VIROLOGY
67,209-218 (19’75)
Influenza
Virus Messenger MARCEL
Department
of Virology,
The Public
Health
Ribonucleoprotein
l
W. PONS
Research Institute New York 10016
Accepted
May
of The
City
of New
York,
Inc., New
York,
6, 1975
Evidence is presented which indicates that influenza virus messenger RNA exists on polysomes, not as free RNA but in the form of a ribonucleoprotein (RNP). The data make it appear extremely unlikely that the presence of RNP on polysomes is an artifact of the extraction procedure. The significance of these findings for translational events is discussed. INTRODUCTION
A previous report from this laboratory dealt with the isolation of influenza virus ribonucleoprotein (RNP) and polysomes from infected cells (Pans, 1972), and it was shown that most, if not all, of the virusspecific messenger RNA (mRNA) associated with polysomes of infected cells was complementary (vcRNA) to the RNA found in virions (vRNA). This work has since been confirmed and extended by Etkind and Krug (1974) who showed that all of the virus-specific mRNA associated with polysomes is vcRNA. Pons (1972) also showed that two virusspecified polypeptides, the nucleocapsid (NP) (Pons et al., 1969) and another polypeptide with a molecular weight of 25,000 which is either nonstructural (Lazarowitz et al., 1971) or makes the membrane protein (M) (Gregoriades, 1973), were associated with polysomes. The function of these two polypeptides on polysomes was unclear, but Krug and Etkind (1973) showed that the 25,000-MW polypeptide was only weakly adsorbed to ribosomes, and they suggested this association was nonspecific. We confirmed these results and also examined the relationship of the NP polypeptide to polysomes of infected cells. It will be ‘This work was supported in part by Grant No. AI-10256 of the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service. Copyright 0 1975by Academic Press,Inc. All rights of reproduction in any form reserved.
209
shown that the NP is firmly associated with vcRNA isolated from polysomes and we could find little to no free vcRNA on polysomes. We conclude that the influenza messenger is not free RNA but is an RNP. The significance of these findings to translational events will be discussed. MATERIALS
AND METHODS
Isolation of polysomes from chick embryo fibroblast monolayers (CFM). Pri-
mary CFM were prepared and used for plaque assay or virus production as previously described (Simpson and Hirst, 1961). The isolation of polysomes and RNP has been reported (Pons, 1972), but, since several modifications have been made, the procedure is described in detail here. Three-day-old primary cultures of CFM on 90-mm plastic petri dishes were infected with the WSN strain of influenza virus at multiplicities of infection (m.0.i.) of 1.0-1.5 PFU/cell. After 0.5 hr at room temperature the cells were overlaid with 5 ml of prewarmed FOEE (Simpson and Hirst, 1961) and incubated at 39” in a 5% CO, atmosphere (zero time). After a suitable incubation period the plates were removed from the incubator and processed in a 41” warm-room to minimize cooling. The FOEE overlay was replaced with 5 ml of prewarmed solution containing radioisotope and the plates returned to the 39” incubator. When pulsing for short intervals
210
MARCEL
of 15-30 min the labeling medium was Earle’s saline; for longer labeling intervals a mixture of Earle’s saline:FOEE (4: 1) was used. We subsequently found that better polysome labeling was achieved using this latter medium than Earle’s saline alone, no matter what the pulse interval was. The details of labeling, such as type of isotope, its concentration, pulse interval etc., will be given for each individual experiment. At the end of the labeling period the medium was removed and the cells washed once with 5 ml of ice-cold MSB (Heywood et al., 1967; Zimmerman and Fried, 1971) (0.25 M KCl, 0.01 M MgCl, in 0.01 M Tris-HCl, pH 7.4). This, and all subsequent operations, were performed at O-4”. The cells were scraped off the plates into 0.5 ml of MSB containing 50 pg/ml of heparin (Hep-MSB) (Grubman and Summers, 1973), homogenized with ten strokes of a tight-fitting Dounce homogenizer, treated with 2% Nonidet P-40 (NP-40) and, after 3 min on ice, centrifuged at 10,000 g for 10 min in a Sorvall RCB-B centrifuge. The supernatant fluid was removed and layered over a discontinuous gradient consisting of 2.5 ml of 2.0 M sucrose and 4.0 ml of 0.5 M sucrose in Hep-MSB, the latter solution also containing 0.2% NP-40. This is a modification of the technique of Kennedy (1972). The discontinuous sucrose gradients were centrifuged in a Spinco 50 Ti rotor at 50,060 rpm for 70 min at 4” in a Spinco L2-65B ultracentrifuge. Monosomes and polysomes were pelleted by this procedure while other components, such as the RNP, collected at the 2.0-0.5 M sucrose interface or remained in the upper portions of the discontinuous gradient. The translucent polysome pellets were gently resuspended in 2.0-3.0 ml of HepMSB and, after standing on ice for approximately 1 hr to ensure good dispersion, applied to 34-ml linear 15-40% sucrose gradients in Hep-MSB. All sucrose solutions were pretreated with diethylpyrocarbonate (diethyloxydiformate, Eastman Kodak Co.) to inactivate RNase by the method of Kumar and Lindberg (1972). After centrifugation at 10,000 rpm for 16.5 hr at 4’ in a Spinco SW 27 rotor, fractions
W. PONS
of 1 ml were collected from the bottoms of the tubes after scanning the OD,,,,, in a Gilford recording spectrophotometer. Radioactivity was usually determined by counting O.l-ml aliquots in a dioxanebased scintillation fluid in a NuclearChicago Mark II liquid scintillation counter. When necessary, polysomes were concentrated from these sucrose gradients by pooling the appropriate fractions and centrifuging at 50,000 rpm for 80 min at 4” in a Spinco 50 Ti rotor. The small clear pellets were further processed as described in the individual experiments. Phenol:chloroform-SDS extraction of polysomes. RNA extractions were carried
out as previously described (Pons, 1972) with the following exception: A 1: 1 mixture of phenol:chloroform was used rather than phenol alone (Perry and Kelly, 1968). Polyacrylamide-gel electrophoresis (PAGE) of RNA and proteins. PAGE
of RNA and proteins was described previously (Pons, 1972). Isotopes. [5-3H]uridine (sp act, 2-5 Ci/ mmole) was obtained from Amersham/ Searle Corporation. A mixture of 3H- or “C-labeled amino acids was obtained from the New England Nuclear Corporation. Annealing of RNA. Annealing experiments were carried out as described by Robinson (1970) and Pons (1971). In every case determinations were made in triplicate using a minimum of 1000 cpm for each sample of the “infected” sets. In several cases uninfected controls contained less than this minimum, but, since no annealing of vRNA to host cell RNA was ever detected, this was not a serious drawback. In every case, enzyme-treated samples were treated with 10 &ml of pancreatic RNase and 1 pglml of RNase T, for 10 min at 37”. The samples were precipitated with an equal volume of 20% TCA, filtered through glass-fiber filter papers, washed with 20 ml of 1% TCA, and counted in a dioxane-based scintillation fluid. No corrections were applied to these data; the values of percent resistance of infected-cell RNA are not corrected for uninfected control cell RNA counts.
INFLUENZA
MESSENGER
211
RNP
Lastly, in every case 15-20-fold excesses of vRNA were added to ensure complete saturation of the system with this RNA.
H x---x
INF CONT.
:’
RESULTS
Nature of the Messenger RNA Associated with Polysomes
Previous results indicated that the major viral polypeptides associated with polysomes were the NP and the 25,000-dalton polypeptide (NS, or M) (Pons, 1972). Krug and Etkind (1973) showed that the latter is only weakly bound to ribosomal subunits and is easily dissociable by washing with hypertonic salt solutions. The function of NP on polysomes is still unknown. Here, evidence is presented which shows that NP is firmly associated with vcRNA isolated from polysomes and, since there does not appear to be any free vcRNA present, it is concluded that the mRNA of influenza virus exists as an RNP. Control and infected CFM were labeled from 1.5-3 hr after infection with 15 &i/ml of [5-3H]uridine after which time cells were harvested and processed as described under Materials and Methods. Figure 1 shows the OD,,,,, and radioactivity profiles of infected and control cell homogenates. The polysome regions of the two gradients shown in Fig. 1 (fractions 4-15) were pooled and centrifuged to pellet the polysomes as described in Fig. 2, where it will be seen that the resuspended polysomes were then disrupted by a combination of puromycin (Blobel, 1971) and EDTA treatment. This combined treatment increased the yield (over that obtained with puromycin or EDTA alone) of material sedimenting in the RNP region (55-22 S) of the gradient shown in Fig. 2 by reducing to almost nil the amount of material which was pelleted due to incomplete dissociation of polysomes. Similarly, the presence of 1 M urea in the gradients was critical, since in its absence virtually all of the RNP was pelleted. This phenomenon has been discussed elsewhere (Pons et al., 1969, 1971). The preparation of the RNP marker from virus particles is also given in the legend to Fig. 2. It will be noted (Fig. 2) that very little
I IO FRACTION
I 20 No.
FIG. 1. Incorporation of [5-3H]uridine into polysomes after a 1.5-hr pulse-labeling interval. Ten 3-day-old CFM were infected with 1.0 PFU of influenza virus/cell while another 10 CFM served as controls. The cultures were labeled with 15 &i/ml of [5-sH]uridine in Earle’s:FOEE (4:l) from 1.5-3 hr after infection. Polysomes were prepared and analyzed as described under Materials and Methods.
free viral RNA (which sediments with a mean sedimentation value of 18 S) appeared in the infected cell gradient (fractions 20-25), while the majority of the labeled RNA appeared in the 55-22 S region of the gradient corresponding to the area where RNP sediments. In another similar experiment, pools were made of the regions corresponding to the RNP region (55-22 S), the 21-11 S region, where free viral RNA sediments, and the 10-5 S regions of infected and control cell gradients. Each pool was phenol:chloroform-SDS extracted and annealed (see Materials and Methods) with 20 pg of vRNA. The percentage of RNase-resistant material formed in each case was 80% (55-22 S), 9% (21-11 S), and 22% (lo-5 S) (Table 1). The control cell pools all annealed less than 0.2% to vRNA. The three controls for each infected group, selfan-
212
MARCEL
W. PONS
a--*
INF. CONT.
o-.-a
RNP
ZOOO~ CPM 0.1
1000.
FIG. 2. Isolation of RNP from polysomes. Fractions 4-15 of the two gradients shown in Fig. 1 were pooled and centrifuged for 80 min at 50,006 rpm in a 50 Ti rotor in a Spinco ultracentrifuge. The pellets were resuspended in 1.8 ml of STE-urea buffer. In addition, a third 15-40% sucrose gradient (not shown in Fig. 1) served as a source of viral RNP which was used as a sedimentation marker. [3H]uridine-labeled WSN was added to uninfected cells. The cells were homogenized, treated with 2.0% NP-40, and analyzed on gradients in parallel with those shown in Fig. 1. The RNP from this gradient was concentrated by acetate precipitation (Pons, 1971), collected by centrifugation, resuspended in 1.8 ml of STE-urea buffer and processed further along with the cellular-derived materials. After standing for 3 hr at O”, 0.2 ml of a 30 mM solution of puromycin was added to each of the three samples and the mixtures incubated for 15 min at 0” and 15 min at 37”. The samples were then centrifuged through 34-ml lo-35% glycerol gradients in STE-urea for 16.5 hr at 5” at 18,000 rpm in an SW 27 rotor. Samples of approximately 1 ml each were collected and O.I-ml aliquots counted in a dioxane-based scintillation fluid.
unheated, nealed, and heated-quick cooled, the latter two with added vRNA present, showed only a small amount, if any, vRNA present in the polysome preparation. The enzyme-resistant RNA present in unheated samples, which was diminished by heating and quick cooling, may represent small amounts of doublestranded RNA (or RNP). The lighter material (lo-5 S) may represent 1) degraded RNP since it also appears in the control RNP marker gradient, 2) free vcRNA which may be partially degraded, or 3)
uncompleted nascent strands of vcRNA. A subsequent report will show that the latter explanation is the most likely one. Lastly, we have already shown that CsCl equilibrium sedimentation centrifugation of gluteraldehyde-fixed material obtained from polysomes had a buoyant density equal to that of viral RNP with no evidence of free RNA being present (Pons, 1972). Similar treatment of the 55-22 S RNP produced similar results, i.e., a major peak at a density of 1.34 gm cm- 3 and no evidence of free RNA.
INFLUENZAMESSENGERRNP In order to purify further and identify the viral RNA associated with polysomes and compare it to RNA obtained from the marker RNP, the marker RNP and corresponding regions of the infected and control cell gradients were pooled and RNA extracted as described in the legend to Fig. 3. As shown in Fig. 3, the RNA extracted from the marker RNP sedimented with an average sedimentation coefficient of 18 S, and this region and corresponding regions from the other two gradients were pooled, the RNA precipitated and subjected to PAGE as described in the legend to Fig. 4. Although only two of the RNA peaks seen in the electropherogram (Fig. 4) of infected-cell RNA can be identified as having
mobilities identical to vRNA, it is likely that all are present since cells infected under the conditions employed here will produce new virus particles. Discrete peaks of smaller RNA segments are not discernible due either to some breakdown of the virus-specific RNA or to the presence of incomplete larger RNA segments (see above). Clearly, there is little to no RNA present in the infected-cell electropherogram smaller than the smallest vRNA segment and only one type which is larger, the 18 S ribosomal RNA. In another similar experiment, using a 90-min pulse-label, polysomal RNA was isolated as described, but instead of PAGE analysis it was annealed to 15 Fg of vRNA.
TABLE SUMMARY
OF ANNEALING
Source
of infected-cell
DATA OBTAINED
Fig. 1, pool of fractions 4-15 Selfannealed With 20 pg of vRNA added
Fig. 2, pools of 55-22 S Selfannealed With 20 pg vRNA added
Fig. 2, pools of 21-11 S Selfannealed With 20 fig of vRNA added
Fig. 2, pools of 10-5 S Selfannealed With 20 pg of vRNA
added
Fig. 3, pools of 15-22 S Selfannealed With 15 pg of vRNA added
Fig. 3, pools of 9-15 S Selfannealed With 15 pg of vRNA added
DURING
1
ISOLATION
RNA
OF
RNA
FROM POLYSOME-ASSOCIATED + RNase
Annealed Heated-quick Unheated
Annealed Heated-quick Unheated
Annealed Heated-quick Unheated
Annealed Heated-quick Unheated
Annealed Heated-quick Unheated
Annealed Heated-quick Unheated
a All determinations were done in triplicate, control, uninfected cell RNA’s are not shown
cooled
- RNase
Percent resistant
15,876 16,091 15,987 17,803
4.5 63.2
224 8,274 64 303
10,644 10,343 10,728 11,323
2.1 80.0 0.6 2.6
12 133 10 21
1,482 1,388 1,517
42 356 7 51
1,755 1,620 1,674 1,876
2.4 22.0 0.4 2.7
33 2,224 10 41
2,499
1.3 83.0 0.4 1.5
25 1,876 8 38
2,008 1,947 2,087 2,308
714 10,170 143
923
cooled
cooled
cooled
cooled
cooled
RNP”
and data reported are uncorrected but percent resistant values never
1,409
2,680 2,503 2,707
0.9 5.2
0.88 9.0 0.72 1.4
1.3
94.6 0.4 1.6
values. Data for annealing exceeded 0.25%.
of
214
MARCEL
I,000
W. PONS
-INF *-*CONT. W.-Q RNP
3 !i ! !;
i
,P y
:, I
i
4
FRACTION
NO.
3. Gradient sedimentation of RNA obtained from RNP isolated from polysomes of infected and control cells. Fractions 7-15 of the three gradients shown in Fig. 2 were pooled and the RNA extracted by diluting with an equal volume of SLA (0.5% SDS, 0.14 M LiCl, in 0.01 M acetate buffer, pH 4.9) and adding 0.05 ml of 2-mercaptoethanol and 10 ml of phenohchloroform (1:l). After precipitation of the extracted RNA with two volumes of 95% ethanol, the RNA’s were dissolved in 2.0 ml of STE buffer and analyzed on 34-ml linear 10-35s glycerol gradients in STE-urea by centrifuging at 24,006 rpm, 16.5 hr, 4”, in an SW 27 rotor. Fractions of approximately 1 ml were collected and O.l-ml aliquots counted in a dioxane-based scintillation fluid. FIG.
Table 1 summarizes the annealing data already referred to at different stages in the isolation of the RNP and RNA from infected cells. In most cases the RNA was not that shown in the figure but from another identical experiment. In the last set (designated in Table 1 as Fig. 3, 15-22 and 9-15 S) annealing to vRNA generated 94.6% enzyme-resistant RNA for the 9-15 S RNA and 83% for the 15-22 S RNA. The latter is lower presumably due to the presence of labeled host 18 S RNA. Is the Protein Associated with Viral mRNA Present as the Result of a Nonspecific Protein-RNA Interaction? It is possible that during the isolation of polysomes mRNA nonspecifically becomes associated with protein (see Baltimore and Huang, (1970) for a comparison to the HeLa cell-poliovirus system). Two types of experiments have been carried out to test this possibility. In the first type of experiment, either [5-3H]uridine-labeled, phenol-SDS extracted vRNA obtained from virions or similarly labeled vcRNA pre-
pared by phenol:chloroform-SDS extraction of isolated polysomes labeled from 1.5-3 hr p.i. (that is, material prepared and isolated from the 55-22 S region of gradients such as those shown in Fig. 2) were added to intact, unlabeled, infected and control cells which were then homogenized in Hep-MSB buffer. In both cases a minimum of lo5 cpm was added to each of the groups. The mixtures, clarified by low speed centrifugation, were analyzed further by centrifugation on 15-40s sucrose gradients to isolate polysomes. In all cases, less than 1% of the added label sedimented in the polysome regions of these gradients while the major portion of labeled RNA sedimented at 18 S. In another similar experiment, the clarified supernatant fluids were analyzed not on polysome gradients but on lo-35% glycerol gradients in STE-urea buffer (23,000 rpm, 16 hr, 5” in an SW 27 Spinco rotor). Although 5-12s of the labeled RNA was found at the top of the gradients, indicating breakdown due to RNase or mechanical shearing during homogenization, the remaining RNA sedi-
INFLUENZA
MESSENGER
mented as did free viral RNA (18 S). In no experiments was there any evidence for RNP being formed. Since Scholtissek and Becht (1971) showed that NP was extremely efficient in binding free influenza v- and vcRNA’s, our results suggest that there is very little free NP polypeptide in the infected cell (see Discussion). As shown previously (Pons, 1972), CsCl equilibrium sedimentation analysis rather than velocity sedimentation analyses as described above also failed to show any sign of RNP being formed by the nonspecific association of vRNA with protein. The second type of experiment involved a different approach. Infected and control CFM were labeled with 20 &i/ml of 3Hlabeled amino acids from 2.25-3.25 hr p.i. Polysomes were isolated as described under Fig. 5, treated with puromycin in STEurea buffer, and analyzed on gradients (see legend to Fig. 2). Fractions lo-17 and 25-29 (Fig. 5) were pooled, precipitated with TCA and prepared for PAGE as previously described (Pons et al., 1969). Although not shown here, fractions 25-29 contained only the 25,000-MW protein. As shown in Fig. 6, fractions lo-17 contained, predominantly, a polypeptide with an electrophoretic mobility identical to that of the NP. The smaller peak represented material that migrated just a bit more rapidly than the M protein. The control was not subjected to PAGE since too little labeled material was present to make the analysis feasible. Previously, we showed that PAGE of isolated polysomes indicated the presence of several polypeptides, the two major ones being the NP and the 25,000-MW polypeptides (Pans, 1972). In the experiment described above, we treated the isolated polysomes with puromycin and urea, isolated RNP from a second gradient, and found, essentially, only the NP present in those regions of gradients where the presumed mRNA (or RNP) was sedimenting. This confirmed the observation of Krug and Etkind (1973) that the 25,000-MW polypeptide was not firmly associated with polysomes or ribosomes. The smaller peak at fraction 97 of Fig. 6 could be either the M or the NS, polypeptide (Lazarowitz et al., 1971). It is impossi-
215
RNP
-
+
CONTROL
200..
INFECTED
CPM
..
r
RNP-RNA
I
P
1,000-
25
50 FRACTION
75 NO.
FIG. 4. PAGE of RNA isolated from RNP associated with polysomes. Fractions 16-21 from each of the gradients shown in Fig. 3 were pooled and precipitated with two volumes of ethanol. The precipitates were collected by centrifugation and subjected to PAGE as described under Materials and Methods.
ble to make this distinction without coelectrophoresis of “C-labeled virion polypeptides and SH-labeled polypeptides isolated from polysomes as described here. This was not feasible, since so little material was present we could not locate it when using a double label. The NP polypeptide isolated from virions and polysomes, as described above, migrate identically when coelectrophoresed. DISCUSSION
The experiments in this report deal with the nature of influenza mRNA on polysomes. We believe that the majority of viral mRNA on polysomes is in the form of RNP. The major objection to this conclusion was that proteinization of RNA might
216
MARCEL
*---I
INE CONT.
28s 1
W. PONS
I8 s 1
FIG. 5. [3H]amino acid-labeled material associated with polysomes of infected and control cells. Ten CFM were infected with 1.0 PFU of virus/cell and 10 were uninfected. At 2.25-3.25 hr p.i. the cultures were pulse-labeled with 20 &i/ml of [SH]amino acids in Earle’s saline. Polysomes were prepared and analyzed as described under Materials and Methods. The polysome regions of the gradients were pooled, pelleted, treated with puromycin and analyzed on glycerol gradients as described under Fig. 2.
have occurred after extraction. We feel this is not the case for reasons already given and summarized here: 1) No evidence of proteinization was found when free labeled viral RNA or vcRNA was added to intact infected and control cultures, the cells treated as described and analyzed for the presence of labeled RNP. 2) Only one polypeptide was found associated with mRNA isolated with polysomes, the NP. If proteinization were nonspecific, one might expect to find other proteins being adsorbed also (Baltimore and Huang, 1970). Since only NP was found associated with mRNA, this nonspecific reaction could occur only if there were large pools of free NP within the cell at the time of extraction. This was not the case. Infected cells were pulse-labeled with 8H-labeled amino acids and the entire cell homogenate (minus nuclei) examined on polysome gradients. Pools of various regions of the gradient were made and examined by
PAGE. There was very little free NP at the top of the gradient where it would be expected (Pons, unpublished results). It is not surprising that little free NP is found within the cell, given its high affinity for RNA (Scholtissek and Becht, 1971). Proteinization of viral RNA most likely occurs as the RNA is being synthesized or immediately afterwards. Another possible objection to the conclusion that viral messenger is RNP arises if vcRNA, which is bound to ribosomes, i.e., free mRNA on polysomes, is preferentially removed or degraded during the initial steps of the isolation. As described in the Results dealing with Fig. 2, 11% of the labeled RNA applied to gradients sedimented at 5-10 S and, of this, 22%, or 2% of the total, annealed to virion RNA. This 2% represented either free vcRNA on polysomes or degraded vcRNP. It is impossible to determine which at this time but, since it represented only 2%, a small amount of
INFLUENZA INFECTED
MESSENGER
+
FIG. 6. PAGE of [3H]amino acid-labeled polypeptides isolated from gradients shown in Fig. 5. Fractions 10-17 of the gradients shown in Fig. 5 were pooled, precipitated with 10% TCA and prepared and analyzed by PAGE as previously described (Pans et al., 1969). The upper panel is the electropherogram of material obtained from infected cells; the lower panel represents the electropherogram obtained from SDSdisrupted [aH]amino acid-labeled purified virions.
the total RNA associated with polysomes, it is unlikely it plays a significant role in virus translational processes. The suggestion that polysomal mRNA occurs as RNP complexes is not new. Other workers (Spirin and Nemer, 1965; Perry and Kelley, 1968; Henshaw, 1968; Kumar and Lindberg, 1972; Bryan and Hayashi, 1973; see Gross (1968) and Spirin (1969) for reviews) have shown that mRNA of eucaryotic cells appears to be associated with proteins. Also, Bukrinskaya et al. (1969) suggested that Sendai virus RNP is associated with polysomes of infected Ehrlich ascites tumor cells. The function of these mRNP’s is unknown and, in some cases, they could have been artifacts of extraction procedures (Baltimore and Huang, 1970). Indeed, it has been suggested that all claims of isolation of mRNP’s are artifactual due to nonspecific association of mRNA with proteins. Here, we have taken
217
RNP
steps to avoid nonspecific RNA-protein interactions by extracting cells in hypertonic buffer in the presence of Nonidet P-40, a nonionic detergent. In addition, our conclusion that the mRNP is not an artifact of extraction is bolstered by the fact that only one polypeptide (the NP) is found associated with mRNA. The role of mRNA-protein complexes or mRNP (also called “informasomes” by Spirin (1969) is unclear. Spirin (1969) suggested that mRNP may be involved in translational controls, in the transport of mRNA from the nucleus or in the stabilization of the labile mRNA. The mode of association of mRNP with polysomes during translation is unknown. The influenza virus system offers great potential for the study of the relationship of mRNP with ribosomes during translation since the complex is stable and easily isolated in a relatively pure form by the methods described here. ACKNOWLEDGMENT lent
The author technical
thanks Ellen assistance.
Goldstein
for her
excel-
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