA species which serve to monitor the nuclear fractionation. In the experiments described in this paper, the pulse label was at 4 hr postinfection (p.i.) (MDCK cells) or at 5.5 hr p.i. (HeLa cells), times at which only virus-specific proteins are synthesized. Similar results were obtained at 5 hr p.i. with MDCK cells and at 4 hr p.i. with HeLa cells. As maximum migration of virus-specific proteins to the nucleus occurs after 10 min of the chase period (Krug, 1972; Krug and Etkind, 1973), any chase period longer than 10 min yielded the same results. For the experiments in which only nuclear RNA was labeled, [3H]adenosine at 20 &i/ml was added to infected or mock-infected cells for the time periods indicated in each figure legend. Cells were collected into reticulocyte standard buffer (RSB) (0.01 MKCl; 0.0015 M MgCl,; 0.01 M Tris-HCl, pH 7.4), and detergent-washed nuclei were obtained as described previously (Krug, 1972). The nuclei were disrupted using electrophoretitally purified deoxyribonuclease in a highsalt buffer: 0.5 M NaCl; 0.05 M MgCl,; 0.01 M Tris, pH 7.4. This high-salt buffer replaced the buffer used previously for the demonstration of intranuclear viral RNP’s (Krug, 1971). The nuclear lysate was lay-
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ered onto a 15-30% sucrose gradient in Materials high-salt buffer and was centrifuged for 15 [35S]Methionine, [3H]adenosine, [3H]min at 17,000 rpm in the SW 27.1 rotor at uridine, and 3H-labeled amino acids in a 4”. The top 3.6 ml of the gradient (containreconstituted protein hydrolyzate were ing 90-95% of the HnRNA) comprised the purchased from New England Nuclear nucleoplasm (Penman, 1969; Soeiro and Corporation, Boston, MA. Renografin Basile, 1973). The pellet, which contained (76%) was obtained from E. R. Squibb and the crude nucleoli, was resuspended in 2.2 Sons, Inc., New York, NY. ml of Renografin-buffer (0.1 M NaCl; 0.001 RESULTS M CaCl, ; 0.01 M Tris-HCl, pH 7.4) containing 1% Triton X-100. This suspension Nuclear Fractionation of Virus-Infected Cells was layered onto a Renografin gradient and was subjected to centrifugation for 16 hr at The nuclei of infected cells were frac34,000 rpm in the SW 41 rotor at 4” (Soeiro tionated using high-ionic-strength buffer as and Basile, 1973). In most experiments, a described in Materials and Methods. Figdiscontinuous Renografin gradient with ure 1 shows the distribution of virus-spetwo layers, 60% and 40% (v/v) (5 ml each) cific proteins and of nuclear RNA’s after was employed. The nucleoli sedimented to centriguation of crude nucleoli from inthe interface between these two layers (see fected cells in preformed, discontinuous Results). In one experiment (Fig. 3) a Renografin gradients. The labeled RNA continuous Renografin gradient (30-50%) was concentrated in one major peak at the was employed. After centrifugation, the interface between the 60% and 40% Renogradients were analyzed for radioactivity. grafin layers. Analysis of the RNA in this The continuous Renografin gradients were peak demonstrated only specific nucleolar analyzed for density by refractive index RNA species (Fig. 2). Ribosomal precurmeasurement (Soeiro, unpublished experisor RNA, sedimenting at 45 S and in the ments). 32 S to 28 S region, were the major species found. Sucrose density-gradient analysis Analysis of Proteins by Polyacrylamide Gel does not adequately resolve the other Electrophoresis RNA’s known to exist in nucleoli, the ribosomal precursor RNA’s intermediate beThe proteins in the various nuclear fractions were concentrated by ethanol precipitween 45 S and 32 S RNA (most evident in tation and were analyzed by electrophoreFig. 2A). Only minimal amounts of RNA sis on 7.5% acrylamide gels as described sedimenting greater than 45 S (putative previously (Krug and Etkind, 1973). HnRNA) were detectable. Thus, as was found previously for uninfected HeLa cells Analysis of RNA’s by Sucrose Density (Soeiro and Basile, 1973), the nuclear maGradients terial collecting at the interface between The nuclear fraction was made 2% in 60% and 40% Renografin is comprised of sodium dodecyl sulfate (SDS) and 0.5 M in purified nucleoli. In addition, the Renografin-banded inurea and warmed to 22”. Marker 28 S and 18 S ribosomal RNA was added as an fected cell nucleoli contained virtually all the ribosomal precursor RNA in the nualiquot of an SDS-urea-treated cytoplascleus: 90-95% of the 45 S RNA and 80mic extract obtained from uninfected HeLa cells labeled with 0.5 pCi/ml of 85% of the 32-28 S RNA in the nucleus [14C]uridine for 24 hr. The mixture was were recovered in the purified nucleoli. layered onto a 15-30% sucrose gradient in Similar results were obtained with unin0.1 M NaCl; 0.01 M Tris-HCl, pH 7.4, fected cell nuclei. Thus, by these criteria, 0.5% SDS; and the gradient was cen- the purity of the nucleoli and nucleoplasm trifuged for 16-17 hr at 18,500 rpm in the obtained from infected cell nuclei was comSW 27 rotor at 22”. The gradients were parable to that obtained for these fractions from uninfected cell nuclei (Soeiro and fractionated and were analyzed for radioBasile, 1973; present study). Therefore, activity.
INTRANUCLEAR
LOCALIZATION
(6) HeLa Cells
4
8
OF NP AND NS1
381
_
12 Tube
16
20
24
28
number
FIG. 1. Analysis of crude nucleolar fractions from infected cells by equilibrium centrifugation in preformed, discontinuous Renografin gradients. The procedures for labeling infected cells with [3H]adenosine and [‘“S]methionine, for isolating crude nucleolar fractions, and for centrifugation of these fractions in preformed, discontinuous Renografin gradients were as described in Materials and Methods. Sedimentation is from right to left.
infected cell nucleoli appeared to be stable to the fractionation procedure. Intranuclear Localization Proteins
of Virus-Specific
As shown in Fig. 1, virus-specific proteins were associated with the purified nucleoli. Sixty to seventy per cent of the virus-specific proteins in the crude nucleolar preparations from both cell lines banded with the nucleoli. The remaining 30-40s were separated from the nucleoli band and appeared predominantly in the fractions at the top of the Renografin gradient. In order to further verify that the virus-specific proteins were indeed banding with the nucleoli, the nucleolar preparation was subjected to equilibrium centrifugation in a continuous, rather than a discontinuous, Renografin gradient (Fig. 3).
The nucleoli, whether obtained from infected or control cells, formed a broad band in the Renografin gradient with the peak at a density of 1.24 g/cc. No change in the distribution of virus-specific proteins was effected by the use of continuous Renografin gradients. Again, 60-70s of the virusspecific proteins banded isopycnically with the nucleoli. The identity of the virus-specific proteins associated with the purified nuclear fractions was determined by PAGE, with the proteins of 3H-labeled amino acidlabeled WSN virions included as internal markers. The virus-specific proteins in the nucleoplasm of infected HeLa cells are presented in Fig. 4. The two major virusspecific proteins were the nucleocapsid protein (NP, molecular weight 60,000) and a protein of approximately 25,000 daltons
382
KRUG AND SOEIRO
which migrates ahead of the virion MP protein in SDS-acrylamide gels (Krug and Etkind, 1973; Lazarowitz et al., 1971; Skehel, 1972). On the basis of this observed difference in migration of MP and the nuclear virus-specific protein, we will denote the latter protein NSI (nonstructural protein 1) as was done in our previous publication (Krug and Etkind, 1973). A recent report has suggested, however, that this protein may not be distinct from MP (Gregoriades, 1973). Proteins NP and NS, were present in the nucleoplasm in approx‘Is-
I
70
MP
x JOE 0 D i wl
15.
: NJ 20
FIG. 2. RNA in Renografin-banded nucleoli from infected cells. Infected MDCK cells and HeLa cells were labeled with [JH]adenosine from 4.25-5.0 hr, and from 5.75-6.5 hr, respectively. The nucleoli were obtained after centrifugation in preformed, discontinuous Renografin gradients as described in Fig. 1. To the nucleoli was added SDS (2%) and urea (0.5 kfl, and “C-labeled ribosomal RNA; and the RNA species were analyzed on sucrose density gradients as described in Materials and Methods.
.
k.
30
40
50
60
70
so
90
100 110 120 130
FIG. 4. Polyacrylamide gel electrophoresis (PAGE) of the proteins in the nucleoplasm of infected HeLa cells. The procedures for labeling infected cells with [%]methionine, for obtaining the nucleoplasm from these cells, and for PAGE analysis were as described in Materials and Methods. *H-Labeled WSN virion proteins were coelectrophoresed with the nucleoplasmic proteins, and the arrows represent the positions of the virion proteins NP and MP. 1130
‘..
\.\ h
\
Tube number
FIG. 3. Analysis of the crude nucleolar fraction from infected HeLa cells by equilibrium centrifugation in a preformed, continuous Renografin gradient. The procedures were the same as described in Fig. 1, except that the Renografin gradient employed was a 30-50s continuous gradient.
383
INTRANUCLEARLOCALIZATIONOFNPANDNS1 imately equimolar amounts. In this and subsequent analyses, we shall not concern ourselves with the two minor virus-specific proteins in the nucleus, NS2 (at 125 mm) and P (at 28 mm) (Krug and Etkind, 1973). The latter protein was somewhat obscured by other large proteins, protein aggregates, and proteins associated with the small amount of plasma membranes which might be contaminating these nuclear preparations (Krug and Etkind, 1973). After purification of the nucleoli from infected HeLa cells by banding in a Renografin gradient (peak A, Fig. 5), essentially only a single virus-specific protein, NS,, remained associated with the nucleoli. A small amount of NP was also present, but NP was distributed approximately equally throughout the main fractions of the Renografin gradient (note that the PAGE of fractions A, B, and C in Fig. 5 have different scales), strongly suggesting that this protein was not specifically associated with any material in the gradient. As NP 60
CA-I
24
was found predominantly in the nucleoplasm (see below), the NP in the Renografin gradient is most probably a contaminant from the nucleoplasm which was incompletely removed from the nucleoli during the initial purification steps. Consequently, NP is most probably totally nucleoplasmic in location. In contrast to NP, protein NS, was concentrated in two peaks in the gradient: in the nucleolus (peak A) and in the top component (peak C) of the Renografin gradient. Table 1 summarizes the distribution of NP and NSI among the nuclear fractions of infected HeLa cells. Purification of the nucleoli clearly removed almost all of the NP protein, leaving NSI as the predominant virus-specific protein in the nucleoli. However, only 35% of the NS, in the nucleus is associated with the nucleolus, with 40% being in the nucleoplasm and 17% being in the top component of the Renografin gradient. The fact that such a large percentage of NS, was nonnucleolar A Nucleolus
t
Ljd
7 1
4
I3
12
I-B-I A 16
Tube number
20
24
28
20
40
60
SO
100
I20
Mobhty, mm
FIG. 5. The virus-specificproteinsassociated with the purified nucleoli from infected HeLa cells and associated with other components separated from these nucleoli by equilibrium centrifugation in a Renografin gradient. Infected HeLa cells were labeled with [*%]methionine, and the crude nucleoli from these cells was subjected to equilibrium centrifugation in a preformed, discontinuous Renografin gradient as described in Materials and Methods. The distribution of virus-specific proteins in the Renografin gradient is shown on the left. On the right is shown the PAGE analysis of fractions from the Renografin gradient, pooled as shown. The PAGE analysis, with ‘H-labeled WSN virion proteins as internal markers, was as described in Materials and Methods.
384
KRUG
AND SOEIRO
suggested the possibility that the association of NS, with the nucleoli might result from the adsorption of nucleoplasmic NS, to the nucleoli, analogous to the known adsorption of NS, to cytoplasmic ribosomes (Krug and Etkind, 1973). To test this possibility, we carried out the following reconstruction experiment. Infected cell nucleoplasm containing radiolabeled NS, (and NP) in high-salt buffer was prepared and was used as the high-salt buffer for the disruption and fractionation of either uninfected or infected nuclei radiolabeled in RNA. The nucleoli were then purified by banding in Renografin gradients. Less than 0.2% of the radiolabeled proteins from the infected cell nucleoplasm were found to be associated with the purified nucleoli. This result strongly suggests that the association of NS, with the nucleolus did not simply result from adsorption. It rather strongly suggests that the NS1 associated with the nucleolus is at its true intranuclear location. The results obtained with the nuclei from infected MDCK cells differ from those obtained with infected HeLa cells in one significant aspect: less of the nuclear NS, was associated with the purified nucleoli (Table 2). Only 6% of the NS, remained associated with the Renografinbanded nucleoli, while a comparable amount of NS1 was found in the top TABLE
1
DISTRIBUTION OF NP AND NS, BETWEEN THE NUCLEOPLASM AND THE NUCLEOLUS OF INFECTED H&A c ELLSa
Nuclear fraction
Nucleoplasm Crude nucleoli Purified nucleoli
% of total in nucleus NP
NS,
76 15 5
40 52 35
a Infected HeLa cells were labeled with [Y?~]methionine, and the nuclear fractions were obtained as described in Materials and Methods. The nuclear fractions included the material sedimenting in the high-salt sucrose gradient between the nucleoplasm and the crude nucleoli. The proteins in each of these fractions were analyzed by PAGE as described in Materials and Methods. The results shown represent the average of four separate experiments.
TABLE DISTRIBUTION NUCLEOPLASM
2
OF NP AND N’S, BETWEEN THE AND THE NUCLEOLUS OF INFECTED MDCK CELLS’
Nuclear fraction
Nucleoplasm Crude nucleoli Purified nucleoli
% of total in nucleus NP
NS,
90 2 1
77 11 6
“Infected MDCK cells were labeled with [,,S Jmethionine, and the proteins in all of the nuclear fractions from these cells were analyzed by PAGE as described in Materials and Methods. The results shown represent the average of three separate experiments.
component of the Renografin gradient. Nonetheless, NS, was the predominant virus-specific protein banding with the nucleoli. The molar ratio of NS1 to NP associated with the nucleolus was approximately 12 to 1. Reconstruction experiments suggested that, as with HeLa cells, the NS, associated with the nucleoli did not become bound during the nuclear disruption procedure. Effect of Infection thesis
on Nucleolar
RNA Syn-
Influenza virus infection reportedly inhibits the processing of ribosomal precursor RNA occurring in the nucleolus, with little effect on the synthesis of 45 S RNA (Stephenson and Dimmock, 1974). To determine whether there was any other difference in the nucleoli from infected HeLa and MDCK cells, we compared the effect of influenza virus infection on the nucleolar ribosomal RNA processing in these two cell lines. As shown in Fig. 6, after infection of HeLa cells, the rate of appearance of 32 S in the nucleolus slowly declined 30% during the first 6.7 hr of infection, with little effect on the synthesis of 45 S RNA. In contrast, in MDCK cells the effects of infection on ribosomal RNA processing were more rapid and drastic (Fig. 7). In uninfected cells, the predominant ribosomal RNA species appearing during a 0.7-hr labeling period was 32-28 S RNA and a distinct 45 S RNA species could not be discerned, suggesting
INTRANUCLEAR
4
6
12
16
LOCALIZATION
4
6
12
OF NP AND NS,
16
4
6
12
16
Tube number
FIG. 6. The effect of influenza virus infection on the nucleolar RNA synthesis of HeLa cells. Mock-infected HeLa cells, and infected HeLa cells at 4 and 5.5 hr were labeled with 20 &i/ml of [SH]adenosine for 1.2 hr. Purified (Renografin-banded) nucleoli were prepared from these cells, and the labeled RNA in the nucleoli was analyzed on sucrose density gradients as described in Materials and Methods. Infected 4-4 7hxrs
Tube number
FIG. 7. The effect of influenza virus infection on the nucleolar RNA synthesis of MDCK cells. Mock-infected and infected MDCK cells (at 4 hr) were labeled with 20 rCi/ml of [*H]adenosine for 0.7 hr. The labeled nucleolar RNA was analyzed on sucrose density gradients as described in Fig. 6.
that the rate of processing was rapid relative to the rate of synthesis of 45 S RNA. In contrast, in infected cells, only a minimal amount of 32-28 S RNA appeared during a 0.7-hr labeling period despite the appearance of a distinct 45 S RNA species, indicating an almost complete inhibition of ribosomal RNA processing. Thus, in MDCK cells, in which less NS, was found to be associated with purified nucleoli, the effect of infection on the nucleolar function of ribosomal RNA processing was more pronounced. DISCUSSION
Uninfected HeLa cell nuclei can tionated into two major fractions based on their RNA content, are ered to represent the nucleoplasm
be fracwhich, considand the
nucleolus (Penman, 1969; Soeiro and Basile, 1973). In the present study, we have used the same procedures to fractionate the nuclei of two different cell lines infected by influenza virus. From these cells we obtained two major nuclear fractions with RNA species distribution similar to that obtained from uninfected HeLa cells, indicating that these procedures may be utilized to yield the nucleoplasm and the nucleolus from influenza virus-infected cells. This result was obtained despite the fact that influenza virus infection reportedly leads to an eventual disaggregation of nucleoli (Compans and Dimmock, 1969). However, at the times of infection studied in the present work, little or no nucleolar breakdown was detectable by electronmicroscopic examination of infected cells
386
KRUG AND SOEIRO
(Krug, unpublished experiments). The distribution of virus-specific proteins in these nuclear fractions suggest specific locations of these proteins in the nucleus of infected cells. The removal of essentially all of the nucleocapsid protein (NP) from the nucleolus during the nuclear fractionation strongly suggests that NP is totally nucleoplasmic in location. This conclusion is consistent with most of the immunofluorescence studies (Dimmock, 1969; Fraser, 1967; Morgan et al., 1961). On the other hand, the finding of the NS, protein in nucleoli after banding in Renografin gradients indicates that at least some of the NS, is tightly associated with the nucleolus. Furthermore, the mixing experiments suggest strongly that the NS1 associated with the nucleoli did not result from simple adsorption of NS, from the nucleoplasm. The finding of NS, in the nucleolus is also consistent with immunofluorescence analysis (Dimmock, 1969). Not all of the nuclear NS, was found to be associated with purified nucleoli. Of the NS, in the nucleus of infected HeLa cells, 35% was found to be associated with the nucleolus whereas much less, only 6%, of the nuclear NS1 was found to be associated with the nucleolus of infected MDCK cells. Whether this distribution of NSI between the nucleoplasm and the nucleolus observed after nuclear fractionation reflects the true distribution of NS, within the infected cell nucleus cannot be ascertained with certainty. It must be taken into consideration that the nucleolar purification procedure employed in the present study has been designed to eliminate loosely bound proteins but to retain all ribosomal precursor RNA species (Soeiro and Basile, 1973). Electron micrographs of nucleolar preparations purified from uninfected HeLa cells by banding in Renografin gradients reveal mainly nucleolar cores (Soeiro, unpublished experiments). Therefore, some loosely bound proteins associated with the granular portion of the nucleolus may be released in an attempt to define only those proteins which are tightly bound to the organelle. Although our results indicate that the nucleoli from infected cells retain all the ribosomal precursor RNA, we
have not examined the effect of influenza virus infection on the stability of the association of proteins, both host and viral, with the nucleolus. For example, it is possible that the more extensive inhibition of ribosomal RNA processing observed in MDCK cells after virus infection may be reflected in a less-stable association of proteins (including NSI) to the nucleolus. Consequently, the percentage of the nuclear NS, associated with nucleoli in uiuo may be higher than that found after this nuclear fractionation. However, the most important observation of the present study is that a significant portion of NS 1remains tightly bound to nucleoli even after extensive purification of this organelle. Further experiments are necessary to determine the functional role of NS1 in the nucleolus during viral infection and to decide whether the nucleolar NS1 protein is involved in the known inhibition of ribosomal RNA processing that occurs during influenza virus infection (Stephenson and Dimmock, 1974; present study). ACKNOWLEDGMENTS We thank Barbara Benson for expert technical assistance. This investigation was supported by Grants CA 08748 (RMK) and CA 10933 (RS) from the National Cancer Institute and Grant AI-11772-01 from the National Institute of Allergy and Infectious Disease (RMK). REFERENCES 1. CHOPPIN, P. W. (1969). Replication of influenza virus in a continuous cell line: high yield infective virus from cells inoculated at high multiplicity. Virology 39, 130-134. 2. CHOPPIN, P. W., and PONS, M. W. (1970). The RNAs of infective and incomplete influenza virions grown in MDBK and HeLa cells. Virology 42,603-610. 3. COMPANS, R. W., and DIMMOCK, N. J. (1969). An electron microscopic study of single-cycle infection of chick embryo fibroblasts by influenza virus. Virology 39, 499-515. 4. DIMMOCK, N. J. (1969). New virus-specific antigens in cells infected with influenza virus. Virology 39, 224-234. 5. FRASER, K. B. (1967). Immunofluorescence of abortive and complete infections by influenza virus: extraction from virus and infected cell and acidic chloroform-methanol. Virology 54, 369-383.
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7. KRUG, R. M. (1971). Influenza viral RNP’s newly synthesized during the latent period of viral growth in MDCK cells. Virology 44, 125-136. 8. KRUC, R. M. (1972). Cytoplasmic and nucleoplasmic viral RNP’s in influenza virus-infected MDCK cells. Virology 60, 103-113. 9. KRUG, R. M., and ETKIND, P. E. (1973). Cytoplasmic and nuclear virus-specific proteins in influenza virus-infected MDCK cells. Virology
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Press, New York. 13. PENMAN,S., SMITH,I., HOLTZMAN,E., and GREENBERG,H. (1966). RNA metabolism in the HeLa cell nucleus and nucleolus. Nat. Cancer Inst. Monogr.
23,489~512.
14. SKEHEL, J. J. (1972). Polypeptide synthesis in influenza virus-infected cells. Virology 49, 23-36.
15. SOEIRO,R., and BASILE,C. (1973). Non-ribosomal 56, 334-348. nucleolar proteins in HeLa cells. J. Mol. Biol. 79, 507-519. 10. LAZAROW~~Z, S. G., COMPANS,R. W., and CHOP16. STEPHENSON, J. R., and DIMMOCK,N. J. (1974). PIN, P. W. (1971). Influenza virus structural and non-structural proteins in infected cells Effect of influenza virus on cellular RNA synthesis. In “Negative Strand Viruses” (B. W. J. and their plasma membranes. Virology 46, Mahy and R. D. Barry, eds.), Academic Press, 830-843. 11. MORGAN,C., Hsu, K. C., RIFKIND,R. A., KNOX, A. New York, in press. W., and ROSE,H. M. (1961). The application of 17. TAYLOR,J. M., HAMPSON,A. W., and WHITE,D. 0. ferritin conjugated antibody to electron micro(1969). The polypeptides of influenza virus. I. scopic studies of influenza virus in infected Cytoplasmic synthesis and nuclear accumulacells. II. The interior of the cell. J. Ezp. Med. tion. Virology 39, 419-425. 114. 833-836. 18. TA~OR, J. M., HAMPSON,A. W., LAYTON,J. E., 12. PENMAN,S. (1969). Preparation of purified nuclei and WHITE, D. 0. (1970). The polypeptides of and nucleoli from mammalian cells. In “Funinfluenza virus. IV. An analysis of nuclear damental Techniques in Virology” (K. Habel accumulation. Virology 42, 744-752. and N. P. Salzman, eds.), pp. 35-48. Academic
