Studies on the relationship between 5′ leader sequences and initiation of translation of adenovirus 2 and simian virus 40 late mRNAs

VIROLOGY

101, 363-375 (1980)

Studies on the Relationship between 5’ Leader Sequences and Initiation of Translation of Adenovirus 2 and Simian Virus 40 Late mRNAs DEBRA J. WOLGEMUTH,’

HO-YUN YU, AND MING-TA HSU

The Rockefeller University, Molmulur Cell Biology Department, New York, New York 10021 Accepted November 13, 1979 The question of whether or not 5’ leader sequences might confer characteristic translational properties to the mRNAs to which they are spliced was studied using two complementary experimental situations with respect to the composition of mRNA molecules. Late in adenovirus type 2 (Ad-2) infection of human cells, a common tripartite 5’ leader sequence is spliced to mRNA sequences coding for at least 13 different late viral proteins. This results in different mRNAs with identical 5’ leader sequences. In the second experimental system, identical coding sequences are spliced to different 5’ leader sequences with different caps. Wild-type SV40 VP1 mRNA (16 S) contains a 5’ leader sequence coded for at position 72-76 on the SV40 genome; in the SV40 mutant dl-808 part of the sequences in this region, including the sequence coding for cap, is deleted. The relative ability of these mRNAs to direct incorporation of radioactive amino acids into their respective polypeptide products was assayed in. vivo under conditions of hypertonic initiation block (HIB) or in the presence of low concentrations of cycloheximide. Messenger RNAs with identical 5’ leader sequences were observed to exhibit very different responses in both experimental situations. Conversely, similar responses to HIH were observed for VP1 mRNAs with different 5’ leader sequences. It thus appears that the presence of specific sequences per se between the 5’ cap and the initiation codon does not alone confer characteristic initiation properties as measured indirectly by perturbation of the in viva environment.

1979). Further, the primary sequence structure at the points where splicing occurs and Although 2 years has passed since the recently of the entire leader sequences in initial observation of spliced leader se- viral mRNAs has received considerable atquences in viral mRNAs (Chow et al., 1977; tention (Ziff and Evans, 1978; Lockard et Berget et al., 197’7; Klessig, 1977; Gelinas al., 1979; Ghosh et al., 1978; Zain et al., and Roberts, 1977; Aloni et al., 1977; Celma 1979; Akusjarvi and Peterson, 1979). As et al., 1977; Lavi and Groner, 1977; Hsu leader sequences are 5’ components of the and Ford, 1977) very little is known about mRNA, immediately adjacent to the cap what role, if any, these sequences play in and 5’ to the initiator codons, we chose to the regulation of expression of the viral pro- address the question of how the leader teins for which such composite mRNAs code. sequences function in the translational Several investigators have focused on process. We wished to determine if these determining how these viral mRNAs are sequences might confer characteristic transgenerated-elucidating the steps in process- lational properties, particularly with reing and searching for the possible enzymes spect to initiation, to the messages to which involved (Blanchard et al., 1978; Chow and they are spliced. Broker, 1978; Horowitz et al., 1978; Lai et Two experimental systems were selected al., 1978; Chow et al., 1979; Manley et al., for these studies. In the first system, adenovirus type 2 (Ad-2) messenger RNAs produced late in Ad-2 infection of HeLa cells ’ To whom reprint requests should be addressed. INTRODUCTION

363

0042-6822/80/040363-13$02.00/O Copyright All rights

0 1980 by Academic Resa, Inc. of reproduction in any form reserved.

364

WOLGEMUTH,YU,ANDHSU

were examined. The majority of the late cytoplasmic mRNAs, coding for a variety of different proteins, are formed from a common major late transcript (Evans et al., 197’7)and contain a common capped tripartite leader sequence (Berget et al., 19’77;Chow et al., 1977; Gelinas and Roberts, 1977; Klessig, 1977). Thus, the same leader sequence is spliced to at least 13 or 14 (Flint, 1977; Nevins and Darnell, 1978) different coding sequences. In the second experimental system, a converse situation with respect to mRNA architecture was provided: identical coding sequences are preceded by different caps and leader sequences. The mRNAs studied were the late 16 S mRNAs coding for VP1 capsid protein in wild-type simian virus 40 (strain SVS) and in a viable deletion mutant, dl808 (Mertz and Berg, 1974), infected CV-1 cells. Wild-type SV40 16 S mRNA has been shown to contain a spliced leader sequence at the 5’ end of the coding sequences for VP1 (Aloni et al., 1977; Celma et al., 1977; Hsu and Ford, 1977; Lavi and Groner, 1977; Ghosh et al., 1978; Haegeman and Fiers, 1978). In dl-808, most of the DNA sequences coding for the 16 S mRNA leader sequence are deleted and the resulting 16 S mRNA coding for VP1 contains a new cap and leader sequence. (Johnson, 1979; J. Mertz, personal communication). We investigated the relative initiation properties of these various mRNAs by using in vivo systems in which initiation efficiency can be probed by conditions of hypertonicity (Saborio et al., 1974) and by partial inhibition of elongation (Lodish, 1971; David, 1976). Differences in sensitivity to hypertonic initiation block (HIB) have been demonstrated between viral and cellular mRNAs (Opperman and Koch, 1976; Nuss and Koch, 1976a; Cherney and Wilhelm, 1979), among cellular mRNAs (Nuss and Koch, 1976b), and among viral mRNAs (Tershak, 1978):the most efficiently initiating messages being most resistant to increasing tonicity (Saborio et al., 1974). As the rate of elongation is thought to be the same for viral and cellular mRNAs (Jen et al., 1978), the use of cycloheximide, an elongation inhibitor, at

concentrations which only partially inhibit protein synthesis permits less efficiently initiating messages to “catch up” to the efficient initiators. With initiation no longer limiting, any differences among mRNAs are reflected by the fasterinitiating messages being most affected compared to no cycloheximide. Although this approach is indirect, it nonetheless has the advantage of measuring in vivo responses. Using these two in vivo perturbations of protein synthesis, we were able to detect clear differences in initiation efficiency among mRNAs which contained identical 5’ leader sequences and conversely, similar responses to HIB among messages with identical coding sequences but with different leader sequences. The implication of these results with respect to the involvement of 5’ leader sequences in regulation of translation is discussed. MATERIALSANDMETHODS

Cells and Viruses HeLa cells, grown in suspension culture, were infected with adenovirus 2 at 2000 particles/cell (Wall et al., 1972) and utilized at 18-24 and 44 hr postinfection. CV-1 cells were grown as confluent monolayers and were infected with SVS strain of SV40 (Takemoto et aZ., 1966) or dl-808 (Mertz and Berg, 1974) at input multiplicity of about 5-10 PFU/cell as previously described (Ford and Hsu, 1978). Hypertonic Initiation

Block

Adenovirus 2: HeLa cells. Procedures were modified from Opperman and Koch (1976) and were carried out at 37” unless otherwise noted. Ad-2 infected HeLa cells at 5 X lo5 cells/ml were spun out of suspension, resuspended in methionine-free medium (Joklik modified MEM, Gibco) containing 2% dialyzed fetal calf serum (dFCS), and incubated for 10 min. Aliquots of cells were removed to sterile centrifuge tubes, centrifuged, resuspended to 4 x lo6 cells/ml in the Meth(-) +2% dFCS medium containing 0 to 200 mM excess NaCl, and incubated 10 min. Then 2 x lo6 cells (0.5 ml) were removed to 1.5-ml Eppen-

5’ LEADERS

AND INITIATION

dorf centrifuge tubes, [35S]-methionine (NEN sp act 2 500 Cilmmol) was added to 50-100 &i/ml, and the incubation was carried out for 12- 15 min in a shaking water bath. The tubes were removed to ice and the cells pelleted by centrifugation for 15 set in an Eppendorf table-top centrifuge at 0”. The cells were resuspended in 200 ~1 of 50 mM Tris-HCl, pH 7.4 and 1 mM phenylmethyl sulfonyl fluoride (PMSF, generously supplied by Dr. Georgio Vidali). Aliquots were taken from this suspension for TCA precipitation -or processed for gel analysis. SV,$O:CV-1cells. Forty-eight hours postinfection, the infected cells in 60-mm dishes were washed with 5 ml warm medium without lysine (Gibco) and supplemented with 2% dFCS and different amounts of NaCl as indicated in the text. After further incubation for 10 min with 5 ml of warm medium as described above, the cells were pulse-labeled with 100 &i of rH]lysine (Amersham, 93 Ci/mmol) in 1 ml of medium for 1 hr. At the end of the labeling period, the plates were transferred to ice and the radioactive medium was replaced by ice-cold TD buffer (0.25 mM Tris-HCl, pH 7.4, 0.136 M NaCl, ‘7 mM KCl, 0.7 mM Na,HPO,. The cells were scraped from the plates with the aid of a rubber policeman and pelleted by centrifugation. The cells were resuspended in 0.5 ml of 50 mM Tris pH 7.4, 0.5 mM PMSF, and divided into 50-~1 aliquots. Cycloheximide Inhibition 2: HeL,a Cells

of Adenovirus

Experimental procedures were modified from Jen et al. (1978) and all manipulations were performed at 37” unless noted. Aliquots of Ad-2 infected HeLa cells, 5 x lo5 cells/ml, at 22 hr p.i. were removed to small sterile beakers with stirring bars, adjusted to appropriate concentrations of cycloheximide (0 to 10 a), and incubated for 20 to 30 min. Then 4 x lo6 cells were removed to sterile 15-ml centrifuge tubes, spun down, and resuspended in 1.0 ml of Meth(-): 2% dFCS medium containing the appropriate concentration of cycloheximide. Aliquots 0.5 ml, of cells were removed to

OF TRANSLATION

365

sterile 1.5-ml Eppendorf centrifuge tubes, brought to 100 Ci/ml r5S]methionine (NEN sp. act 2 500 Ci/mmol), and incubated with shaking for 30 min. The reactions were terminated and processed as described for the HIB experiment with Ad2:HeLa cells. Gel Electrophoresis Ad-2:HeLa. Aliquots, 50 ~1 of the TrisHCl:PMSF cell suspension (5 x lo5 cells) or purified Ad-2 virions in 20 ~1 of Tris-HCl: PMSF buffer were brought to 2% SDS and 0.28 M 2-mercaptoethanol and boiled for 10 min. Then 13% polyacrylamide (30% acrylamide: 0.8% bisacrylamide) gels with 5% stacking gels were run at 4” according to Laemmli (1970). Gels were photographed and then assayed for radioactivity by slicing into l-mm fractions (-185 slices), dissolving in 0.5 ml Protosol, and counting in toluene-based scintillant. SV40:CV-1. SV40 or dl-808 infected cells prepared as described above were adjusted to 2% SDS, 0.28 M mercaptoethanol, and boiled for 10 min. Samples were analyzed in 12% SDS-polyacrylamide gel with 5% stacking gel as described by Laemmeli (1970). Quantitation

of Viral Proteins

With the aid of a computer, total counts per minute per sample and the percentage of total counts per minute per sample represented by each gel slice were obtained. The percentage of total r5S]methionine (Ad-2: HeLa) or rH]lysine (SV4O:CV-1) incorporated per sample represented by a specific viral peak was calculated by planimetry of computer-generated plots of percentage of total counts per minute versus gel slice number (Ad-2:HeLa) or by summing the percentage of total counts per minute contained in individual peaks (SV4O:CV-1). RESULTS

Late mRNAs in Ad 2 Infected HeLa Cells The addition of NaCl to suspension cultures of Ad 2 infected HeLa cells resulted

366

WOLGEMUTH,

in an overall decrease in [35S]methionine incorporation into TCA-precipitable counts. A range of excess NaCl concentrations from 0 to 200 mil4 consistently resulted in a decrease in total protein synthesis from 100% (control) to
FIG. 1. Effect of hypertonic medium on the incorporation of [s5S]methionine into total proteins of Ad-2 infected HeLa cells. In this experiment, 22 hr postinfection with Ad-2 at 2000 particles/cell, HeLa cells were concentrated to 4 x lo8 cells in methioninefree media and labeled for 13 min with 100 &i/ml of P5S]methionine (sp act 2 500 Ci/mol) as described under Materials and Methods in the presence of excess NaCl concentrations noted on the abscissa. Aliquots, 50 ~1, were subjected to polyacrylamide gel electrophoresis and incorporated radioactivity was determined by scintillation counting of gel slices. The total cpm recovered at 0 m&f excess NaCl (which was 5.65 x 10’ cpm in this experiment) was designated the control level or 100% synthesis. The incorporation of [3JS]methionine for each NaCl concentration is expressed as a percentage of the control value (ordinate).

YU, AND HSU

a

bed

e

f

-II -III ‘:y -VI -VII

-IX FIG. 2. Polyacrylamide gel electrophoresis of polypeptides in Ad-2 infected HeLa cells. In this experiment, 20 h postinfection with Ad-2, HeLa cells were concentrated to 4 x lo6 cells/ml, and experimental procedures were as described under Materials and Methods and the legend to Fig. 1. Aliquots, 50 ~1, were run on a 13% polyacrylamide gel (Laemmli, 1970), 18 cm in length, with a 5% stacking gel, at 15 mA for 18 hr at 4”. Lanes a, b, d, e, and f contain total proteins from Ad-2 infected HeLa cells incubated in different excess NaCl concentrations. Lane c contains purified Ad-2 virions as markers. Roman numerals in the margin correspond to adenovirion proteins as designated by Weber et al. (19’77)and Anderson et al. (1973).

determined by scintillation counting of gel slices. With the aid of .a computer, the amount of radioactivity in each slice was expressed as a percentage of the total counts in each sample (one slot contained. one sample). The results of one such experiment, at 20 hr postinfection, are shown in Fig. 3 (A series). Peaks of radioactivity representing specific viral proteins were identified by comparison with Ad-2 virion markers (Fig. 2) and by their electrophoretic mobility and were assigned according to Everitt et al. (19’73)and Weber et al. (19’77). The amounts of proteins synthesized at different NaCl concentrations were plotted as percentages of control incorporation (no additional NaCl), thus permitting direct comparison of the relative sensitivity to HIB for given proteins as measured by the changes in area under a specific peak. This was also useful since the exact percentage of total protein synthesis represented by a given protein at a

5’ LEADERS

AND INITIATION

specific NaCl concentration varied from experiment to experiment (e.g., at 0 mM excess NaCl, the percentage of total 35S cpm incorporated into hexon protein ranged from 20 to 35%). Very distinctive classes of responses to HIB among the Ad-2 proteins were observed: (a) sensitive -the percentage of total counts per minute represented by these proteins decreased with increasing NaCl; (b) resistant -the percentage represented by these proteins remained about the same or decreased only slightly; and (c) highly resistant -the percentage of total counts incorporated into these proteins actually increased. An example of which proteins fell into each of the above classes is represented in Fig. 4. In this figure, the percentage of incorporation for a given protein is expressed as a percent of the control, i.e., resistant proteins would remain close to the 100% baseline while sensitive proteins would progressively decrease with increasing NaCl. The production of protein II (hexon) and also of IV (fiber) was relatively resistant to increasing NaCl while the precursor molecules for proteins VI and VII were sensitive. At the higher concentrations, peaks of radioactivity corresponding to pV1 and pVI1 were almost undetectable (Fig. 3 (A series) and data not shown). Protein V was less sensitive than pV1 and pVI1 but more sensitive than II, IV, or IX. Most dramatic was the resistance of protein IX production to HIB. This unusually high resistance was observed whether the experiments were conducted at 18-24 hr (note Fig. :3 (A series)) or 40 hrs (Fig. 4) postinfection and is of the same level of resistance to HIB recently reported by Cherney and Wilhelm (1979) for protein IX production in adenovirus type 5 infected KB cells. While there was variability in the exact amount of resistance or sensitivity (as reflected by percentage of control) from experiment to experiment, the Ad-2 proteins we studied clearly and consistently displayed characteristic resistance or sensitivity. These observations are summarized in ‘Table 1. In order to alleviate the possibility -that the different responses of mRNAs con-

367

OF TRANSLATION TABLE

1

RESPONSES TO HIB OF SPECIFIC Ad-2 LATE POLYPE~TIDES Sensitive PVI pv11 V Resistant II IV 1OOK Highly IX

Resistant

taming identical 5’ leader sequences which we observed would only occur under conditions of hypertonicity, we examined the patterns of synthesis of these same proteins using low doses of cycloheximide (Lodish, 1971), an elongation inhibitor. The range of doses of cycloheximide necessary to only partially inhibit protein synthesis in Ad-2 infected HeLa cells was determined (Fig. 5). The late (24 hr postinfection) Ad-2 proteins produced under these conditions were analyzed by gel electrophoresis and scintillation counting, an example of which is shown in Fig. 3 (B series). Using this experimental approach, efficiently initiating mRNAs would be more affected by the elongation block and would subsequently make up a lower percentage of total protein synthesis than if no cycloheximide were present (David, 1976; Jen et al., 1978). As seen in the results of a typical experiment, shown in Fig. 6, mRNAs with identical 5’ leader sequences can have very different responses to elongation inhibitors (compare, for example, proteins II and pVI1). These different responses are quite characteristic and consistent and are summarized in Table 2. VP1 Synthesis in SVS and dl-808 Infected CV-1 Cells In order to investigate the converse experimental system, i.e., the same mRNA coding sequences spliced to different leader sequences, we compared the synthesis of

368

WOLGEMUTH,

YU. AND HSU

4

83

3 2

L 50Kx)150 FIG. 3. Series A and B. Computer-generated plots of radioactivity in polyacrylamide gel slices from Ad-2 infected HeLa cells labeled with [SsS]methionineunder conditions of hypertonicity (Series A) or cycloheximide treatment (Series B). (A) Ad-2 infected HeLa cells, 44 hr postinfection, were concentrated to 4 x lo6 cells/ml and incubated under conditions of hypertonicity as described under Materials and Methods in the presence of [?S]methionine (sp act 2 566 Ci/mmol) for 15 min. Labeled proteins were assayed on a 13% polyacrylamide gel sliced into l-mm fractions. Incorporation of psS]-

5’ LEADERS AND INITIATION

OF TRANSLATION

369

the major late protein of SV40, VPl, in wild-type and a deletion mutant, dl-808 (Mertz and Berg, 1974), infected ,CV-1 cells. The DNA segment missing in dl-808 has been located at 0.723-0.758 on the SV40 map (Mertz and Berg, 1974), the region which encodes the major portion of the 202 nucleotides-long leader sequence of wildtype VP1 mRNA (Ghosh et al., 1978). The new 5’ end of the resulting leader sequence in the dl-808 VP1 mRNA is in fact encoded for by DNA sequences upstream from those which code for the leader sequences in wild-type SV40 VP1 mRNA (J. Mertz, personal communication). Efficiency of initiation of these two mRNAs for VP1 was evaluated using HIB FIG. 4. Effect of hypertonicity on incorporation at concentrations of additional NaCl wherein of [%]methionine into specific Ad-2 proteins. In this overall protein synthesis fell to -39% of experiment, Ad-2 protein synthesis was assayed 20 hr the control levels (Fig. 7). Proteins were postinfection (2000 particles/cell) as described under analyzed on 12% acrylamide gels and in- Materials and Methods and in the legends to Figs. 2 and corporation of [3H]lysine was assayed by 3. Areas under identified peaks were quantitated by scintillation counting of the gel slices and planimetry and expressed as percentage of control expressed as percentage of total incorpora- synthesis (ordinate). Concentration of excess NaCl is tion. Figure 8 shows the patterns of in- depicted on the abscissa. Roman numerals refer to the corporation by both SVS and dl-808 at individual Ad-2 proteins identified by electrophoretic mobility compared with proteins from purified virions. various NaCl concentrations. Quantitation The mRNAs for all proteins except protein IX contain a of the peak heights corresponding to VP1 common tripartite leader sequence. indicated that (a) mRNA coding for VP1 was very resistant to increasing concentrations of NaCl, and (b) VP1 mRNA from both SVS and dl-808 was similarly highly we wished to measure in viva properties of resistant. This resistance to HIB by VP1 the various mRNAs in question, we were mRNA from both SVS and dl-808 is seen restricted to using the somewhat indirect approach of examining the relative efficienquite clearly in Fig. 9. cies of initiation of the mRNAs as reflected by their responses to inhibitors of initiation DISCUSSION and elongation. Using this approach very In our experiments two complementary different patterns of sensitivity to the inproducts of splicing events were examined: hibitors were found among the late Ad-2 in the first, identical leader sequences are mRNAs which contained an identical 5’ spliced to different coding sequences; in the leader sequence. Conversely, very similar second, different leader sequences are responses to inhibition to initiation were spliced to identical coding sequences. Since observed between VP1 mRNAs comprised methionine per slice is expressed as percentage of total counts in the sample-one sample per slot (ordinate). Direction of migration is from right to left, gel slice number is labeled on the abscissa. Identilled peaks of Ad-2 proteins are labeled with Roman numerals. A,, 0 mM excess NaC1; Al, 50 mM; A,, 75 mM; A,, 100 mM; A,, 125 mM. (B) Ad-2 infected HeLa cells, 22 hr postinfection, concentrated to 4 x 106cells/ml and incubated for 30 min in the presence of r%]methionine and cycloheximide (as described under Materials and Methods) as follows: B,, 0 @; B,, 1 &f; B3, 2 pM; Bd, 4 pM; Bg, 8 pM. Direction of migration is from left to right. Identified Ad-2 proteins are indicated by Roman numerals, abscissa and ordinate as in (A).

370

WOLGEMLJTH, YU, AND HSU

FIG. 5. Inhibition of overall incorporation of [9!]methionine by Ad-2 infected HeLa cells in the presence of increasing concentrations of cycloheximide. In this experiment, Ad-2 infected HeLa cells, 22 hr postinfection (2000 particles/ml) were concentrated to 4 x 109 cells/ml and incubated for 30 min in the presence of [35S]methionine (as described under Materials and Methods) and concentrations of cycloheximide as noted along the abscissa. Aliquots, 10 ~1, of each labeled cell suspension were precipitated by TCA and counted on filters. The cpm for 0 /&f (control) was designated as 100% (6.18 x lo5 cpm). Incorporation for samples at each of the designated concentrations (abscissa) was expressed as percentage of control incorporation (ordinate).

of different 5’ caps and leader sequences. These results suggest that, at least at the primary structural level, 5’ leader sequences are not the major factor affecting initiation of translation of the viral mRNAs to which they are spliced. That is, the presence or absence of given sequencesper se does not confer characteristic initiation properties under the experimental conditions described. These results are of interest in light of other studies on (a) the role of splicing in regulation of coordinate expression of different genes, and (b) the role of the 5’ ends of mRNAs in regulation of translation. Ad-2 mRNAs provide a possible model system for studying a series of genes which are coordinately expressed in time: late in infection of human cells, virtually all radioactive amino acids are incorporated into virus-specific proteins (Bell0 and Ginsberg, 1964; White et al., 1969; Russel and Skehel, 1972; Anderson et al., 1973). The majority of these proteins are translated from mRNAs produced from a single late transcription unit (Evans et al., 1977) whose promoter is at map position 16.4 (Ziff and Evans, 1973) and whose 5’ leader sequences are identical (Berget et al., 1977; Chow et al., 1977; Gelinas and Roberts, 1977;

Klessig, 1977). Thus, it was tempting to speculate that the common 5’ ends could function in ensuring their coordinate expression by confering a common translational property. Although Beltz and Flint (1979) have recently suggested that the switch from cellular to viral protein synthesis is mediated primarily by the failure of newly synthesized cellular mRNAs to enter the cytoplasmic pool late in infection, thereby implying that viral protein synthesis is favored by virtue of its increasing concentrations of mRNAs, other evidence suggests that preferential translation of viral over cellular mRNAs may also be a factor in this regulatory phenomenon. First, although >90% of the newly synthesized mRNA to enter the cytoplasm is viral (Beltz and Flint, 1979), as much as 98% of the steady-state cytoplasmic mRNA may still be cellular at, for example, - 18 hr postinfection (Philipson et al., 1974; Flint and Sharp, 1976). Second, Cherney and Wilhelm (1979) have recently

FIG. 6. Effect of increasing concentrations of cycloheximide on the incorporation of [35S]methionine into specific Ad-2 proteins. In this experiment, incorporation of [3sS]methionine into Ad-2 proteins synthesized by Ad-2 infected HeLa cells (2000 particles/cell) 22 hr postinfection (as described under Materials and Methods and the legends to Figs. 3 and 4) in the presence of increasing concentrations of cycloheximide (abscissa) was determined. Peaks of radioactivity as plotted with the aid of a computer (Fig. 3, B series) were measured by planimetry and expressed as percentage of the control, 0 @f cycloheximide, 100% synthesis. Roman numerals refer to specific Ad-2 proteins identified by electrophoretic mobility as compared with proteins from purified Ad-2 virions. 106X protein identification is tentative because of lower resolution of high molecular weight proteins on this particular gel. All identified proteins except protein IX are synthesized from mRNAs with a common tripartite 5’ leader sequence.

5’ LEADERS AND INITIATION

OF TRANSLATION

371

eucaryotic mRNAs in a eucaryotic cellfree synthesizing system (Paterson and SUMMARY OF RESPONSES OF SPECIFIC Ad-2 LATE Rosenberg, 1979). The presence of a cap PROTEINS TO Low DOSES OF CYCLOHEXIMIDE structure does not of itself guarantee translation of coding sequences downIn order of increasing sensitivity pVI1 < IV, III,= IX < lOOK,” pV1 < II stream from nontranslated 5’ nucleotides: Least sensitive Most sensitive coding sequences whose initiator codon AUG is -330 nucleotides to the 3’ of the o Identification tentative. cap are not translated (Rosenberg and Paterson, 1979). The authors suggested reported a preferential translation of Ad-5 that it may be the relative proximity of mRNAs late in infection of KB cells under the translation start site to the capped 5’ conditions of hypertonicity, a response sim- end of the transcript that was important ilar to that seen in other virus infections for efficient translation of the protein, in where preferential initiation of viral mRNA this case, A cro protein. has been shown (Opperman and Koch, 1976; However, other published observations David, 1976; Tsershak, 1978; Jen et al., would tend to preclude a simple nonsequence 1978). Third, in our own experiments with specific “distance between cap and initiation Ad-2 infected HeLa cells, higher con- codon” model. Examination of the secentrations of excess NaCl reduced the quences of the SV40 VP1 mRNA 5’ end incorporation of [35S]methionine into non- (Ghosh et al., 1978) reveals the presence viral peaks to almost undetectable levels. And of two AUG sequences in the spliced leader conversely, with the use of an elongation sequence at positions 11 and 61 (where the inhibitor, an increase in the “background” m7GpppmsAmis defined as position l), both incorporation across the gel was observed. of which occur before the AUG at position One interpretation is that this nondiscrete 240 which is the initiation codon for in vivo background represents a spectrum of cellular proteins translated from cellular mRNAs still present in the cytoplasm. Thus, translational regulation may be involved, if only secondarily to transcriptional regulation (Beltz and Flint, 1979). However, it appears that a major role in this regulation cannot be ascribed to the primu?-y sequence structure of the 5’ leader regions. It is also important to consider how the 5’ ends (defined here as sequences to the left of the active initiation codon) of 0100 mRNAs are involved in the initiation mM excess NaCl process, particularly with respect to ribosome binding. Recent studies on the funcFIG. 7. Effect of hypertonicity on incorporation tional level have produced interesting but of [3H]lysine into total protein synthesized by SV40 somewhat confusing results. On one hand, infected CV-1 cells. Confluent monolayers of CV-1 the presence of a cap structure has been cells, 48 hr postinfection with wild-type SV40 (10 PFU/ shown to be integral for proper trans- cell), were assayed for incorporation of [3H]lysine in lation: its removal or chemical modifica- the presence of increasing concentrations of NaCl as described under Materials and Methods. Aliquots, tion greatly affects the translational ef- 50 ~1, (approximately 5 x 105cells) were analyzed on ficiency of the mRNA in question (for 12% polyacrylamide gels which were fractionated into reviews see Shatkin, 1976; Kozak, 1978). 2-mm slices and radioactivity was assayed by scintilIn vitro capping of procaryotic mRNAs lation counting. Total cpm per electrophoresed sample confers upon them a translational efficiency is shown (ordinate) at concentrations of excess NaCl equal to or even surpassing that of noted on the abscissa. TABLE 2

WOLGEMUTH,YU,ANDHSU

372

AZ

1

L A3

1

6

L!!d I L 4

I

4

A4

5’ LEADERS AND INITIATION

VP1 (Kozak, 1978). Further, according to Dunn et al. (1978) the presence of as many as 181 extra nucleotides comprising a fourth spliced leader segment (Zain et al., 1979) between the cap and AUG of Ad-2 fiber mRNA sequences does not affect the in vitro translational efficiency of this message as compared with fiber mRNA species containing a tripartite 5’ leader segment. This may be compared with the opposite situation where the 5’ ends of mRNAs are deleted. Subramamian (1979) recently reported DNA sequence studies on a series of SV40 deletion mutants that focus on the region of map units 0.72 to 0.76, which contains the VP1 mRNA leader sequence (Ghosh et al., 1978).As few as six nucleotides of the sequences which code for the 5’ leader of wild-type VP1 mRNA remain and yet there is functional VP1 mRNA produced, as evidenced by the fact that these are viable mutants. While the presence of an alternative cap and 5’ leader sequence might be required to generate a necessary secondary structure for translation of the mRNA, it would appear that there is not an absolute requirement for a given cap and leader sequence to ensure translation of the VP1 mRNA. Yet there is considerable evidence that suggests a requirement for specific sequences between the cap and the initiation codon. Most compelling is the complementarity of sequences at the 5’ ends of mRNAs to a purine-rich tract of sequences at the 3’ end of 18 S rRNA of eucaryotes (Hagenbuchle et al., 1978). In fact, in both the late Ad-2 mRNAs and wild-type SV40 VP1 mRNAs which were the subjects of our study, such regions of putative 18 S 3’-end complementarity can be seen in the leader sequences (Ziff and Evans, 1978; Contreras

OF TRANSLATION

373

FIG. 9. Effect of incorporation of [3H]lysine into VP-l from wild-type SV40 and dl-808 in the presence of increasing hypertonicity. The amount of radioactivity present in each of the VP-l peaks shown in Fig. 8 was determined and expressed as percentage of the total cpm per sample (ordinate) at the concentration of excess NaCl as noted (abscissa). (0 0) Wild-type SV40; (0 - - - 0) dl-808.

et uZ., 1978; Hagenbuchle et al., 1978). Their role in initiation of translation is speculative to date in that there is no direct evidence of their involvement in ribosome binding. However, the fact that an analogous region of 5’ mRNA: 16 S rRNA sequence homology in procaryotes has been shown to be involved in initiation of translation (Shine and Dalgarno, 1974; Steitz and Jakes, 1975) coupled with the fact that procaryotic mRNAs when capped can be efficiently recognized by eucaryotic protein synthesizing machinery (Paterson and Rosenberg, 1979) argue strongly for further study of specific sequences in this 5’ region. ACKNOWLEDGMENTS We wish to thank Dr. James E. Darnell for helpful discussions, Drs. Warren Jelinek and MingChu Hsu for help in the computer analysis, Dr. Janet Mertz for supplying mutant virus and for communicating unpublished observations, and Audrey

FIG. 8. Computer-generated plots of [3H]lysine incorporation into polyacrylamide gel electrophoresis-separated proteins from CV-1 cells infected with wild-type SV40 (Series A) or dl-808 (Series B) under conditions of hypertonicity. Samples, 50 ~1, were obtained and analyzed as described under Materials and Methods and the legend to Fig. 7. Incorporation of [SH]lysine in each fraction slice (abscissa) is expressed as percentage of the total cpm per sample (ordinate). Series A shows wild-type SV40 infected CV-1 cells at: A,, 0 mi%!excess NaCI; A,, 25 mM; Al, 50 mM; &, 75 mM; As, 100 mM. Series B: CV-1 cells infected with SV40 mutant dl-808. B,, 0 mM excess NaC1; Bf, 25 mM; B,, 50 mM; B,, 75 mM; Bg, 100mM. Direction of migration is from left to right for A and B. The peak corresponding to the major late protein VP-l, identified by electrophoretic mobility, is indicated in the top panels as are the regions of the gel corresponding to the cellular histones Hza, HZb, H,, and H,.

374

WOLGEMUTH,

English and Lois Cousseau for secretarial assistance. This work was supported in part by Public Health Service National Cancer Institute Grant CA19073-3. D.J.W. is a Fellow in Cancer Research supported by Grant DRG-F230 of the Damon RunyonWalter Winchell Cancer Fund. REFERENCES AKUSJ~RVI, G., and PETTERSSON, U. (1979). Sequence analysis of adenovirus DNA: Complete nucleotide sequences of the spliced 5’ noncoding region of adenovirus 2 hexon mRNA. Cell 16, 841850. ALONI, Y., DHAR, R., LAUB, O., HOROWITZ, M., and KHOURY, G. (1977). Novel mechanism for RNA maturation: The leader sequences of simian virus 40 mRNA are not transcribed adjacent to the coding sequences. Proc. Nat. Acad. Sci. USA 74, 3686-3690.

BELLO, L. J., and GINSBERG,H. S. (196’7).Inhibition of host protein synthesis in type 5 adenovirusinfected cells. J. Virol. 1, 843-850. BELTZ, G. A., and FLINT, S. J. (1979). Inhibition of HeLa cell protein synthesis during adenovirus infection: Restriction of cellular mRNA sequences to the nucleus. J. Mol. Biol. 131, 353-374. BERGET, S., MOORE, C., and SHARP, P. A. (1977). Spliced segments at the 5’ terminus of adenovirus 2 late mRNA. Proc. Nat. Acad. Sci. USA 74,31713175. BLANCHARD, J. -M., WEBER, J., JELINEK, W., and DARNELL, J. E. (1978). In vitro RNA-RNA splicing in adenovirus 2 mRNA formation. Proc. Nat. Acad. Sci. USA 75, 5344-5348. CELMA, M., DHAR, R., PAN, J., and WEISSMAN, S. M. (1977). Comparison of the nucleotide sequences of the messenger RNA for the major structural protein of SV40 with the DNA sequence encoding the amino acids of the protein. Nucleic Acids Res. 4, 2549-2559. CHERNEY, C. S., and WILHELM, J. M. (1979). Differential translation in normal and adenovirus type 5-infected human cells and cell-free systems, J. Viral.

30, 533-542.

CHOW, L. T., and BROKER, T. R. (1978). The spliced structures of adenovirus 2 fiber message and the other late mRNAs. Cell 15, 497-510. CHOW, L. T., GELINAS, R. E., BROKER, T. R., and ROBERTS, R. J. (1977). An amazing sequence arrangement at the 5’ ends of adenovirus 2 messenger RNA. Cell 12, 1-8. CHOW, L. T., KILLPATRICK, B. A., LEWIS, J. B., GRODZICKER,T., SAMBROOK,J., GELINAS, R. E., and BROKER, T. R. (1979). Splicing patterns and pathways of adenoviral RNA transcripts. J. Supramol.

Struet. Suppl. 3, 45.

CONTRERAS,R., RIGIERS, R., VAN DE VOORDE,A., and FIERS, W. (1977). Overlapping of the VP,-VP,

YU, AND HSU and the VP1 gene in the SV40 genome. Cell 12, 529-538. DAVID, A. E. (1976). Control of vesicular stomatitis virus protein synthesis. Virology 71, 217-229. DUNN, A. R., MATHEWS, M. B., CHOW, L. T., SAMBROOK,J., and KELLER, W. (1978). A supplementary adenoviral leader sequence and its role in messenger translation. Cell 15, 511-526. EVANS, R. M., FRASER, N., ZIFF, E., WEBER, J., WILSON, M., and DARNELL, J. E. (1977). The initiation sites for RNA transcription in Ad2 DNA. Cell 12, 733-739. EVERI~, E., SUNDQUIST,B., PETTERSSON,U., and PHILIPSON, L. (1973). Structural proteins of adenovirus. X. Isolation and topography of low molecular weight antigens from the virion of adenovirus type 2. Virology 52, 130-147. FLINT, J. (1977). The topography and transcription of the adenovirus genome. Cell 10, 153-166. FLINT, S. J., and SHARP, P. ,A. (1976). Adenovirus transcription V. Quantitation of viral RNA sequences in adenovirus-infected and transformed cells. J. Mol. Biol. 106, 749-771. FORD, J. P., and HSU, M. -T. (1978). Transcription pattern of in viva-labeled late simian virus 40 RNA: Equimolar transcription beyond the mRNA 3’-terminus. J. Virol. 28, 795-801. GELINAS, R. E., and ROBERTS, R. J. (1977). One predominant 5’-undecanucleotide in adenoviurs 2 late messenger RNAs. Cell 11, 533-544. GHOSH, P. K., REDDY, V. B., SWINSCOE, J., CHOUDARY,P. V., LEBOWITZ, P., and WEISSMAN, S. M. (1978). The 5’-terminal leader sequence of late 16 S mRNA from cells infected with simian virus 40. J. Biol. Chm. 253, 3643-3547. HAEGEMAN, G., and FIERS, W. (1978). Evidence for “splicing” of SV40 16s mRNA. Nature (London) 273, 70-73. HAGENBUCHLE, O., SANTER, M., STEITZ, J. A., and MANS, R. J. (1978). Conservation of the primary structure at the 3’ end of 18s rRNA from eucaryotic cells. Cell 13, 551-563. HOROWITZ,M., LAUB, O., BRATOSIN, S., and ALONI, Y. (1978). Splicing of SV40 late mRNA is a posttranscriptional process. Nature (London) 275, 558-559.

HSU, M. -T., and FORD, J. P. (1977). Sequence arrangement of the 5’ ends of simian virus 40 16s and 19s mRNAs. Proc. Nat. Acad. Sci. USA 74, 4982-4985.

JEN, G., BIRGE, C. H., and THACH, R. E. (1978). Comparison of initiation rates of encephalomyocarditis virus and host protein synthesis in infected cells. J. Viral. 27, 640-647. JOHNSON, A. (1979). Masters Degree Thesis, University of Wisconsin. KLESSIG,D. F. (1977). Two adenovirus 2 mRNAs have a common 5’ terminal leader sequence encoded at

5’ LEADERS

AND INITIATION

least 10 kb upstream from their main coding regions. Cell 12, 9-21. KOWUC, M. (1978). How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell 15, 1109-1123.

LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-684. LAI, C. -J., DHAR, R., and KHOURY, G. (1978). Mapping the spliced and unspliced late lytic SV40 RNAs. Cell 14, 971-982. LAVI, S., and GRONER, Y. (1977). 5’-Terminal sequences and coding regions of late simian virus 40 mRNAs are derived from noncontiguous segments of the viral genome. Proc. Nat. Acad. Sci. USA 74, 5323-%327.

LOCKARD, R. E., BERGET, S. M., RAJ BHANDARY, U. L., and SHARP, P. A. (1979). Nucleotide sequence at the 5’ terminus of adenovirus 2 late messenger RNA. J. Biol. Chem. 254, 587-590. LODISH, H. F. (1971). Alpha and beta globin messenger ribonucleic acid. Different amounts and rates of initiation of translation. J. Biol. Chem. 246, 7131-7138. MANLEY, J. L., SHARP, P. A., and GEFTER, M. L. (1979). Synthesis of adenovirus 2 mRNA in vitro. J. Supramol. Struct. Suppl. 3, 65. MERTZ, J. E., and BERG, P. (1974). Viable deletion mutants of simian virus 40: Selective isolation by means of a restriction endonuclease from Hemphilus paminfluenzae. USA 71, 4879-,4883.

Proc. Nat. Acad. Sci.

NEVINS, J. R., and DARNELL, J. E., JR. (1978). Steps in the processing of AD2 mRNA: Poly(A)+ nuclear sequences are conserved and poly(A) addition precedes splicing Cell 15, 1477- 1493. NUSS, D. L., and KOCH, G. (1976a). Differential inhibition of vesicular stomatitis virus polypeptide synthesis by hypertonic initiation block. J. Viral.

17, 283-286.

NUSS, D. L., and KOCH, G. (1976b). Variation in the relative synthesis of immunoglobulin G and non-immunoglobulin G proteins in cultures of MPC-11 cells with changes in the overall rate of polypeptide chain initiation and elongation. J. Mol. Biol.

102, 601-612.

OPPERMAN, H., and KOCH, G. (1976). Individual translational efficiencies of SV40 and cellular mRNAs. Arch. Viral. 52, 123-134. PATERSON, B. M., and Rosenberg, M. (1979). Efficient translation of prokaryotic mRNAs in a eukaryotic cell-free system requires addition of a cap structure. Nature (London) 279, 692-696. PHILIPSON, L., PETTERSSON, U., LINDBERG, U., TIBBEITS, C., VENNSTROM,B., and PERSSON,T. (1974). RNA synthesis and processing in adenovirus infected cells. Cold Spring Harbor Symp. Qua&. Biol. 39, 447-456.

OF TRANSLATION

375

ROSENBERG, M., and PATERSON, B. M. (1979). Efficient cap-dependent translation of polycistronic prokaryotic mRNAs is restricted to the first gene in the operon. Nature (London) 279, 696-701. RUSSELL, W. C., and SKEHEL, J. J. (1972). The polypeptides of adenovirus infected cells. J. Gem. Viral. 15, 45-57. SABORIO,J. L., PONG, S. -S., and KOCH, G. (1974). Selective and reversible inhibition of initiation of protein synthesis in mammalian cells. J. Mol. Biol. 85, 195-211. SHATKIN, A. J. (1976). Capping of eukaryotic mRNAs. Cell 9,645-653. SHINE, J., and DALGARNO, L. (1974). The 3’ terminal sequence of E. coli 16s ribosomal RNA: Complementarity to nonsense triplets and ribosome binding sites. Proc. Nat. Acad. Sci. USA 71, 13421346. STEITZ, J. A., and JAKES, K. (1975). How ribosomes select initiator regions in mRNA: Base pair formation between the 3’ terminus of 16s rRNA and the mRNA during initiation of protein synthesis in E. coli Proc. Nat. Acad. Sci. USA 72, 4734-4738.

SUBRAMANIAN, K. N. (1979). Segments of simian virus 40 DNA spanning most of the leader sequence of the major late viral messenger RNA are dispensable. Proc. Nat. Acad. Sci. USA 76, 2556-2560.

TAKEMOTO,K. R., KIRSCHSTEIN, R. L., and HABEL, K. (1966). Mutants of simian virus 40 differing in plaque size, oncogenicity and heat sensitivity. J. Bacterial. 92, 990-994. TERSHAK, D. R. (1978). Peptide-chain initiation with LSc poliovirus is intrinsically more resistant to hypertonic environment than is peptide-chain initiation with Mahoney virus and deletion mutants of Mahoney virus. J. Viral. 28, 1006-1010. WALL, R., PHILIPSON,L., and DARNELL, J. E. (1972). Processing of adenovirus specific nuclear RNA during virus replication. Virology 50, 27-34. WEBER, J., BEGIN, M., and CARSTENS,E. B. (1977). Genetic analysis of adenovirus type 2. IV. Coordinate regulation of polypeptides 80K, IIIa, and V. Virology

76, 709-724.

WHITE, D. D. SCHARFF, M. D., and MAIZEL, J. V. (1969). The polypeptides of adenovirus. III. Synthesis in infected cells. Virology 3, 395-406. ZAIN, S., SAMBROOK,J., ROBERTS, R. J., KELLER, W., FRIED, M., and DUNN, A. R. (1979). Nucleotide sequence analysis of the leader segments in a cloned copy of adenovirus 2 fiber mRNA. Cell 16, 851-861. ZIFF, E. B., and EVANS, R. M. (1978). Coincidence of the promoter and capped 5’ terminus of RNA from the adenovirus 2 major late transcription unit. Cell 15, 1463-1475.