Location of the sequences coding for capsid proteins VP1 and VP2 on polyoma virus DNA

Cell, Vol . 9, 481 -487, November 1976, Copyright © 1976 by MIT

Location of the Sequences Coding for Capsid Proteins VP1 and VP2 on Polyoma Virus DNA

Alan E . Smith, Robert Kamen, Walter F . Mangel,* Helen Shure,t and Tricia Wheeler Departments of Molecular Virology and Cellular Regulation Imperial Cancer Research Fund London WC2A 3PX, England

Summary The 19S and 16S polyoma virus late mRNAs have been separated on sucrose-formamide density gradients and translated in vitro . The 16S RNA codes only for polyoma capsid protein VP1, while the 19S RNA codes' In addition for capsid protein VP2 . Since the 19S and 16S species have been previously mapped on the viral genome, these results allow us to deduce the location of the sequences coding for VP1 and VP2 . Comparison of the chain lengths of the capsid proteins with the size of the viral mRNAs coding for them suggests that VP1 and VP2 are entirely virus-coded . Purified polyoma 19S RNA directs the synthesis of very little VP1 in vitro, although it contains all the sequences required to code for the protein . The initiation site for VP1 synthesis which is located at an internal position on the messenger is probably inactive either because it is inaccessible or because it lacks an adjacent "capped" 5' terminus . Similar inactive internal initiation sites have been reported for other eucaryotic viral mRNAs (for example, Semliki forest virus, Brome mosaic virus, and tobacco mosaic virus), suggesting that while eucaryotic mRNAs may have more than one initiation site for protein synthesis, only those sites nearer the 5' terminus of the mRNA are active . Introduction The productive cycle of polyoma virus replication proceeds through two phases : the early phase which occurs before and the late phase which occurs after the onset of viral DNA synthesis . During the late phase, large quantities of viral RNA appear in the cytoplasm, and at the same time, viral capsid proteins are synthesized and transported into the nucleus where progeny virions are assembled . Late cytoplasmic viral RNA has been separated into two species with sedimentation constants of 19S and 16S (Buetti, 1974) . These have been positioned on the physical map of polyoma DNA by hybridization to specific restriction endonuclease ''Present address : Department of Biochemistry, University of Illinois, Urbana, Illinois 61801 . Present address : Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel .

fragments . 19S RNA comprises a transcript of nearly all the late region of the viral DNA, while the 16S RNA corresponds to the 3' terminal half of the 19S RNA (Kamen and Shure, 1976) . Polyoma virus particles consist of a viral DNA molecule complexed with an equal weight of cellular histone and one major (VP1) and two minor (VP2 and VP3) capsid proteins . The molecular weights of the viral proteins are 45,000, 34,000, and 23,000 daltons, respectively . Tryptic peptide analysis suggests that although VP1 and VP2 are different proteins with non-overlapping sequences, VP2 and VP3 contain common peptide sequences (Gibson, 1974 ; Fey and Hirt, 1974 ; Hewick, Fried, and Waterfield, 1975) . We have previously established conditions for the cell-free synthesis of polyoma capsid proteins VP1 and VP2, using total mRNA isolated from the cytoplasm of lytically infected cells (T . Wheeler, S . T . Bayley, R . Harvey, L . V . Crawford, and A. E . Smith, manuscript submitted for publication) . We also showed that the active mRNA species could be purified by preparative hybridization to singlestranded polyoma DNA immobilized on cellulose nitrate filters . It is therefore probable that the capsid proteins were translated from the 19S and 16S viral mRNA species described above . Here we have separated the two polyoma RNAs, translated them in vitro, and identified the products made . Using the known location of the viral RNAs on the polyoma genome, the data obtained enabled us to deduce the position of the coding sequences for the viral capsid proteins . Results The translation studies described here were conducted in parallel with hybridization experiments, the results of which have already been presented (Kamen and Shure, 1976) . These experiments analyzed the virus-specific polyadenylated RNA molecules present in the cytoplasm of polyoma-infected 3T6 cells late during the productive cycle . Total polyadenylated cytoplasmic RNA was fractionated by sedimentation through sucrose-formamide density gradients (Lewis et al ., 1975) . Polyoma-specific mRNA was detected and mapped on the viral genome by hybridizing the nonradioactive RNA in the gradient fractions to the 32 P-labeled, single-stranded DNA derived from different specific restriction endonuclease fragments . In this way, two major viral late mRNAs, transcribed from the L DNA strand and sedimenting at 19S and at 165, were characterized . In several experiments, mRNA in the sucrose gradient fractions was also analyzed by cell-free translation . Preliminary experiments showed that the mRNA encoding capsid protein VP1 co-sedimented

Cell 482

predominantly with the 16s viral RNA, while the mRNA coding for VP2 sedimented with the 19s peak detected in the hybridization assays. To achieve a better separation of the two viral species, the 16s and 19s regions of such a sucrose gradient were separately pooled, concentrated by ethanol precipitation, and refractionated by sedimentation through identical sucrose-formamide gradients. Figure 1 shows an autoradiograph of an SDS-polyacrylamide gel separation of the products made in the wheat germ cell-free system in response to mRNA resolved on the second gradient. mRNA purified from the 16s region of the first sucrose gradient coded for a number of proteins (slots l-3) the major one of which co-migrated with polyoma VP1 and was coded for by a messenger which resedimented between 14s and 18s. No discrete protein band with the gel mobility of polyoma VP2 was present in the 16s RNA-directed product, but the background was too high in this region of the gel to be certain of its absence. Slots 4-9 show the results of translation across the gradient refractionating 19s RNA. The 14S-17s region of this gradient (slot 4) contained RNA encoding a protein with the gel mobility of VPI. Since the initial gradient con-

I----16s1 2

3

~19s-----l 4 5

6

7

tained about 4 times more 16s viral RNA than 19s RNA as determined by hybridization, we attribute this activity to contaminating 16s messenger which was resolved by the second centrifugation step. RNA from the 18S-20s region (slots 5 and 6) directed the synthesis of some VPl, as well as larger amounts of a protein which co-migrated with polyoma capsid protein VP2. These results suggested that the 16s mRNA codes only for VPl, whereas 19s mRNA codes in addition for VP2. To eliminate the possibility that some VP2 is made by 16s mRNA but obscured in the host protein background in this region of the gel, we fractionated total infected cell mRNA on a sucrose-formamide gradient, pooled and translated RNA from adjacent fractions, and immunoprecipitated samples of the products with two different antisera, one raised against purified intact virions and the other against polyoma VP2 purified from disrupted virions. Figure 2 shows a polyacrylamide gel analysis of the proteins immunoprecipitated from each fraction. We have previously shown that the anti-virion serum reacts with a protein similar, if not identical, to polyoma VPl, whereas the antiVP2 serum reacts with all three capsid proteins (T.

8

9

VP 1 VP 2 VP 3

Figure

1. Polyacrylamide

Gel Electrophoresis

of Proteins

Made

in Response

to Polyoma

19s

and 16s

mRNAs

Cytoplasmic polyadenylated RNA (15 pg) purified from 3T6 cells 30 hr after infection with polyoma virus was denatured and centrifuged on a 5-20% sucrose gradient containing 50% formamide as described in Experimental Procedures. An internal marker containing approximately 2000 Cerenkov cpm of 32P-labeled cytoplasmic RNA from uninfected 3T6 was included. 15 drop fractions were collected. After centrifugation, aliquots of the gradient fractions were hybridized with polyoma total L strand DNA. Tubes corresponding to the peaks of 19s and 16s RNA were thus located (see Figure 5, Kamen and Shure, 1976); these were pooled, precipitated, and recentrifuged under identical conditions. Fractions from the second gradient were pooled (as indicated below), precipitated, and translated. The autoradiograph shows the proteins made in response to pooled samples of refractionated 16s RNA sedimenting at (1) lOS-13S, (2) 14S-18S, (3) 19S-25s; and pooled samples of refractionated 19s RNA sedimenting at (4) 14S-17S, (5) 18S-19S (6) 19S-ZOS, (7) ZOS-255, (8) 25S-3OS, (9) no added RNA. Material from the 19s RNA peak track (5) was rerun (11) next to purified polyoma virus (10). Autoradiography was for 4 days.

Location 483

of Sequences

Coding

for Polyoma

Proteins

Wheeler et al., manuscript submitted for publication). Figure 2 shows that RNA from the 16s region of the gradient codes exclusively for VPl, whereas that from the 19s region codes for both VP1 and VP2. In several experiments of the kind described above, we found that the amount of methionine incorporated into VP1 in response to 19s RNA was less than that into VP2. This result suggested that the VP1 cistron of 19s RNA is not translated efficiently. The small amount of synthesis of VP1 on RNA from the 19s region of the gradient could in fact be due to residual contamination by 16s RNA. We therefore purified further batches of RNA using sucrose gradients containing higher concentrations of formamide (Macnaughton, Freeman, and Bishop, 1974). Figure 3 shows the proteins made in response to fractions from such a gradient. RNA from the region slower than the 18s rRNA marker coded for a number of peptides including a large quantity of VPl, whereas the fraction corresponding to 19s RNA synthesized VP2 but very little VPl. This finding was further substantiated by reacting the cell-free product with VP2 antiserum and characterizing the proteins present in the immunoprecipitate. Figure 3 shows that a major band co-migrating with polyoma VP2 is present in the imI

1

2a 2b

19s 3a 3b

munoprecipitate of the 19s RNA product, but VP1 is not detected. We have already shown that the antiserum cross-reacts with VP1 sufficiently well to bring down at least a substantial portion of this protein if it is present (compare, for example, Figure 2, slot 3b). We interpret this result to mean that the initiation site for VP1 synthesis present on 19s RNA is virtually inactive. To show that the inactivity of the VP1 initiation site on 19s mRNA is not an artifact introduced when a mouse virus mRNA is translated in the heterologous wheat germ cell-free system, we translated RNA from various sucrose gradient fractions in extracts from mouse ascites cells: the mouse is, of course, a permissive host for polyoma virus. The experiments (data not shown) gave results similar to those obtained in the wheat germ system. 16s mRNA directed the synthesis of VPI, while purified 19s RNA did not. The cell-free products were not affected by the presence or absence of S-adenosyl methionine. Presumably, therefore, cleavage of the 19s RNA followed by capping and methylation to give active 16s RNA does not occur in the mouse cell-free system under the present conditions. The cell-free synthesis of VP2 in the mouse extracts using 19s RNA was difficult to establish because

I6a 6b

I

4a 4b

5

16s I 7a 7b 8a

8b

9

VP1 T VP2 -

VP3 J

Figure 2. lmmunoprecipitation mRNAs

of in Vitro

Products

with Anti-Virion

and Anti-VP2

Serum

of Proteins

Made

in Response

to Purified

Poiyoma

50 pg cytoplasmic polyadenylated mRNA from polyoma infected cells was centrifuged on a 5-20% sucrose gradient containing 50% formamide essentially as described in Figure 1. The gradient fractions were pooled in pairs and precipitated. The peak fractions of polyoma 19s and 16s RNA were localized by hybridization and translation (see Figure 10, Kamen and Shure, 1976). Samples of RNA from around the 19s RNA peak (slots 2, 3, and 4) and 16s RNA peak (6, 7, and 8) were translated, and the products were separately immunoprecipitated with anti-virion serum (labeled a) and with anti VP2 serum (labeled b). The autoradiograph shows the proteins present in the immunoprecipitates electrophoresed next to purified polyoma virus (slots 1, 5, and 9). The viral capsid proteins are indicated. Autoradiography was for 14 days. Note that the sample in slot 4a was lost during processing.

Cell 484

of the high background of endogenous thesis in the VP2 region of the gel.

protein

syn-

Discussion Coding Properties of 19s and 16s RNA In previous work, we isolated total poly(A)-containing RNA from mouse cells productively infected with polyoma virus, and translated it in a wheat germ cell-free system. We identified polyoma proteins VP1 and VP2 among the proteins made in vitro by polyacrylamide gel electrophoresis, specific immunoprecipitation, and tryptic peptide mapping (T. Wheeler et al., manuscript submitted for publication). Here we have fractionated the active viral mRNA into 19s and 16s species, and have shown that 19s RNA directs the synthesis of VP2 and 16s RNA the synthesis of VPl. The molecular weights of the three polyoma capsid proteins are shown in Table 1. Since it is probable that VP3 is derived from VP2 by proteolytic cleavage (Hewick et al., 1975) the sequence information required to encode VP3 is probably contained within that coding for VP2. The total molecular weight of unique polyoma capsid protein sequences is therefore about 79,000 daltons. This is equivalent to about 42% of the total theoretical coding capacity of the polyoma genome (Table 1).

28s 1 12345678

It is possible to estimate the approximate coding capacity of those regions of the late cytoplasmic mRNAs which are coded for by the viral genome. Such estimates are based on the saturation levels of hybridization between purified mRNAs and radioactively labeled viral DNA fragments. Using this approach, Kamen and Shure (1976) calculated that 19s RNA is complementary to 42-43% of the viral genome. This could code for about 85,000 daltons of protein. 16s RNA was estimated to be complementary to 22% of the genome corresponding to a protein of 44,000 daltons. These numbers are summarized in Table 2 and are in good agreement with the theoretical values determined above (Table 1). They indicate that sufficient virus-coded information is present in the mRNAs from the late region to code for both of the capsid proteins. The sedimentation coefficients of the 19s and 16s mRNAs on denaturing sucrose gradients also give some indication of their molecular weights. Using ribosomal RNAs of known sedimentation coefficient and molecular weight as standards and the equation derived by Boedtker (1968), we estimate that 19s RNA would have a molecular weight of approximately 760,000 daltons (2200 nucleotides) and 16s RNA 500,000 daltons (1500 nucleotides). After subtracting about 100 nucleotides for the nontranscribed poly(A) tracts, these values for the total mo-

18s 1 9

10

11

12

13

VPl’

VP2 VP3

Figure

3. Proteins

Made

in Response

to Polyoma

mRNAs

Separated

on an 85% Formamide

Sucrose

Gradient

10 pg late polyoma mRNA was separated by centrifugation on a 2-10% sucrose gradient containing 95% formamide. 28 drop fractions were collected, precipitated, and translated. The positions of 185 and 285 RNA markers run in a parallel gradient are indicated. The autoradiograph shows the translation of each gradient fraction (slots l-10); (11) no added RNA; (12) purified polyoma virus; (13) immunoprecipitation with anti-VP2 serum of the product made in response to the peak tube of purified 19s RNA (as in slot 5).

Location of Sequences Coding for Polyoma Proteins 485

Table 1 . Estimate of Number of Nucleotides Required to Code for Polyoma Capsid Proteins VP1

VP2

VP3

VP1 + VP2

45,000

34,000

23,000

79,000

Estimated Number of Amino Acids

410

310

210

720

Estimated Number of Nucleotides

1,230

930

630

2,160

24

18

12

42

Molecular Weight (Daltons)

Genome

Molecular weight determinations were those of R . Hewick et al . (manuscript in preparation) ; the average molecular weight of an amino acid was assumed to be 110 daltons, and the polyoma genome 5200 base pairs (Robberson and Fried, 1974) .

lecular weight of the mRNA species are reasonably similar to those deduced above for the virus-coded portion of the mRNAs and are not indicative of the presence of extensive nonviral sequences . Of course, from these calculations, it is not possible to exclude the existence of short host-coded sequences within the viral mRNAs . This is made less probable, however, by our finding that in the wheat germ cell-free system, polyoma cRNA, made in vitro using highly purified polyoma superhelical DNA and E . coli RNA polymerase, directs the synthesis of complete polyoma VP1 and VP2 (Smith et al ., 1975a, 1975b ; W . F . Mangel, R . Hewick, M . D . Waterfield, R . Harvey, T . Wheeler, and A . E . Smith, manuscript in preparation) . To summarize, we have shown by in vitro translation that polyoma late cytoplasmic 16S RNA codes in vitro for VP1 and 19S RNA codes, in addition, for VP2 . Theoretical calculations of the coding sequences necessary to code for proteins of this size are consistent with these allocations . Furthermore, estimates of the extent of the virus-coded sequences and of the molecular weights of the mRNAs are consistent with the view that both messenger species and consequently both viral proteins are entirely virus-coded . It should be emphasized, however, that implicit in all the arguments presented above is the assumption that the additional sequences present in the 19S RNA but not in the 16S RNA code for VP2, and that a particular linear sequence of nucleotides in polyoma mRNA is read in one phase, the other two being nonsense . Location of Coding Sequences on Polyoma DNA Since the late cytoplasmic mRNAs have been accurately positioned on the physical map of polyoma DNA (Kamen and Shure, 1976), it is possible to locate the coding sequences for the two capsid proteins . Figure 4 shows a map of polyoma DNA drawn in linear fashion with a break in the DNA near the site where DNA replication terminates . VP1 is encoded predominantly in the Hpall fragment 1 region, extending into Hpall fragment 6 . This corresponds to the sequences contained between 47

Table 2 . Estimated Coding Capacity of Polyoma Virus mRNAs

Genome Estimated Number of Nucleotides Estimated Number of Amino Acids Estimated Protein Molecular Weight

19S RNA

16S RNA

42-43

21-23

2,300

1,200

770

400

85,000

44,000

Data taken from Kamen and Shure (1976) ; the assumptions made are the same as in Table 1 .

and 25 map units on the viral DNA . VP2 is coded for by the DNA corresponding to the additional sequences in 19S, comprising much of Hpall fragment 3 and part of fragment 1 . This region extends from 68 to 47 map units on the DNA . Figure 4 also indicates the suspected location of sequences coding for polyoma VP3 and T antigen, although no evidence supporting the latter allocations has been presented in the work described here . Miller and Fried (1976) have recently used the marker rescue method to map polyoma late temperature-sensitive mutants ts10, ts1260, and tsC, as well as the plaque morphology phenotype, into the DNA region between 45 and 27 map units known to code for the 16S mRNA . From our assignment of this RNA as the VP1 messenger, we conclude that these markers lie in the VP1 gene . This conclusion agrees with the recent protein chemical studies of R . Hewick, L . Miller, M . D . Waterfield, and M . Fried (manuscript in preparation) . Similar studies to those reported here have been reported by other investigators using SV40 virus (Prives et al ., 1974a, 1974b) . The corresponding 19S RNA and 16S late cytoplasmic mRNAs have been translated and the 16S RNA shown to code for VP1 . More recently, the SV40 16S RNA has been mapped and shown to be located at the equivalent position on SV40 DNA as polyoma 16S RNA (May, Kopecka, and May, 1975 ; Khoury et al ., 1976) . Furthermore, Fiers and his colleagues (Fiers et al ., 1975b) have established the precise location of the coding sequence for the N terminal region of SV40



Cell 486

a--LATE

4 REGION

60

80

0

20

EARLY REGION OR

Hpa 11

3 5 8,



EC R1 7,l, 2 1

6

i6

5.

20S ,

16S 5 .

T antigen VP1

VP3

Figure 4. Location of Sequences Coding for Viral Proteins on Polyoma DNA

VP1 and have shown it to be at the end of restriction endonuclease Hind fragment K at 95 map units . SV40 B, C, and BC cistron mutations have been assigned to the region of the SV40 genome between 96 and 18 map units, which determines 16S RNA (Lai and Nathans, 1974) . Thus there is little doubt that the gene for SV40 VP1 is located at a position corresponding to that which we have established for polyoma . The identity of the proteins coded for by SV40 19S RNA is less certain . Purified 19S RNA codes for VP1, a polypeptide called X, and several minor products (Prives et al ., 1974a) . Polypeptide X is reported not to be present in purified virions, but is found specifically in virus-infected cells . It is probably coded for by the region of SV40 DNA corresponding to that shown here to be the polyoma VP2 gene . Indeed, it seems probable that SV40 polypeptide X is analogous to polyoma VP2 . SV40 19S DNA separated on nondenaturing sucrose gradients also codes for VP1 (Prives et al ., 1974a), but by analogy with polyoma 19S RNA, it seems probable that the VP1 initiation site on purified SV40 19S RNA would be inactive . Inactive Internal Initiation Site on 19S RNA The coding sequence for VP2 in polyoma 19S RNA is present toward the 5' end of the messenger and that for VP1 toward the 3' end . Some viral mRNAs containing sequence information for more than one protein are translated into a precursor polyprotein which is subsequently cleaved to the different daughter proteins (Korant, 1975) . It is improbable that 19S RNA is translated to give a precursor containing both VP2 and VP1 sequences for the following reasons : first, no such precursor has been detected either in vivo or in vitro ; second, purified 16S RNA is translated directly to give VP1, and it is improbable that a single protein is made by two different mechanisms ; third, we have found that polyomaVP1 can be labeled with formyl-methionine from initiator tRNA, demonstrating directly that VP1 has its own site for the initiation of protein synthesis (S .

T . Bayley, R . Harvey, and A . E . Smith, unpublished observations) . Together these results argue that 19S RNA contains an initiation site for VP1 synthesis at an internal position in the molecule . Our finding that virtually no VP1 is synthesized by highly purified 19S RNA suggests that the internal initiation site is inactive . We do not know the molecular basis of this inactivity, but it is possible that the internal initiation site is made inaccessible by extensive secondary structure, as in the synthetase cistron of phage MS2 RNA (Fiers et al ., 1975a) . Alternatively, the presence of a capped 5' terminus adjacent to the ribosome binding site may be essential for the activity of the VP1 mRNA as in other viral mRNAs (Both et al ., 1975) . Aloni, Shani, and Reuveni (1975) have argued that SV40 19S RNA is a cytoplasmic precursor to 16S RNA ; perhaps it is only after the cleavage of 19S and capping of the resulting 16S RNA that the VP1 mRNA is activated . The inherent instability of 19S RNA predicted by the precursor model and the ratio of 19S to 16S RNA present in cells (about 1 :4) may also account for the different amounts of VP1 and VP2 made in infected cells . Although we do not know the physiological significance of the inactive internal initiation site on polyoma RNA, this observation is not unique . Cells infected with Semliki forest virus contain two major viral RNAs : 42S RNA which corresponds with the viral genomic RNA, and a polysomal 26S RNA . 26S RNA corresponds to the 3' one third of 42S RNA and codes for a precursor to the structural proteins . 42S RNA codes for nonstructural proteins, but initiation from the internal site equivalent to that in 26S RNA is not detected in vitro (Glanville et al ., 1976) . Similarly, the plant viruses, Brome mosaic virus (Shih and Kaesberg, 1973), and tobacco mosaic virus (Hunter et al ., 1976) have inactive internal initiation sites which are present toward the 3' end of larger RNA species and which contain coding information for viral coat proteins . In both cases, a smaller RNA species has been detected which is active in the synthesis of coat protein . In none of these cases is it known whether the internal initiation sites are active in vivo . Experimental Procedures Isolation of infected cell RNA, translation in vitro, and analysis of the in vitro product by SDS polyacrylamide gel electrophoresis and immunoprecipitation have all been described in detail (T . Wheeler et al ., manuscript submitted for publication) . Formamide Sucrose Gradient Centrifugation mRNA was fractionated on denaturing sucrose density gradients containing either 50% (v/v) or 85% (v/v) formamide (BDH Analar) . 50% formamide gradients (Lewis et al ., 1975) contained 5-20% sucrose in 100 mM LiCI, 5 mM EDTA, 0 .2% SDS, 10 mM Tris-HCI (pH 7 .5) . Centrifugation was for 17 .5 hr at 35,000 rpm in a Beckman

Location of Sequences Coding for Polyoma Proteins 487

SW40 rotor. Sample preparation was described by Kamen and Shure (1976) . 85% formamide gradients (Macnaughton et al ., 1974) contained 2-10% sucrose in 10 mM Tris-HCI (pH 7 .5), 1 mM EDTA . Centrifugation was at 25°C for 16 hr at 40,000 rpm in a Beckman SW56 rotor . 10-25 µg quantities of total poly(A) RNA in 10-25 gl H 2 O were mixed for 10 min at room temperature with an equal volume of formamide-containing buffer before centrifugation . Fractions (10-30 85% formamide, 30-75 50% formamide) were collected through the bottom of the tube and precipitated with alcohol after the addition of 10 Ng carrier wheat germ tRNA . Fractions were reprecipitated 3 times before translation . Acknowledgments We thank Linda Straus for help in running 85% formamide sucrose gradients . Received June 9, 1976 ; revised August 3, 1976 References Aloni, Y ., Shani, M ., and Reuveni, Y . (1975) . Proc . Nat . Acad . Sci . USA 72, 2587 . Boedtker, H . (1968) . Methods Enzymol . 12B, 429 . Both, G . W., Furuichi, Y ., Muthukrishnan, S ., and Shatkin, A . J . (1975) . Cell 6, 185 . Buetti, E . (1974) . J . Virol . 14, 249 . Fey, G ., and Hirt, B . (1974) . Cold Spring Harbor Symp . Quant. Biol . 39, 235 . Fiers, W ., Contreras, R ., Duerinck, F., Haegmean, G ., Merregaert, J ., Min Jou, W ., Raeymakers, A ., Volckaert, G ., Ysebaert, M ., Van der Kerckhwe, J ., Nolf, F ., and Van Montagu, M . (1975a) . Nature 256, 273 . Fiers, W ., Rogiers, R ., Soeda, E ., Van de Voorde, Van Heuverswyn, H ., Van Herreweghe, J ., Volckaert, G ., and Young, R . (1975b) . FEBS Symp . 39, 17 . Gibson, W . (1974) . Virology 62, 319 . Glanville, N ., Ranki, M . Morser, J ., Kaariainen, L ., and Smith, A . E . (1976) . Proc . Nat . Acad . Sci . USA 73, 3059 . Griffin, B ., Fried, M ., and Cowie, A . (1974) . Proc . Nat . Acad . Sci . USA 71, 2077 . Hunter, T. R ., Hunt, T ., Knowland, J ., and Zimmern, D . (1976) . Nature 260, 759 . Kamen, R ., and Shure, H . (1976) . Cell 7, 361 . Khoury, G ., Carter, B . J ., Ferdinand, F . J ., Howley, P . M ., Brown, M ., and Martin, M . (1976) . J . Virol . 17, 832 . Korant, B . D . (1975), In Proteases and Biological Control (New York : Cold Spring Harbor Laboratory), p . 621 . Lai, C . J ., and Nathans, D . (1974) . Cold Spring Harbor Symp . Quant . Biol . 39, 53 . Lewis, J . B ., Atkins, J . F ., Anderson, C . W ., Baum, P . R ., and Gesteland, R . F . (1975) . Proc . Nat . Acad . Sci . USA 72, 1344 . Macnaughton, M ., Freeman, K . B ., and Bishop, J . 0 . (1974) . Cell 1, 117 . May, E ., Kopecka, H ., and May, P . (1975) . Nucl . Acid Res . 2, 1995 . Miller, L ., and Fried, M . (1976) . J . Virol ., 18, 824 . Prives, C . L ., Aviv, H ., Gilboa, E ., Revel, M ., and Winocour, E . (1974a) . Cold Spring Harbor Symp . Quant . Biol . 39, 309 . Prives, C. L ., Aviv, H„ Paterson, B . M ., Roberts, B ., Rozenblatt, S ., Revel, M ., and Winocour, E . (1974b), Proc . Nat. Acad . Sci . USA 71, 302 . Robberson, D . L ., and Fried, M . (1974) . Proc. Nat . Acad . Sci . USA 71, 3497 .

Shih, P . S ., and Kaesberg, P . (1973) . Proc . Nat . Acad . Sci . USA 70, 1799 . Smith, A. E ., Bayley, S . T ., Mangel, W . F ., Shure, H ., Wheeler, T ., and Kamen, R . I . (1975a) . FEBS Symp . 39, 151 . Smith, A . E„ Bayley, S . T., Wheeler, T., and Mangel, W . F . (1975b) . INSERM 37, 331-338 .